Prolonged effect of a single serotonin treatment in adult age on the serotonin and histamine content of white blood cells and mast cells of rats

Hormonal imprinting was provoked by serotonin treatment in adult age. Three weeks after treatment with 100 microg serotonin, the serotonin and histamine content of peritoneal cells (mast cells, lymphocytes and the monocyte-macrophage-granulocyte group), white blood cells (lymphocytes, granulocytes and monocytes) and thymic lymphocytes was studied by flow cytometry. The content of both amines was significantly higher in the mast cells of males and lower in females. Blood lymphocytes contained a higher serotonin and histamine level in males, and a lower serotonin level in females. The peritoneal monocyte-macrophage-granulocyte group contained less serotonin in both males and females. Thymocytes contained higher levels of both amines in females and higher histamine level in males. The experiments demonstrate that a single treatment at adult age can provoke imprinting, which alters-in the present case-the serotonin and histamine content of immune cells durably.     

Single treatment (hormonal imprinting) of newborn rats with serotonin increases the serotonin content of cells in adults

Hormonal imprinting takes place perinatally at the first encounter between the hormone and its target receptor, causing the finishment of the maturation of receptor-signal transduction system. In the presence of an excess of the target hormone or related molecules faulty imprinting develops with life-long consequences. In earlier experiments single neonatal treatment with minute dose of IL-6 caused also prolonged stimulation of IL-6 production. In the present experiment newborn female and male rats were treated with 20 microg serotonin (hormonal imprinting) and were studied for serotonin content of different cell types in adult age. Serotonin content was measured by flow cytometry and its localization was determined by confocal microscopy. Serotonin content was detected in white blood cells (lymphocytes, monocytes and granulocytes); in lymphocytes, monocytes (macrophages), granulocytes and mast cells of peritoneal fluid and thymic lymphocytes. Serotonin was present in all cell types of control animals studied. Serotonin content extremely elevated in the white blood cells and also increased in the peritoneal cells of neonatally treated female animals. There was no elevation in thymic lymphocytes. The mean values of male animals remained at the control level. The experiments call attention to the life-long effect of the perinatal hormonal imprinting manifested presently in the elevation of serotonin content and point to the gender differences of serotonin imprinting. Considering the role of serotonin in mood and psychiatric diseases, the observations could have some clinical importance.     

Effect of the inhibition of triiodothyronine (T3) production by thiamazole on the T3 and serotonin content of immune cells

Triiodothyronine (T3) and serotonin are present in the cells of immune system (blood, peritoneal and thymic lymphocytes, monocytes and granulocytes of the blood and peritoneal fluid, mast cells). In the present experiments the effect of thiamazole, an antithyroid drug was studied on the content of these two hormones in immune cells after one and two weeks of continuous treatment (by drinking water, containing 30 mg/100 ml thiamazole, ad libitum) in adult male rats, using flow cytometric and confocal microscopic analysis. In thymic lymphocytes both hormone contents significantly increased in both time points. A significant decrease of T3 was observed in peritoneal monocytes and granulocytes also in both time points, in peritoneal mast cells after one week and in blood lymphocytes after two weeks. Serotonin content in addition to the elevated thymic values (in both measurements) was significantly reduced in blood lymphocytes after two weeks. Confocal microscopy demonstrated heterogeneous cell populations with disparate hormone content and mostly diffuse localization The experiments call attention to the presence of T3 in the immune cells and to its variable concentration under the effect of a thyrostatic drug as well, as to the T3-serotonin relationship in the cells of the immune system.     

Effect of a single neonatal endorphin treatment on the hormone content of adult rat white blood cells and mast cells

Using flow cytometry and confocal microscopy, the presence of endorphin, serotonin and chorionic gonadotropin (hCG) was demonstrated in rat white blood cells and peritoneal mast cells. After a single neonatal treatment with beta-endorphin (hormonal imprinting), the mast cells of female rats reaching adulthood contained significantly less endorphin and serotonin, as well as slightly less hCG, than control cells. There was no change in the hormone content of the mast cells of males. The lymphocytes, monocytes and granulocytes of both sexes also contained the three hormones, but endorphin imprinting had no effect on these cells.     

