Pituitary hormones modulate cell–cell interactions between thymocytes and thymic epithelial cells

Journal of Neuroimmunology 76 Ž1997. 39–49

Pituitary hormones modulate cell–cell interactions between thymocytes and thymic epithelial cells Valeria ´ de Mello-Coelho a, Dea ´ Maria Serra Villa-Verde a, Mireille Dardenne b, Wilson Savino a,) a

Laboratory on Thymus Research, Department of Immunology, Institute Oswaldo Cruz, Foundation Oswaldo Cruz, AÕe. Brasil 4365-Manguinhos, 21045-900 Rio de Janeiro, Brazil b Hopital Necker, CNRS URA-1461, Paris, France Received 15 August 1996; revised 24 December 1996; accepted 26 December 1996

Abstract The thymic microenvironment plays a key role in the intrathymic T-cell differentiation. It is composed of a tridimensional network of epithelial cells whose physiology is controlled by extrinsic circuits such as neuroendocrine axes. Herein we show that the expression of extracellular matrix ligands and receptors by cultured thymic epithelial cells is upregulated by prolactin ŽPRL. and growth hormone ŽGH., the latter apparently occurring via insulin-like growth factor I ŽIGF-I.. Thymocyte release from the lymphoepithelial complexes, thymic nurse cells, as well as the reconstitution of these complexes are enhanced by PRL, GH or IGF-I. Treatment of a mouse thymic epithelial cell line with these hormones induced an increase in thymocyte adhesion, an effect significantly prevented in the presence of antibodies to fibronectin, laminin or respective receptors VLA-5 and VLA-6. Our data suggest that the in vitro changes in thymocyterthymic epithelial cell interactions induced by pituitary hormones are partially mediated by the enhancement of extracellular matrix ligands and receptors. Keywords: Thymic epithelium; Thymocytes; Prolactin; Growth hormone; Insulin-like growth factor-I; Extracellular matrix; Integrins

1. Introduction The thymus is a primary lymphoid organ in which bone marrow-derived T-cell precursors undergo a complex process of maturation, eventually leading to the migration of mature thymocytes to the T-dependent areas of peripheral lymphoid organs. This differentiation process involves sequential expression of a variety of membrane proteins and rearrangements in T-cell receptor genes. Most thymocytes thought to be self-reactive are negatively selected by clonal deletion, whereas some appear to be rescued from death through positive selection, eventually yielding the vast majority of the T-cell repertoire Žvan Ewijk, 1991; Anderson et al., 1996.. Key events of intrathymic T-cell differentiation are driven by the influence of the thymic microenvironment, a tridimensional network composed of distinct cell types such as epithelial cells, macrophages and dendritic cells, as well as extracellular matrix ŽECM. elements ŽBoyd et al., 1993.. ) Corresponding author. Tel.: q55-21-2801486r5984327; fax: q5521-2801589r5909741; e-mail: [email protected]

The thymic epithelium is the major component of the thymic microenvironment and has an important and multifaceted influence on early events of T-cell differentiation, by secretion of various polypeptides including thymic hormones and cytokines ŽSavino and Dardenne, 1984; Le et al., 1988. and cell–cell contacts, such as the interactions involving major histocompatibility complex gene products expressed by thymic epithelial cells ŽTEC. with the T-cell receptor Žvan Ewijk, 1991. and those occurring through classical adhesion molecules ŽPatel and Haynes, 1993.. Lastly, TEC can bind to and interact with maturing thymocytes by means of ECM ligands and respective receptors ŽSavino et al., 1993.. Interestingly, it is possible that supramolecular ECM arrangements function as a conveyor belt, allowing an ordered migration of thymocytes within the organ ŽSavino et al., 1996.. From a physiological viewpoint, it is increasingly apparent that extrinsic factors Žendogenous and exogenous. can also influence thymic functions. In particular, numerous recent studies point to a neuroendocrine control of thymus physiology ŽDardenne and Savino, 1994.. Data from different laboratories have demonstrated that thymic

