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Cell Biology International (2004) 28, 487–490 (Printed in Great Britain)
Immunologically demonstrable hormones and hormone-like molecules in rat white blood cells and mast cells
G Csaba*, P Kovács and Éva Pállinger
Department of Genetics, Cell and Immunobiology, Semmelweis University and Molecular Immunological Research Group, Hungarian Academy of Sciences, H-1445 Budapest POB 370, Hungary


Abstract

The presence or absence of four biologically active hormone or hormone-like molecules was studied in rat immune cells using specific antibodies with flow cytometry and confocal microscopy. Epidermal growth factor (EGF) was not demonstrable at all, digoxin was present only in blood lymphocytes, and insulin was found in the monocyte-macrophage-granulocyte group in peritoneal fluid and thymic lymphocytes. Immunologically demonstrable triiodothyronine (T3) was present in all cells studied (lymphocytes, mast cells and monocyte-macrophage-granulocytes in peritoneal fluid and blood, and thymic lymphocytes). While there is no explanation of the presence of digoxin and insulin, it is assumed that T3 is an extrathyroidal source of the hormone that is needed for maintaining cell proliferation and normal status in the immune system, particularly as it is absent in the case of transitional or durable thyroid deficiency.


Keywords: Triiodothyronine, Insulin, Digoxin, Immune system, Thyroid, White blood cells, Mast cells.

*Corresponding author. Tel./fax: +36-1-210-2950.


1 Introduction

White blood cells and mast cells produce and secrete cytokines, as well as histamine and serotonin, materials which have a direct role in immune reactions (Nicola, 1994; Selye, 1965; Csaba et al., 2003). Recently, it was demonstrated that β-endorphin is also produced by lymphocytes (Panerai and Sacerdote, 1997; Jessop, 1998) and granulocytes (Csaba et al., 2002), which alter the status of cytokine secretion and modulate immune reactions, suggesting that it could have a role in inflammatory processes. In addition, it was also demonstrated that hormones that do not have a well known influence on immunity, such as chorionic-gonadotropin (Alexander et al., 1998), luteinizing hormone (Sabharwal et al., 1992) and growth hormone (Lantinga van Leeuwen et al., 2000), are also present in different immune cells. This is why we wanted to study the presence of other hormones or hormone-like molecules in white blood cells and mast cells. We chose three molecules to study: a small peptide hormone (epidermal growth factor (EGF)), a polypeptide hormone (insulin), and an amino-acid-type hormone (triiodothyronine (T3)). Digoxin was chosen as a fourth molecule for the study as it is an endogeneous product in mammals (Castaneda-Hernandez, 1989; Ghione et al., 1993), with a steroid hormone-like chemical structure.

2 Materials and methods

2.1 Experimental animals

Two and a half-month-old female Wistar rats from our closed breed were used in the experiments. Isotonic sodium citrate solution was injected (during ether anesthesia) into the peritoneal cavity, which was regained after 30s. Blood was then taken by heart puncture (into sodium citrate), and cells were also obtained from the thymus. Four animals were used for observation and the experiments were repeated twice.

2.2 Flow cytometric analysis

Before labeling, erythrolysis was performed (with Becton Dickinson FACS Lysing Solution). The lysed red blood cell remnants were removed by washing with PBS and the cells were fixed in EDAC (1-ethyl-3[3-dimethylamino-propyl] carbodiimide; Sigma, USA). After that, the cells were permeabilized with 0.1% saponin. The hormone content of permeabilized cells was detected with the following primary antibodies: antihuman EGF (produced in mouse, E-2520, Clone EGF-10, Sigma, USA), monoclonal anti-insulin (produced in mouse, I-2018, Clone K360C10, Sigma), anti-3,3′,5-triiodo-l-thyronine (anti-T3, produced in rabbit, T-2777, Sigma), and monoclonal anti-digoxin (produced in mouse, D-8156, Clone Di 22, Sigma), and the following secondary antibodies: FITC-labeled anti mouse IgG (F-8771, Sigma) or anti-rabbit FITC-IgG (developed in goat, F-9887, Sigma). Autofluorescence of the cells and aspecificity of the secondary antibodies were also detected. 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 for data analysis.

During the evaluation, cell populations were separated on the basis of size and granulation and defined by gating. The hormone content (concentration) inside the cells was compared in identical cell populations. The numerical comparison of detected values was performed using percentage changes in geometric mean channel values (Geo-mean) with respect to the control groups treated only with secondary antibody.