Three-generation investigation on serotonin content in rat immune cells long after beta-endorphin exposure in late pregnancy.

Female rats were treated with beta-endorphin on the 19th day of pregnancy. Serotonin content of immune cells (peritoneal lymphocytes, monocyte-macrophage-granulocyte group (mo-gran), mast cells, blood lymphocytes, granulocytes and monocytes, thymus lymphocytes) were studied in the mothers (P-generation four weeks after delivery), in the male offspring (F1) generation (at seven weeks), in the female offspring (four weeks after their own delivery) and in their offspring (F2 generation, at seven weeks). P-mother cells' serotonin content was not influenced by endorphin treatment, while F1 generation's mo-gran and blood lymphocyte serotonin content was reduced (in contrast, histamine content of mo-gran increased). Four weeks after delivery, an increase in serotonin content was observed in the F1 generation in the peritoneal lymphocytes and mast cells as well as in blood lymphocytes. In contrast, serotonin content was reduced in blood granulocytes and monocytes. In the F2 (grandson) generation, a reduction in mast cell serotonin content and sensitization of blood and thymic lymphocytes to repeated endorphin treatment was provoked. The significant changes were more expressed in the F2 generation compared to F1, also appearing earlier. The results unequivocally suggest that the increase in endorphin levels during late pregnancy can cause permanent changes in the F1 and F2 generations, which means that the imprinting effect can be transgenerationally transmitted.     

Effect of single neonatal or repeated benzpyrene exposure on the serotonin content of immune cells in young male rats

In earlier experiments single benzpyrene treatment of newborn rats caused strong alterations in the endorphin content of adult rats' immune cells. In the present experiments young (4-6 weeks old) male rats were studied for demonstrating the effect of the single neonatal or repeated (neonatally and at weanling) benzpyrene exposure on the serotonin content of immune cells (blood lymphocytes, monocytes, granulocytes; peritoneal fluid lymphocytes, mast cells, monocytes and granulocytes, thymic lymphocytes). Flow cytometric analysis showed that 50 microg benzpyrene treatment of five-week-old animals was ineffective after 5 days and this was the situation four weeks after single neonatal (20 microg) benzpyrene exposure. However, the repeated treatment of neonatally benzpyrene exposed 4 weeks old animals after 5 days resulted in elevated blood and thymic lymphocyte serotonin amount and in one index (peritoneal monocyte-granulocyte group) reduced serotonin content. This means that neonatal benzpyrene treatment does not influence directly the serotonin content (production or transport) of immune cells (unlike to the endorphin content) however, sensitizes them to a following benzpyrene exposure. The results widen the list of harmful effects (influencing steroid receptor binding, sexual behavior and immune cells' endorphin content) of perinatal benzpyrene exposure.     

Effects of deprenyl on monoamine oxidase and neurotransmitters in the brains of MPTP-treated aging mice

Deprenyl is a selective monoamine oxidase B (MAO-B) inhibitor and has been used in the treatment of Parkinson's disease. However, it is not known whether deprenyl effects are symptomatic or pharmacological. Aging mice were partially lesioned with MPTP. Control and MPTP-treated mice were given deprenyl in drinking water for 14 days. Brain tissue (including the striatum, olfactory tubercle and cerebral cortex) was assayed for MAO-B and neurotransmitter levels. The results show that deprenyl treatment, given alone or after MPTP, reduced MAO-B activity in all the three regions. No change was seen in dopamine (DA), 3,4-dihydroxyphenyl acetic acid (DO-PAC), and homovanillic acid (HVA) content in any of the three areas. Cortical norepinephrine (NE) levels were also unaltered. However, striatal serotonin (5-HT) levels were decreased while its metabolite, 5-HIAA levels were significantly increased in the olfactory tubercle in animals receiving deprenyl alone. These data suggest that deprenyl treatment reduces MAO-B activity in regions in addition to the striatum without affecting norepinephrine, dopamine (DA) and its metabolites.     

