Cell Biology International 27 (2003) 423–427
Cell Biology International www.elsevier.com/locate/cellbi
Effect of a single neonatal endorphin treatment on the hormone content of adult rat white blood cells and mast cells Gyo¨rgy Csaba 1*, Pe´ter Kova´cs 1, E´va Pa´llinger 2 1
Department of Genetics, Cell and Immunobiology, Semmelweis University, H-1445 Budapest, POB 370, Hungary 2 Molecular Immunological Research Group of the Hungarian Academy of Sciences, Budapest, Hungary Received 5 September 2002; revised 21 November 2002; accepted 28 January 2003
Abstract 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 -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. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Endorphin; Serotonin; White blood cells; Mast cells; Hormonal imprinting
1. Introduction In addition to their specific functions, white blood cells and mast cells produce, contain and secrete many hormones or hormone-like molecules, such as endorphin (Blalock, 1998; Panerai and Sacerdote, 1997; van Woudenberg et al., 1992), histamine, serotonin (Selye, 1965), chorionic gonadotropin (Alexander et al., 1998; Hotakainen et al., 2000) and growth hormone (Recher et al., 2001). Some of these hormones have known functions, e.g. in the mechanism of defense against the inflammatory process (Blalock, 1998; Panerai and Sacerdote, 1997; van Woudenberg et al., 1992; Selye, 1965), however, the role of others is unknown. Certain interactions between these hormones have also been demonstrated. Beta-endorphin, for example, influences growth hormone or chorionic gonadotropin secretion (Cemerikic et al., 1991), as well as serotonin secretion (Sacerdote et al., 1991; Pini et al., 1997). The first perinatal encounter between the developing receptor and its target hormone is the initial step in hormonal imprinting. This is essential for the normal maturation of receptors (Csaba and Nagy, 1987), which * Corresponding author. Tel.: +36-12102950; fax: +36-13036968 E-mail address:
[email protected] (G. Csaba).
reach adult binding capacity after imprinting (Csaba, 1980, 1994, 2000). An absence or excess of hormone during that period, as well as the presence of molecules different from the target hormone, but still able to bind to the receptor (e.g. members of the same hormone family, synthetic hormones, drugs or environmental pollutants), leads to life-long receptorial, morphological, biochemical, behavioral, and even genetic consequences (Bern et al., 1975, 1987; Csaba, 1980; Csaba, 1994, 2000; Gibson et al., 1991; Gray-Nelson et al., 1994; Iguchi, 1992; Mirzahosseini et al., 1996; Tchernitchin and Tchernitchin, 1992). Imprinting may also cause altered production of the imprinter hormone for life, e.g. a single neonatal treatment of interleukin-6 or serotonin increases their levels in peritoneal and blood cells for life (Csaba and Kova´cs, 2001; Csaba et al., 2002a). Bearing these points in mind, we thought it reasonable to study the effect of a single neonatal endorphin treatment on the hormone content of mast cells and white blood cells in adult rats. 2. Materials and Methods 2.1. Rats Newborn male and female animals from our closed Wistar breed (of Charles River origin) were given 3 µg
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00034-9
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G. Csaba et al. / Cell Biology International 27 (2003) 423–427
Table 1 Hormone content of white blood cells of neonatally endorphin treated adult female rats.
Table 2 Hormone content of white blood cells of neonatally endorphin treated adult male rats.
Geo-means.d.
Geo-means.d.
Hormone
Lymphocytes
Monocytes
Granulocytes
Hormone
Lymphocyte
Monocyte
Granulocyte
End in treated End in control 5HT in treated 5HT in control HCG in treated HCG in control
28463 31855 16049 16718 5529 4921
31667 37787 21733 24037 7934 6727
21953 22047 32871 27165 9047 7026
End in treated End in control 5HT in treated 5HT in control HCG in treated HCG in control
4724 5724 6511 8223 3213 3414
5226 5719 7314 8929 35115 3815
11888 11468 292276 236256 8576 6660
End=endorphin; 5HT=serotonin; HCG=chorionic gonadotropin.
endorphin (Sigma, USA) subcutaneously 24 h after birth. When the animals were 2 months old, isotonic sodium citrate was injected (under ether anesthesia) into the peritoneal cavity, which was recovered after 30 s. Blood samples were then obtained by cardiac puncture into isotonic sodium citrate solution. 2.2. Flow cytometry Erythrolysis was performed before labeling, using Becton Dickinson FACS Lysing Solution. The lysed red blood cell remnants were removed by washing in PBS, and the cells were fixed in 4% paraformaldehyde solution. The cells were then permeabilized with 0.1% saponin. The hormone content of permeabilized cells was measured with primary antibodies to rat endorphin (produced in rabbit, Sigma, USA), serotonin (produced in rabbit, Sigma, specific to rat), hCG (anti-human chorionic gonadotropin -subunit, produced in mouse, Sigma), or human growth hormone (produced in rabbit, Sigma). FITC-labelled anti-rabbit or mouse IgGs (Sigma) were used as secondary antibodies. To control for the specificity of the antibodies, the autofluorescence of the cells and aspecificity of the secondary antibodies were also noted. Measurements were performed in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, USA), using 10,000 cells for each measurement. The CellQuest 3.1 software program was used. During the evaluation, cell populations were separated (‘gated’) on the basis of size and granulation (mast cells are richer in granules and much larger than the others). The hormone content of identical cell populations was compared. 2.3. Confocal microscopy After the flow cytometric analysis, the cells were subjected to confocal microscopic analysis in a BioRad MRC 1024 confocal laser scanning microscope, equipped with krypton-argon mixed gas-laser as a light source, at an excitation wavelength of 480 nm. Confocal microscopy supported the flow cytometric data and the accuracy of gating. All experiments were repeated twice.
End=endorphin; 5HT=serotonin; HCG=chorionic gonadotropin. Table 3 Hormone content of peritoneal mast cells of neonatally endorphin treated (imprinted) adult rats. Hormone
Geo-mean s.d. female
Geo-means.d. male
Endorphin in treated Endorphin in control Serotonin in treated Serotonin in control HCG in treated HCG in control
39362† 607180 24448* 37983 7235 11388
8951 8245 105131 13042 4219 4010
† *
Significance=P