2.3 Confocal microscopy

After flow cytometry, the cells were subjected to confocal microscopy 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 480nm.

3 Results and discussion

The presence or absence of four biologically active molecules was studied in rat immune cells. Although EGF has an immunomodulatory role (Elitsur et al., 1991; Freitas et al., 1998) and its presence has been demonstrated in mast cells (Artuc et al., 2002) and in the membrane of thymocytes (Freitas et al., 1998), we did not find it at all in the cells studied. Digoxin or digoxin-like material is also present in other cell types of several mammalian species (Castaneda-Hernandez, 1989), including humans (Ghione et al., 1993). However, using immunological methods, we only found this heart glycoside in blood lymphocytes (16.27±0.64 vs. 13.75±1.19; significance p<0.01), while the other cells studied were free of digoxin. Exogeneous digoxin has some effects on the immune system, but they are negative influences (Esposito et al., 1989; Gentile et al., 1997), so these do not explain its endogeneous presence in blood lymphocytes.

Immunologically demonstrable insulin was found in significant amounts in the monocyte-macrophage-granulocyte group of peritoneal fluid, and also in thymic lymphocytes (Table 1). However, we did not find it in peritoneal or blood lymphocytes, or in mast cells. It is not known whether this insulin was a product of the cell itself or was internalized from the circulation (Carpentier et al., 1981). The latter cannot be excluded, as insulin receptors can be found in the membrane of immune cells (Schwartz et al., 1975; Chen et al., 1983). Immunologically demonstrable insulin has been found in many cell types, but we have no explanation of its function in these cells. We also do not know why lymphocytes from different localizations behave differently, however, it is known that their origin or function is also different, so this could be connected with their different hormone content.


Table 1. Fluorescence of immune cells treated with anti-insulin and secondary antibodies (1+2) compared to secondary antibody alone (control)

Image

mo-gran = monocyte-macrophage-granulocyte group; n.s. = not significant.

The most surprising result was seen by studying the T3 content of the cells. All the cell types studied showed highly significant T3 positivity (Table 2). It is known that endodermal organs (such as thyroid and thymus) are able to take up iodine (Csaba et al., 1973 and Csaba and Prohaszka, 1978), which is needed for T3 production. This could explain why lymphocytes and mast cells contain T3, however, the T3 content of myeloid cells remains unknown. There are data in the literature on thyroid hormone–immune system interrelationships (Arpin et al., 2000 and Johnson et al., 1992). Physiological levels of thyroid hormones are needed to maintain the weights of thymus and spleen and normal levels of circulating lymphocytes ( Bachman and Mashaly, 1987).

Table 2. Fluorescence of immune cells treated with anti-triiodothyronine (T3) and secondary antibodies (1+2) compared to secondary antibody alone (control)

Image

mo-gran = monocyte-macrophage-granulocyte group.

A bidirectional communication between the hypothalamo-thyroid and immune systems is also assumed (Klecha et al., 2000), which points to the importance of T3 in the maintenance of immune status and proliferation of immune cells, particularly lymphocytes. T3 is also known to enhance the expression of interleukin-2 receptor (Nakanishi et al., 1999). On the basis of these data, the production (and/or accumulation) of immunologically demonstrable T3 could be an extrathyroidal source of the hormone, which is needed for a well-balanced T3 milieu to avoid thyroid insufficiency. The paracrine (and/or autocrine) secretion of the hormone could positively influence the immune status in a deficiency of thyroid-derived hormones. These ideas are supported by the observation that, in hypophysectomized mice, a significant increase in T4 is observed in parallel with extrapituitary regulation by TSH produced by dendritic cells (Bagriacik et al., 2001).

The confocal microscopic observations support the results obtained by flow cytometry. Using this technique, digoxin, as well as T3 fluorescence, seems to be dispersed in the cytoplasm without specific localization (Fig. 1).



Full-size image (28K) - Opens new windowFull-size image (28K)


The most surprising result was seen by studying the T3 content of the cells. All the cell types studied showed highly significant T3 positivity (Table 2). It is known that endodermal organs (such as thyroid and thymus) are able to take up iodine (Csaba et al., 1973; Csaba and Prohaszka, 1978), which is needed for T3 production. This could explain why lymphocytes and mast cells contain T3, however, the T3 content of myeloid cells remains unknown. There are data in the literature on thyroid hormone–immune system interrelationships (Arpin et al., 2000; Johnson et al., 1992). Physiological levels of thyroid hormones are needed to maintain the weights of thymus and spleen and normal levels of circulating lymphocytes (Bachman and Mashaly, 1987).