Effect of endorphin exposure at weaning on the endorphin and serotonin content of white blood cells and mast cells in adult rat

Hormonal imprinting takes place perinatally at the first encounter between the developing receptor and its target hormone, resulting in the accomplishment of normal receptor development. In the presence of an excess of target hormone or the absence of it, or an excess of related molecules which can be bound by the receptor, faulty imprinting develops with life-long consequences. In previous experiments neonatal endorphin exposure caused a decrease in endorphin and serotonin content of peritoneal mast cells of adult animals. In the present experiment 25-day-old (weaned) female rats received 2 microg endorphin, and the endorphin as well as serotonin content of adult mast cells and white blood cells was studied by flow cytometry and confocal microscopy. Peritoneal lymphocytes and blood monocytes contained significantly (p<0.01) less endorphin and peritoneal mast cells less serotonin (p<0.07, i.e. of questionable significance) than the untreated control. The results bring attention to the possibility of durable imprinting of differentiating cells later in life and to the durable (possibly life-long) effect of an endorphin excess (perhaps caused by injury) manifested in the change of endorphin and serotonin content of immune cells.     

Prolonged impact of pubertal serotonin treatment (hormonal imprinting) on the later serotonin content of white blood cells

The first encounter between the developing receptor and its target hormone establishes the hormonal imprinting which is needed for the normal function of the cell. In the presence of foreign-however able to bind-molecules, faulty imprinting develops with lifelong consequences. Hormonal imprinting influences not only the receptors, but also the later hormone production of cells. The critical time of hormonal imprinting is the perinatal period, however it can be executed sometimes (in continuously differentiating cells) also at puberty. As in earlier experiments single neonatal serotonin treatment caused a life-long alteration of white blood serotonin content in female rats, the early (10-19 day) and late (8 weeks) effect of single pubertal serotonin treatment was studied presently, by using flow cytometry. In contrast to the earlier (neonatal) results, pubertal treatment caused a radical reduction of serotonin content in male's lymphocytes, monocytes, granulocytes and mast cells, independent on the time of study. The effect in females was rather increasing, however uncertain. The experiments call attention to the possible different effects of neonatal and pubertal hormonal imprinting and to the imprintability of blood cells in adolescence.     

Effect of concanavalin A (Con-A) on the hormone production of the unicellular Tetrahymena and the immune cells of the rat. A comparative study

Tetrahymena populations were treated with 10(-15) g ml(-1) or 10(-6) g ml(-1) concanavalin-A (Con-A) in tryptone-yeast medium for 1 h. Rat peritoneal immune cells (mast cells, lymphocytes, monocyte-granulocyte group) were also treated with 10(-6) g ml(-1) Con-A, for 1 h. The cells' hormone (ACTH, histamine, serotonin, endorphin, triiodothyronine (T(3))) content was measured by using immunocytochemistry and flow cytometry. The extremely low dose of Con-A universally and significantly elevated the hormone contents, while the result of higher dose was uncertain. In the immune cells, Con-A significantly decreased the ACTH level in each cell type and histamine level in mast cells. The results demonstrate the very high sensitivity of Tetrahymena receptors for a non-hormone (lectin) molecule, which can bind to the insulin receptors and mimics the effect of insulin. The results also show that Tetrahymena receptors are more sensitive to lower concentrations of molecules than to higher ones. The universal hormone-production stimulating effect of Con-A-which is observed in Tetrahymena-is specified in rat.     