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endocrine function, particularly thymulin secretion, can be modulated by hormones, including steroid and thyroid hormones, which upregulate thymulin secretion ŽSavino et al., 1984, 1988; Fabris et al., 1986.. Moreover, classical pituitary hormones, namely prolactin ŽPRL. and growth hormone ŽGH. are able to enhance thymulin secretion in different species including mice, dogs and humans ŽDardenne et al., 1989; Goya et al., 1992; Goff et al., 1987; Timsit et al., 1989, 1992.. Interestingly, insulin-like growth factor I ŽIGF-I., known to mediate several GH-related biological activities, induces similar effects ŽTimsit et al., 1992.. In addition to the influence of neuroendocrine circuits upon thymic endocrine function, other aspects of TEC physiology can be modulated by hormones. For example, PRL upregulates the expression of high molecular weight cytokeratins by medullary TEC ŽDardenne et al., 1989.. Furthermore, epithelial growth is increased in vitro following PRL or GH treatment ŽDardenne et al., 1989; Timsit et al., 1992.. Finally, ECM ligands and receptors expressed by TEC can be enhanced by triiodothyronine ŽVilla-Verde et al., 1993.. Herein, we evaluated the role of pituitary hormones upon the expression of these molecules as well as ECM-mediated interactions between thymocytes and thymic epithelial cells.

2. Material and methods 2.1. Animals Female BALBrc and Swiss mice aged 3–4 weeks, or Swiss pregnant animals with 16–17 days of gestation were obtained from the Foundation Oswaldo Cruz animal facilities, Rio de Janeiro. 2.2. Chemicals o-Phenylenediamine, 3-amine-9-ethyl-carbazole, bovine serum albumin, RPMI 1640 culture medium, penicillin and streptomycin were Sigma products ŽSt. Louis.. Perhydrol was purchased from Merck ŽRio de Janeiro. and fetal calf serum was obtained from Cultilab ŽSao ˜ Paulo.. Ovine PRL was obtained from the National Institute of Diabetes and Digestive and Kidney Diseases ŽCA, USA., recombinant ovine growth hormone was provided by A.F. Parlow ŽPituitary Hormones and Antisera Center, Bethesda. whereas recombinant human insulin-like growth factor-I

was from Genzyme ŽBoston. and the streptavidin–biotinylated horseradish–peroxidase complex was provided from Amersham Int. ŽBuckinghamshire.. 2.3. Antibodies Rabbit polyclonal antisera against laminin, fibronectin or type-IV collagen were obtained from the Pasteur Institute ŽCentre de Radioanalyse, Lyon.. These reagents were previously shown to label corresponding ECM proteins in the human and mouse thymus, both in situ and in vitro ŽBerrih et al., 1985; Lannes-Vieira et al., 1991.. The anti-VLA-5 rabbit antiserum was purchased from Telios Pharmaceuticals ŽSan Diego. and the anti-VLA-6 monoclonal antibody ŽmAb, clone GoH3. was provided by Immunotech ŽMarseille.. We previously showed that these antibodies specifically recognize receptors for fibronectin and laminin in cultured thymic epithelial cells ŽLannesVieira et al., 1993; Villa-Verde et al., 1993, 1994.. Aliquotes of each anti-VLA reagent were directly coupled with fluoresceinisothiocyanate in our laboratory and used for cytofluorometry. The anti-Thy 1.2 mAb Žclone 30H12. was obtained by culturing the corresponding hybridoma in vivo using BALBrc nude mice and lastly purifying the resulting ascitis. The anti-PRL mAb was from Immunotech ŽMarseille. and the anti-PRL receptor mAb Žclone U5. was kindly provided by Dr. Paul Kelly. It has been characterized previously and recognizes the PRL receptor on TEC ŽDardenne et al., 1991.. The anti-GH rabbit antiserum was obtained from Biosys ŽCompiegne, France., whereas the anti-IGF-I and anti-IGF-I receptor mAb Žclones 82-9A and a IR3, respectively. were purchased from Oncogene Science ŽNew York.. The mAb irrelevant mouse IgG1 mAb was a gift from Dr. J. Lannes-Vieira ŽFIOCRUZ, Rio de Janeiro., whereas purified irrelevant rat and rabbit immunoglobulins were from WL Immunochemicals ŽRio de Janeiro. and the Biomanguinhos sector of the Foundation Oswaldo Cruz ŽRio de Janeiro., respectively. The biotinylated sheep anti-rat Ig and sheep anti-mouse Ig antibodies were obtained from Amersham Int. ŽBuckinghamshire., whereas the fluorescein-coupled goat anti-rat Ig and goat anti-rabbit Ig sera were purchased from Biosys ŽCompiegne, France.. 2.4. Immunocytochemistry and cytofluorometry Cultures of epithelial cells were submitted to an indirect immunofluorescence technique according to the routine

Fig. 1. Immunofluorescence detection of fibronectin Žpanels A, C, E and G. and fibronectin receptor VLA-5 Žpanels B, D, F and H. in TNC-derived epithelial cell cultures, treated or not with pituitary hormones. Basal levels of fibronectin and VLA-5 in untreated cultures can be seen in panels A and B, respectively. Treatments with PRL ŽC and D., GH ŽE and F. or IGF-I ŽG and H. induced an enhancement of the expression of both fibronectin and VLA-5. The insert in panel A represents a negative control in which the anti-fibronectin antibody was replaced by rabbit purified immunoglobulins =600. Results were confirmed in five experiments and were similar in relation to the detection of laminin and the laminin receptor VLA-6. Additionally, similar data was obtained treating the mouse TEC line Žsee Table 1..

