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.     

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.     

Prolonged effect of endorphin treatment during pregnancy in the rat on the histamine content of immune cells of F1 and F2 offspring generations

Female rats were treated with beta-endorphin on the 19th day of pregnancy and the histamine content of immune cells (blood lymphocytes; peritoneal lymphocytes, monocyte-macrophage-granulocyte group, mast cells; thymic lymphocytes) of the 7-week-old progenies (F1 generation) was studied using a flow-cytometric immunocytochemical technique. In an other group, female F1 progenies of endorphin-treated mothers were mated with control males and the F2 generation was monitored for histamine content similar to the F1. In the F1 generation each cell type, except peritoneal and blood lymphocytes, contained significantly more histamine than the control cells. In the F2 generation only mast cells contained significantly more histamine relative to the appropriate control. This means that the effect of endorphin (hormonal) imprinting is transmitted transgenerationally, but with decreasing intensity however. Mast cells retained the effect of imprinting for longer than the other cells. The results are compared with the levels of serotonin in similarly treated animals, studied in earlier experiments. As the endorphin level can be elevated during pregnancy (by pain, traumatization, or other stress conditions) this can the set biogenic amine content of adult immune cells.     

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.     

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.     

Impact of neonatal imprinting with vitamin A or D on the hormone content of rat immune cells

Neonatal single vitamin A (retinol; 3 mg) or vitamin D3 (cholecalciferol; 0.05 mg) treatment (imprinting) was done in male and female rats and 2 months later the endorphin, triiodothyronine (T3) and ACTH content of immune cells (peritoneal lymphocytes, monocyte-granulocyte-macrophage group [mo-gran], mast cells and thymic lymphocytes) were studied immunocytochemically by using flow cytometry and confocal microscopy. The T3 content was significantly decreased in peritoneal lymphocytes and in mo-gran and the endorphin content decreased in thymocytes of male animals, while ACTH was decreased in female lymphocytes and mo-gran. Vitamin D treatment was absolutely ineffective. The imprinting effects of vitamins A and D and their differences are discussed. The results call attention to the possible harmful effect of vitamin treatments during the perinatal critical period.     

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.     

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.     

Beta-endorphin in granulocytes

The literature indicates that beta-endorphin can be found in the mononuclear cells of peripheral blood. In the present experiments the endorphin content of granulocytes was studied, compared to lymphocytes as reference cells. Granulocytes as well, as lymphocytes contain endorphin. The granulocytes' endorphin content is much higher. Both lymphocytes and granulocytes are also able to take up endorphin from the milieu.     

Influence of paraformaldehyde and EDAC fixation on the demonstrability of hormones (histamine, endorphin, triiodothyronine) in rat immune cells: an immunocytochemical comparative analysis

The amount and localization of three hormones (histamine, endorphin and triiodothyronine [T(3)]) was measured in male and female rat peritoneal cells (lymphocytes, mast cells, monocyte-macrophage-granulocyte group [mo-gran]) using flow cytometry as well as confocal microscopy after paraformaldehyde (PFA) or EDAC fixation. In the EDAC fixed lymphocytes and mo-gran of female animals two-magnitude higher levels of histamine were measured after EDAC fixation and one magitude higher in mast cells. The amount of T(3) was almost four-fold in lymphocytes and 2.5-4-fold in mast cells and mo-gran. Endorphin content was not altered by the type of fixation. In each cell type in males one magnitude higher levels of histamine and T(3) were measured after EDAC fixation and a small, but significant, elevation of endorphin. Confocal microscopy supports the quantitative data. The results show that (1) the fixation with the crosslinking molecule, EDAC, is more suitable for immunocytochemical studies of amino-acid type hormones in immune cells, (2) more histamine and T(3) are present in the immune cells than it was supposed previously when studying PFA-fixed preparations, (3) the estimation of the amount of peptide hormones seems to be accurate after PFA fixation, (4) there is a quantitative difference comparing the results of PFA and EDAC fixation between males and females.     

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.     

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.     

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).     

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.     

In vitro effect of biogenic amines on the hormone content of immune cells of the peritoneal fluid and thymus. Is there a hormonal network inside the immune system?

Immune cells contain different hormones and hormone-like molecules, such as insulin, endorphin, triiodothyronine (T3) histamine, serotonin. In earlier in vitro experiments insulin down-regulated histamine, serotonin and T3 content of thymus cells. Now we studied the effect of biogenic amines on the endorphin, T3, serotonin and histamine content of rat peritoneal and thymic cells. Cells were obtained from male rats of 100g body weight. 100 ng/ml serotonin or 300 ng/ml histamine was added for 30 min. After that the cells were prepared for flow cytometric analysis with antibodies to endorphin, T3, histamine and serotonin as primary antibodies and anti-rabbit IgG as secondary antibody. Finishing the measurements the cells were also studied by confocal microscopy. T3 concentration (binding of anti-T3 antibody) increased in peritoneal mast cells after serotonin treatment and in the monocyte-macrophage-granulocyte group after histamine treatment. Thymocytes' T3 content radically decreased after both treatments. Serotonin and histamine treatment also radically reduced the amine content of each other. Endorphin level was resistant to hormonal treatments. The results call attention to a possible hormonal network inside the immune system in which hormones produced by the immune cells themselves can influence each other.     