Gender differences in the histamine and serotonin content of blood,peritoneal and thymic cells: a comparison with mast cells

The serotonin and histamine content of mast cells and white blood cells in adult male and female rats was compared, using a flow cytometric immunological method. Serotonin was significantly higher in female peritoneal mast cells, peritoneal monocyte-ganulocyte-macrophage cells, blood lymphocytes and blood thymocytes. Histamine was significantly higher in female peritoneal monocyte-granulocyte-macrophage cells, and blood lymphocytes, monocytes and granulocytes, but was significantly less in thymocytes. Peritoneal lymphocytes and the monocyte-granulocyte-macrophage group contained significantly more histamine than mast cells. These experiments call attention to gender differences in the levels of biogenic amines in cells participating in defence reactions, and to the possible non-unique role of mast cells in serotonin and histamine supply.     

Prolonged impact of five imprinters on the serotonin content of white blood cells and mast cells of weanling rats: outstanding effect of benzpyrene and chlorpheniramine

In previous experiments, treatment at weaning or adult age with endorphin, serotonin or an antihistamine (late hormonal imprinting) durably influenced the serotonin content of white blood cells and mast cells of rat. In the present experiments, five molecule (approved imprinters in other indexes) were studied for imprinting effect of immune cells, 3 weeks after a single treatment at weaning. Three steroid hormone-like molecule (vitamin D3, mifepristone and dexamethasone) were ineffective (except dexamethasone in 1/4 indexes), while benzpyrene (aromatic hydrocarbon) and chlorpheniramine (H1-receptor blocker antihistamine) were highly effective (5/6 and 4/4 respectively). The results indicate: (1) a prolonged (late imprinting) effect of a single treatment with certain molecules acting at receptor level; (2) non-generality of late imprinting, and (3) the very extensive effects of benzpyrene, which in earlier experiments was one of the strongest imprinter at receptorial and behavioral level at any periods of life studied.     

Effect of serotonin-acting agents on the serotonin content of immune cells. A peculiar observation

The effect of the tryptophan hydroxylase inhibitor, PCPA methylester, the serotonin reuptake inhibitor fluoxetine and MAO-A inhibitor clorgyline on the serotonin content of rat immune cells was studied, using labelled antibodies and flow cytometry. Each molecule significantly increased in males the serotonin concentration of peritoneal lymphocytes and the monocyte-macrophage-granulocyte group (mo-gran), however the agents were ineffective towards mast cells. In females fluoxetine and clorgyline increased the serotonin concentration in peritoneal lymphocytes and mo-gran. Fluoxetine also increased the serotonin level in mast cells. Thymus was absolutely resistant to the drugs in both genders. The results call attention (1) to the reverse effect of serotonin-acting agents on immune cells, (2) to the influence of the milieu where the cell is located and (3) the effect of gender.     

Phenotypic and functional changes of circulating monocytes and polymorphonuclear leucocytes from elderly persons

The function and phenotype of monocytes and granulocytes in the elderly is consistently remodelled. Because leucocyte adhesion molecules play important roles in mediating a wide variety of leucocyte functions, age-related changes in their expression on granulocyte and monocyte surfaces could be partially responsible for immune dysfunctions during senescence. Considering the central role of innate immunity in the process of immunosenescence and the involvement of cell adhesion molecules (CAM) in the great majority of leucocyte functions, we studied the expression of CD50 and CD62L adhesion molecules in peripheral blood granulocytes and monocytes from healthy elderly and young subjects. We show here that the percentage of granulocytes and monocytes expressing CD62L is decreased in the elderly, whereas its density expression is unchanged on both cell types. A downregulation of the density expression of CD50 at a per cell level characterizes granulocytes in the elderly, whereas CD50 expression on monocytes from old subjects shows a peculiar attitude: its density expression decreases whereas the number of positive cells is expanded. The downregulation of this receptor on granulocytes from aged people could determine a state of hyperactivation contributing to the proinflammatory status of the elderly, while the lower expression on monocytes could therefore contribute to the impaired antigen presentation in the elderly. On the other hand, the increased number of CD50 positive monocytes in the elderly, despite its decreased density expression at a per cell level, could be interpreted as an attempt to counteract the inability to mount strong immune responses. Both CD50 and CD62L changes in ageing polymorphonuclear (PMN) cells allow recognition as non-self or senescent self to permit macrophages in the liver and spleen to remove them from the circulation. The increased proportion of granulocytes and monocytes lacking CD62L and the downregulation of CD50 intensity expression on both cell types may suggest a state of in vivo activation. Therefore, CD50 and CD62L shedding from the cell surface of activated granulocytes and monocytes could be interpreted as a tentative to counteract the dangerous effects of an excessive chronic inflammation in the elderly. However, the increased proportion of CD62L negative granulocytes in the elderly leads to an impairment in cell adhesion which is the first line of response to acute inflammatory stimuli. This phenomenon likely contributes to the increased susceptibility to acute infections of elderly people.     