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technique ŽVilla-Verde et al., 1993, 1994.. Briefly, material was incubated with a given anti-ECM or anti-ECM receptor primary antibody for 1 h, washed with phosphate-buffered saline ŽPBS. and subjected to appropriate fluorescent-coupled antibodies Ždiluted 1:100. for 1 h. Following a further PBS washing, the specimens were mounted and analyzed under a Leitz Ortoplan fluorescence microscope. Detection of Thy1.2 on thymocytes was performed by the immunoperoxidase technique, as detailed previously ŽLannes-Vieira et al., 1991, 1993; Villa-Verde et al., 1993, 1994.. In brief, TECrthymocyte co-cultures were fixed in absolute ethanol, incubated with the anti-Thy1.2 mAb for 1 h, washed in PBS and revealed with corresponding biotinylated second antibody followed by a streptavidin– peroxidase complex. Enzymatic activity was developed with aminoethylcarbazole in the presence of H 2 O 2 . In both immunocytochemistry assays, negative controls in which primary antibodies were replaced by unrelated immunoglobulins did not generate any significant labeling. Cytofluorometric analysis of the mouse TEC line was performed using untreated or hormone-treated cultures according to current protocol. Briefly, control, PRL- or GH-treated cultures were gently trypsinized, resuspended and subjected to one step fluorescence labeling using fluorescein-coupled anti-VLA-5 or anti-VLA-6 antibodies. Following PBS washing, cells were fixed in 0.5% formaldehyde and analysed using an Epics 751 flow cytometer ŽCoulter Electronics, Hialeah, Florida..

Swiss mice were enzymatically dissociated using collagenase, dispase and DNAse. By various sedimentations in a 50% fetal calf serumrPBS density gradient, complexes were obtained and were then settled in culture as described for the murine TEC lines. 2.6. Thymocyte release from thymic nurse cell complexes One experimental model to analyse the ability of pituitary hormones to modulate TECrthymocyte interactions is the TNC complex. When settled in culture, TNCs gradually release their thymocytes, a process which depends on metabolism and cytoskeleton integrity ŽAndrews and Boyd, 1985. and that can be semi-quantitatively evaluated ŽLannes-Vieira et al., 1993; Villa-Verde et al., 1993, 1994; Lagrota-Candido et al., 1996.. For such evaluation, ˆ freshly-isolated lympho-epithelial complexes were plated on tissue culture dishes Ž2.5 = 10 4 cellsrdish. and cultured for 42 h, then fixed with absolute ethanol for 5 min at room temperature. Percentages of adhered rounded TNCs without lymphocyte release, spread TNCs still containing

2.5. Cultures of thymic epithelial cell The BALBrc mouse and Wistar rat TEC lines, IT-76M1 and IT-45R1, respectively, were spontaneously obtained after continuous cultures of thymic stromal cells. The epithelial nature of these TEC lines was characterized by the presence of cytokeratin filaments and desmosomes ŽItoh et al., 1982; Cirne-Lima et al., 1993.. Additionally, their ability to produce ECM proteins was demonstrated ŽSavino et al., 1986; Lannes-Vieira et al., 1991.. All cultures were settled in RPMI 1640, pH 7.2, supplemented with 5% fetal calf serum, 100 IUrml penicillin and 100 m grml streptomycin, at 378C in an atmosphere containing 5% CO 2 . Semiconfluent cultures were treated with 0.25% trypsin–0.02% EDTA in Ca2qrMg 2q-free solution for 5 min and replated in 8-well Labtek chambers ŽNunc, Roskilde, Denmark.. In some experiments, thymocytes freshly isolated from 3–5 week old BALBrc mice were co-cultured with TEC for 30 min, in a ratio of 50 thymocytes per TEC. In addition to TEC lines, we used primary cultures of thymic nurse cells ŽTNC.. These are lymphoepithelial complexes in which one epithelial cell harbours 20–200 thymocytes and were isolated herein as originally described ŽWekerle et al., 1980.. Briefly, fragments from 30–50 pooled thymuses of

Fig. 2. Cytofluorometric profiles for the expression of laminin receptor in cultures of the mouse thymic epithelial cell line. As compared to control untreated cultures ŽC., one can see an upregulation of VLA-6, evidenced by the shift of fluorescence intensity to the right, following prolactin ŽPRL. or growth hormone ŽGH. treatment. A total of 5,000 cells were analysed for each profile. This figure is representative of two separate experiments. In both, the percentage of positive cells varied from 95–97% independently of the cultures being hormone-treated or not.