Table 2. Fluorescence of immune cells treated with anti-triiodothyronine (T3) and secondary antibodies (1+2) compared to secondary antibody alone (control)

Image

mo-gran = monocyte-macrophage-granulocyte group.

A bidirectional communication between the hypothalamo-thyroid and immune systems is also assumed (Klecha et al., 2000), which points to the importance of T3 in the maintenance of immune status and proliferation of immune cells, particularly lymphocytes. T3 is also known to enhance the expression of interleukin-2 receptor (Nakanishi et al., 1999). On the basis of these data, the production (and/or accumulation) of immunologically demonstrable T3 could be an extrathyroidal source of the hormone, which is needed for a well-balanced T3 milieu to avoid thyroid insufficiency. The paracrine (and/or autocrine) secretion of the hormone could positively influence the immune status in a deficiency of thyroid-derived hormones. These ideas are supported by the observation that, in hypophysectomized mice, a significant increase in T4 is observed in parallel with extrapituitary regulation by TSH produced by dendritic cells (Bagriacik et al., 2001).

The confocal microscopic observations support the results obtained by flow cytometry. Using this technique, digoxin, as well as T3 fluorescence, seems to be dispersed in the cytoplasm without specific localization (Fig. 1).



Full-size image (28K) - Opens new windowFull-size image (28K)


A bidirectional communication between the hypothalamo-thyroid and immune systems is also assumed (Klecha et al., 2000), which points to the importance of T3 in the maintenance of immune status and proliferation of immune cells, particularly lymphocytes. T3 is also known to enhance the expression of interleukin-2 receptor (Nakanishi et al., 1999). On the basis of these data, the production (and/or accumulation) of immunologically demonstrable T3 could be an extrathyroidal source of the hormone, which is needed for a well-balanced T3 milieu to avoid thyroid insufficiency. The paracrine (and/or autocrine) secretion of the hormone could positively influence the immune status in a deficiency of thyroid-derived hormones. These ideas are supported by the observation that, in hypophysectomized mice, a significant increase in T4 is observed in parallel with extrapituitary regulation by TSH produced by dendritic cells (Bagriacik et al., 2001).

The confocal microscopic observations support the results obtained by flow cytometry. Using this technique, digoxin, as well as T3 fluorescence, seems to be dispersed in the cytoplasm without specific localization (Fig. 1).


Fig. 1

Presence of digoxin (a) and T3 (c) in the cells of the peritoneal fluid. (b) and (d) are the respective controls (only secondary antibody added). 800×.


Acknowledgments

This work was supported by the National Research Fund (OTKA-T-042513) of Hungary. The authors thank Ms. Katy Kallay and Ms. Angela Kozák for their expert technical assistance.

References

Alexander H, Zimmermann, G, Lehmann, M, Pfeiffer, R, Schone, E, Leiblein, S, Ziegert, M. HCG secretion by peripheral mononuclear cells during pregnancy. Domest Anim Endocrinol 1998:15:377-87
Crossref   Medline   

Arpin C, Pihlgren, M, Fraichard, A, Aubert, D, Samarut, J, Chassande, O, Marvel, J. Effects of T3R alpha 1 and T3R alpha 2 gene deletion on T and B lymphocyte development. J Immunol 2000:164:152-60
Medline   

Artuc M, Steckelings, UM, Henz, BM. Mast cell–fibroblast interactions: human mast cells as source and inducers of fibroblast and epithelial growth factors. J Invest Dermatol 2002:118:391-5
Crossref   Medline   

Bachman SE, Mashaly, MM. Relationship between circulating thyroid hormones and cell mediated immunity in immature male chickens. Dev Comp Immunol 1987:11:203-13
Crossref   Medline   

Bagriacik EU, Zhou, Q, Wang, HC, Klein, JR. Rapid and transient reduction in circulating thyroid hormones following systemic antigen priming: implications for functional collaboration between dendritic cells and thyroid. Cell Immunol 2001:212:92-100
Crossref   Medline   

Carpentier JL, Halban, AE, Renold, AE, Orci, L. Internalization of 125I-insulin by IM-9 cultured human lymphocytes: a comparison between A14-monoiodo-pork and biosynthetic human insulin. Diabetes Care 1981:4:220-2
Crossref   Medline   