Prolonged effect of stress (water and food deprivation) at weaning or in adult age on the triiodothyronine and histamine content of immune cells.

We used two days of total water and food deprivation as stress for female rats at weaning (three weeks old) and at adult age (two and a half months old). Triiodothyronine (T3) and histamine content of immune cells (lymphocytes, mast cells and monocyte-macrophage-granulocyte group in peritoneal fluid; lymphocytes, granulocytes and monocytes in blood; and lymphocytes in thymus) were studied three weeks after stress application using specific antibodies for flow cytometry and confocal microscopy. The stress at weaning increased T3 content of thymus lymphocytes. In case of adult T3, there was a cell type independent significant effect of stress, decreasing values in peritoneal fluid and slightly increasing effect in the blood. Histamine content of granulocytes was also significantly elevated. The experiments demonstrate that not only fetal or neonatal stress has long-lasting consequences, but also stress events in later periods of life in cells (organs) that are continuously differentiating. We will go on to discuss the importance of T3 and histamine in connection with stress and immunity.     

Endorphin content of white blood cells and peritoneal cells in neonatally benzpyrene treated adult rats

White blood cells of rats (lymphocytes, monocytes, macrophages, granulocytes and mast cells) contain beta-endorphin. Two months after a single neonatal benzpyrene treatment (imprinting) there is an elevated level of immunoreactive endorphin in the blood and peritoneal cells of female animals and blood cells of males. The endorphin content decreased in the peritoneal cells of males. In the blood, the granulocytes of female, and the lymphocytes of male rats contained the highest amount of endorphin. In the peritoneal fluid also the granulocytes of females contained the highest amount of endorphin, in contrast to males, where the endorphin content of cells decreased and the lowest level of it was present in the lymphocytes. The experiments justify that benzpyrene treatment can durably influence endorphin levels of white blood cells and gives new data to the already known lifelong health destroying effects of perinatal benzpyrene exposition (alterations of hormone receptor binding capacity and sexual behavior).     

Structure-activity studies leading to (-)1-(benzofuran-2-yl)-2-propylaminopentane, ((-)BPAP), a highly potent, selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain

The catecholaminergic and serotoninergic neurons in the brain change their performance according to the physiological need via a catecholaminergic/serotoninergic activity enhancer (CAE/SAE) mechanism. Phenylethylamine (PEA), tyramine and tryptamine are the presently known endogenous CAE/SAE substances which enhance the impulse propagation mediated release of catecholamines and serotonin in the brain. A PEA derivative, (-)deprenyl (selegiline), known as a selective inhibitor of MAO-B, is for the time being the only CAE/SAE substance in clinical use. Aiming to develop a selective CAE/SAE substance much more potent than (-)deprenyl, a series of new 1-aryl-2-alkylaminoalkanes, structurally unrelated to PEA and the amphetamines, was designed and prepared. Among them, (-)1-(benzofuran-2-yl)-2-propylaminopentane ((-)BPAP) was selected as a promising candidate substance for further studies. (-)BPAP significantly enhanced in rats the impulse propagation mediated release of catecholamines and serotonin in the brain 30min after acute injection of 0.36nmol/kg sc. In the shuttle box, (-)BPAP was in rats about 130 times more potent than (-)deprenyl in antagonizing tetrabenazine induced inhibition of performance. (+/-)BPAP protected cultured hippocampal neurons from the neurotoxic effect of beta-amyloid in 10(-14)-10(-15)M concentration.     