V. de Mello-Coelho et al.r Journal of Neuroimmunology 76 (1997) 39–49

thymocytes or spread lymphocyte-free epithelial cells were counted blind by two different observers. 2.7. Reconstitution of thymic nurse cell complexes A second aspect concerning the in vitro manipulation of TNCs is the possibility of reconstituting lymphoepithelial complexes using co-cultures of fetal thymocytes plus epithelial cells derived from primary cultures of TNCs ŽDefresne, 1986.. Five day TEC cultures derived from TNCs Žtreated or not with hormones. were trypsinized and cocultured with freshly-isolated fetal thymocytes Ž10 5 thymocytesr10 4 TNC per well. in inverted Terasaki plates for 6 h, being fixed with 0.05% formol in PBS solution for 10 min. Percentages of newly formed complexes were then evaluated by direct counting using a light microscope. 2.8. Thymocyter thymic epithelial cell adhesion assays In addition to the TNC model, hormonal influences on TECrthymocyte interactions were tested using the mouse TEC line. The ability of untreated or hormone-treated TEC to bind thymocytes was evaluated by two distinct techniques. One approach was an ELISA using the anti-Thy1.2 mAb, a pan-T-cell marker able to reveal the adhered thymocytes ŽVilla-Verde et al., 1993.. Briefly, nonspecific binding sites of TECrthymocyte co-cultures were blocked with a PBS solution containing 5% dry milk during 1 h. Cells were then subjected to the anti-Thy1.2 mAb and

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washed in a PBSrtween 0.05% solution. Successive incubations with biotinylated anti-rat IgG and streptavidinrperoxidase complex were used as a revealing system, each one being followed by washing procedures. Peroxidase activity was assessed by treatment with ophenylenediamine in the presence of H 2 O 2 . Reaction was stopped using 4 M H 2 SO4 and resulting optical density was read in an ELISA Titertek Multiskan apparatus, model MCCr340 ŽLabsystems, Helsinki, Finland.. A second approach to evaluate thymocyterTEC adhesion was by counting the numbers of adhered thymocytes to TEC cultures that were trypsinized, replated to 8-well labtek chambers Ž10 4 cellsr0.5 ml in each chamber., exposed to thymocytes under a mild shaking condition Ž60 rpm at 378C., fixed in absolute ethanol and labeled by means of immunocytochemistry with the anti-Thy1.2 mAb. Countings were integrated in the form of an association index ŽAI., and calculated using the following formula, previously validated for this type of analysis ŽLannes-Vieira et al., 1993; Villa-Verde et al., 1993, 1994; LagrotaCandido et al., 1996.: ˆ AI s

number of TEC with thymocytes total TEC number =

number of thymocytes bound to TEC total TEC number

= 100

At least 300 TEC Žcontaining or not adhered thymocytes. were counted per well. Triplicates to octoplicates

Fig. 3. Effects of pituitary hormones on thymic nurse cells. In panel A, from left to right the three stages of the in vitro TNC release are demonstrated: rounded, spread lymphocyte-rich and spread lymphocyte-free TEC. Lymphoepithelial complexes were cultured in the absence ŽI. or presence of PRL Ždiagonally striped area., GH Žcross-hatched area. or IGF-I Ždotted area.. Results are expressed in percentages of means " SE and the differences between control and hormone-treated TNCs were statistically significant, as ascertained by the x 2 test Ž p - 0.01.. Panel B shows two Žout of five. representative experiments of reconstitution of thymic nurse complexes from 5-day cultures of TNC, untreated or treated with those hormones. In both experiments hormone-treated cultures were statistically different from corresponding controls Ž p - 0.05..