Acute and delayed effect of (-) deprenyl and (-) 1-phenyl-2-propylaminopentane (PPAP) on the serotonin content of peritoneal cells (white blood cells and mast cells)

Acute and delayed (hormonal imprinting) effect of (-) deprenyl and its derivative without MAO-B inhibitory activity (-) PPAP, were studied on cells of the peritoneal fluid (lymphocytes, monocytes, granulocytes and mast cells) by flow cytometric and confocal microscopic analysis. Thirty minutes after treatment of 6-week-old female animals, deprenyl was ineffective while PPAP significantly increased the serotonin level of these cells. Three weeks after treatment at weaning, deprenyl drastically decreased the serotonin level of each cell type, while PPAP moderately but significantly increased the serotonin level of monocytes, granulocytes and mast cells. This means that the two related molecules have different effects on the immune cells, which seem to be independent of MAO-B inhibition. The experiments emphasize the necessity of studying the prolonged effects of biologically active molecules, even if they are without acute effects. As serotonin is a modulator of the immune system, the influence on immune cells of the molecules studied can contribute to their enhancing effect.     

Single neonatal treatment with beta-endorphin (hormonal imprinting) extremely enhances nocistatin level of cerebrospinal fluid in adult rats

In earlier experiments endorphin treatment of newborn rats caused the decrease of brain serotonin content, increasing aggressivity, enhanced sexual activity of females and changes in the binding capacity of uterine estrogen receptors at adult age, however nociceptin content of the cerebrospinal fluid was not changed. In the present experiment neonatal treatment of male and female rats was done with a single dose of 3 microg beta-endorphin and in five months old rats the level of nociceptin antagonist nocistatin was determined by radioimmunoassay in the cerebrospinal fluid. In both genders the amount of nocistatin was one magnitude higher in the endorphin treated groups. There was also a significant difference between the male and female nocistatin level in the treated and non-treated groups alike, with the advantage of females. The results call attention to the possibility of influencing pain-tolerance for life, by the pain-provoked endorphin levels during delivery.     

Effect of neonatal beta-endorphin imprinting on sexual behavior and brain serotonin level in adult rats

A single dose (3 microg) beta-endorphin was administered to newborn female and male rats (hormonal imprinting). In adult age (at 5 months) sexual behavior, steroid hormone binding capacity and brain serotonin content was studied. Females' sexual activity (lordosis quotient) significantly decreased and more animals protested against mounting (ratio of kicking and crying 21/24 vs. 8/24; p < 0.001). Males' sexual activity did not change, however more males were aggressive (4/10 vs. 1/10). Uterine estrogen receptor density significantly increased and affinity decreased. There was no change in the binding capacity of thymic glucocorticoid receptors. In the brain, five regions were studied for serotonin content. There was a gender difference in serotonin level and the intragroup differences were also high. In the endorphin treated males the serotonin level was significantly lower than in the controls. In the endorphin treated females the intragroup scattering has been significantly reduced. Nociceptin content of the cerebrospinal fluid was not changed. The experiments call attention to the possibility of adjustment of sexual and behavioral sphere by the individually different endorphin surge during labor.     

Glycosphingolipid patterns of peripheral blood lymphocytes, monocytes, and granulocytes are cell specific

We analyzed the amounts and types of glycosphingolipids (GSLs) from peripheral blood lymphocytes, monocytes, and granulocytes isolated by counter-current elutriation. The three cell types contained different amounts of neutral and acidic GSLs. The highest amount of neutral GSLs (109 micrograms/10(8) cells) was found in granulocytes, with considerably less found in monocytes (11 micrograms/10(8) cells) and lymphocytes (4 micrograms/10(8) cells). The neutral GSLs were composed of four types of lipids, GL1 through GL4 (mono-, di-, tri-, and tetraosylceramide). The highest percentage of GL1 was detected in lymphocytes and the lowest percentage in granulocytes, with the reverse order observed for GL2. GL3 and GL4, which were minor components of the neutral GSLs, were highly cell specific, with lymphocytes containing GL3 and GL4 of the globo series, granulocytes containing GL3 and GL4 of the lacto or neolacto series, and monocytes containing GL3 and GL4 of both types. The acidic GSL, sialosyl hexaosylceramide (lacto-series), was abundant in granulocytes but not in monocytes or lymphocytes. Another ganglioside, GM3, although present in all three cell types, was most abundant in monocytes and lymphocytes, whereas sialosyl paragloboside was higher in granulocytes than in lymphocytes and monocytes. These results indicate that peripheral blood lymphocytes, monocytes, and granulocytes have distinct "GSL fingerprints."