Castaneda-Hernandez G. Evidence for the existence of the same endogeneous digitalis-like factor in several mammalian species. Comp Biochem Physiol C 1989:94:49-53
Crossref   Medline   

Chen PM, Kwan, SH, Hwang, TS, Chiang, BN, Chou, CK. Insulin receptors on leukemia and lymphoma cells. Blood 1983:62:251-5
Medline   

Csaba G, Kiss, J, Nagy, SU. Comparative studies on the 125I uptake of the thyroid and thymus. Experientia 1973:29:357-8
Crossref   Medline   

Csaba G, Prohaszka, J. 131I uptake of one-day chicken endodermal organs. Acta Physiol Hung 1978:51:385-8

Csaba G, Kovács, P, Pállinger, É. β-Endorphin in granulocytes. Cell Biol Int 2002:26:741-3
Crossref   Medline   

Csaba G, Kovács, P, Pállinger, É. Prolonged effect of a single serotonin treatment in adult age on the serotonin and histamine content of white blood cells and mast cells. Cell Biochem Funct 2003:21:191-4
Crossref   Medline   

Elitsur Y, Majumdar, AP, Sakr, WA, Luk, GD. Epidermal growth factor regulation of DNA synthesis in human colonic lamina propria lymphocytes. Dig Dis Sci 1991:36:335-40
Crossref   Medline   

Esposito AL, Poirier, WJ, Clark, CA. The cardiac glycoside digoxin disrupts host defense in experimental pneumococcal pneumonia by impairing neutrophil mobilization. Am Rev Respir Dis 1989:140:1590-4
Medline   

Freitas CS, Dalmau, SR, Kovary, K, Savino, W. Epidermal growth factor modulates fetal thymocyte growth and differentiation. Dev Immunol 1998:5:169-82
Crossref   Medline   

Gentile DA, Henry, J, Katz, AJ, Skoner, DP. Inhibition of peripheral blood mononuclear cell proliferation by cardiac glycosides. Ann Allergy Asthma Immunol 1997:78:466-72
Medline   

Ghione S, Balzan, S, Decollogne, S, Paci, A, Pieraccini, L, Montali, U. Endogeneous digitalis-like activity in the newborn. J Cardiovasc Pharmacol 1993:22:Suppl. 2:S25-8
Medline   

Jessop DS. β-Endorphin in the immune system—mediator of pain and stress? Lancet 1998:351:1828-9
Crossref   Medline   

Johnson BE, Marsh, JA, King, DB, Lillehoj, HS, Scanes, CG. Effect of triiodothyronine on the expression of T cell markers and immune function of thyroidectomized White-Leghorn chickens. Proc Soc Exp Biol Med 1992:199:104-13
Medline   

Klecha AJ, Genaro, AM, Lysionek, AE, Caro, RA, Coluccia, AG, Cremaschi, GA. Experimental evidence pointing to the bidirectional interaction between the immune system and the thyroid axis. Int J Immunopharmacol 2000:22:491-500
Crossref   Medline   

Lantinga van Leeuwen IS, Teske, E, van Garderen, E, Mol, JA. Growth hormone gene expression in normal lymph nodes and lymphomas of the dog. Anticancer Res 2000:20:2371-6
Medline   

Nakanishi K, Taniguchi, Y, Onji, M. Triiodothyronine enhances expression of the interleukin-2 receptor alpha chain. Endocr J 1999:46:437-42
Crossref   Medline   

Nicola NA. An introduction to the cytokines. Guidebook to cytokines and their receptors 1994:1-7

Panerai AE, Sacerdote, P. β-Endorphin in the immune system: a role at last. Immunol Today 1997:18:317-9
Crossref   Medline   

Sabharwal P, Varma, S, Malarkey, WB. Human thymocytes secrete luteinizing hormone: an autocrine regulator of T-cell proliferation. Biochem Biophys Res Commun 1992:187:1187-92
Crossref   Medline   

Schwartz RH, Bianco, AR, Handwerger, BS, Kahn, CR. Demonstration that monocytes rather than lymphocytes are the insulin binding cells in preparation of human peripheral blood mononuclear leukocytes: implications for studies of insulin-resistant states in man. Proc Natl Acad Sci U S A 1975:72:474-8
Crossref   Medline   

Selye H. The mast cells. 1965:


Received 9 February 2004/11 March 2004; accepted 29 March 2004

doi:10.1016/j.cellbi.2004.03.013


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)