Prolonged effect of an H1-receptor blocker antihistamine on the histamine content of white blood cells and mast cells

Hormonal imprinting usually takes place perinatally at the first encounter between the developing receptor and its target hormone, determining the future binding capacity of the receptor for life. Molecules similar to a hormone can cause faulty imprinting also with life-long consequences. Hormone production of the imprinted cell is also durably influenced. In cytogenic organs imprinting can also be provoked in adulthood. At present the effect of a single terfenadine treatment in adult rats on the histamine content of peritoneal cells (lymphocytes, mast cells and the monocyte-macrophage-granulocyte group), white blood cells (lymphocytes, granulocytes, monocytes) and thymic lymphocytes was studied 3 weeks after treatment to clarify the effect of prolonged treatment with an antihistamine in adulthood.The cells were studied by flow cytometric analysis. Peritoneal mast cells contained significantly more and thymic lymphocytes significantly less histamine than controls. In the other cells the differences were not significant. The results support earlier observations on the effect of antihistamines on mast cell histamine release (inhibition) and call attention to the fact that this effect is durable (hormonal imprinting provoked in adults).     

Recombinant human (rh) stem cell factor and rhIL-4 stimulate differentiation and proliferation of CD3+ cells from umbilical cord blood and CD3+ cells enhance FcepsilonR1 expression on fetal liver-derived mast cells in the presence of rhIL-4

We previously reported that rhIL-4 induced apoptosis and rhIL-6 mediated protection of human mast cells derived from cord blood mononuclear cells. Based on the result, we attempted to obtain the phenotypes and differentiation of CD3+ cells from cord blood by investigating their cell surface markers in the presence of rhSCF plus rhIL-4. The effect of co-cultured CD3+ cells on fetal liver mast cells (FLMCs) was also determined. Phenotypes from cord blood-derived cells were analyzed by flow cytometry and cell numbers were determined. Fetal liver mast cells were cultured with cord blood-derived cells (mainly CD3+) in the presence of rhSCF and/or rhIL-4 and were analyzed to determine cell number and expression of Kit+ and FcepsilonR1. The percentage of CD3+ cells from cord blood-derived cells on day 0 was about 41 +/- 13.5%, following monocytes and granulocytes. CD3+ cells increased in number (1.5-fold) and purity (90%), whereas other cell types did not survive. More than 60% of CD3+ cells from cord blood at day 0 were CD4(-)CD8-. These double-negative cells dramatically decreased by 1 week of culture, while CD4+CD8+ cells increased in number and purity through 3 weeks of culture, and then decreased as greater numbers of single-positive T cells emerged. We also found that FcepsilonR expression on FLMC increased in the presence of rhIL-4, but was not affected by the T cells that developed from cord blood mononuclear cells. The results indicate that IL-4, a Th2 type cytokine, together with rhSCF, can induce T cell proliferations, differentiation, and maturation from cord blood progenitor cells.     

Recovery of MAO-B enzyme activity after (-)deprenyl (selegiline) pretreatment, measured in vivo

The recovery of MAO-B activity after a single dose of 0.25 mg/kg s.c. (-)deprenyl was measured in rat, by following the time course of the changes in the PEA (phenyl-ethylamine)-induced hyperactivity. One hour after its administration (-)deprenyl enhanced both the PEA-induced locomotion and stereotypy, however, its effect on the latter was more marked. At 24 h and at later time-points only stereotype was enhanced. The results show that a single small dose of (-)deprenyl which selectively blocks MAO-B, causes a long-lasting inhibition of PEA metabolism, and the enzyme activity needs more than one week to restore completely.