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were used in the various experiments; each of them being repeated at least three times. In most cases, countings were performed blind by two distinct observers. In both assays, freshly-isolated thymocytes were co-cultured with TEC Ž50 thymocytes per epithelial cell. that had been replated 24 h before. Following 30 min of co-culture, wells were gently washed to eliminate non-adherent thymocytes and thymocytes were processed for immunocytochemical detection of the Thy1.2 molecule. 2.9. Protocols for hormone treatment in the absence or presence of Õarious antibodies Prolactin, GH or IGF-I were applied at the concentrations ranging from 10y6 to 10y1 0 M, although in most cases, the dose of 10y8 M was used. For the detection of

Fig. 4. Adhesion of thymocytes Žarrows. to the mouse thymic epithelial cell line is stimulated by pituitary hormones. In panel A, representative microscopic fields of TECrthymocyte adhesion are shown in control untreated TEC Žleft., or after treatment with 10y8 M PRL Žcenter. or 10y8 M GH Žright.. This effect also was evaluated by direct counting and determination of adhesion index Žpanel B. or by ELISA Žpanel C.. Results expressed by the association index or mean". Differences were statistically evaluated by Mann–Whitney test for the ELISA or by Student’s t-test, for the association indexes. Data are representative of three separate experiments and in both assays, values of the hormonetreated cultures were statistically different from the corresponding untreated control Ž p- 0.01.. Magnification in panel A: =960.

ECM ligands and receptors by the thymic epithelium, 2-day cultures of the mouse and rat TEC lines, as well as 5-day primary cultures of lymphocyte-free TNC-derived epithelial cells were submitted to 24 h of treatment with PRL, GH or IGF-I, washed in PBS and processed for immunocytochemistry or cytofluorometry. Regarding the hormonal effects on the degree of thymocyte release from TNC complexes, freshly-isolated TNCs were settled in culture for 42 h, being subjected to PRL, GH or IGF-I during the last 18 h. Cells were then fixed and the differential stages of thymocyte release were counted. Hormonal influence on TNC reconstitution was studied by treating 5-day cultures of lymphocyte-free TNC-derived epithelial cells for 16 h with PRL, GH or IGF-I. Cell were then trypsinized, co-cultured with fetal thymocytes and fixed for further counting of reconstituted lymphoepithelial complexes. Modulation of TECrthymocyte adhesion was ascertained by treating the TEC line with a given hormone for 8 h and co-culturing with thymocytes. TEC cultures were trypsinized and replated in 8-well labtek chambers Ž10 4 cellsr0.5 mlrwell. for 16 h. The adhesion degree was evaluated by immunocytochemistry and cell counting, or by ELISA. To avoid a direct effect of each hormone upon thymocytes, cultured TEC were washed with PBS at least three times before thymocytes were added. As regards the effects of pituitary hormones on the degree of thymocyte release from TNC complexes, freshly-isolated TNCs were settled in culture for 42 h; being subjected to PRL, GH or IGF-I during the last 18 h of culture. Cells were then fixed and percentages of TNCs at differential stages of thymocyte release were counted. The specificity of PRL effects was ascertained by simultaneous incubation of cells with PRL q anti-PRL antibody or pre-treatment of TEC cultures with the anti-PRL receptor mAb during 30 min, followed by PRL incubation, co-culture with thymocytes, immunolabeling and cell counting. Cultures were also treated by GH in the presence of anti-GH, or anti-IGF-I or anti-IGF-I receptor antibodies prior to evaluation of TECrthymocyte adhesion. Similar blocking experiments with the anti-IGF-I or anti-IGF-I receptor antibodies were performed in TEC cultures treated with IGF-I. Lastly, the various anti-hormone or antihormone receptor antibodies were also applied onto untreated TEC cultures. A possible involvement of ECM ligands and receptors in the effects of pituitary hormones on TECrthymocyte adhesion was approached by incubating PRL-, GH- or IGF-I-treated TEC with antibodies specific for distinct ECM ligands and receptors, prior to co-culturing with thymocytes. 2.10. Statistical analysis In order to assess the statistical significance of differences between control and experimental groups, the

V. de Mello-Coelho et al.r Journal of Neuroimmunology 76 (1997) 39–49

Mann–Whitney test or the Student’s t-test were applied for the analysis of TECrthymocyte adhesion, whereas the x 2 test was applied to the evaluation of thymocyte degree from TNCs.

3. Results 3.1. Effect of pituitary hormones upon the expression of ECM ligands and receptors by thymic epithelial cells

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Table 1 Increase of extracellular matrix ligands and receptors on thymic epithelial cells subjected to prolactin, growth hormone or insulin-like growth factor-I treatment abcd

Type IV collagen Fibronectin VLA-5 Laminin VLA-6

No treatment

PRL

GH

IGF-I

q qq q q q

qqq qqqq qqqq qqq qqq

qq qqqq qq qqq qq

qqq qqqq qqq qqqq qqq

a

The mouse TEC line was used in these experiments. Distinct molecules were detected by indirect immunofluorescence. c Degrees of immunofluorescence staining: q, very weak; qq weak; qqq strong; qqqq very strong. d Hormones were applied at 10y8 M concentration. b

Initially we investigated whether pituitary hormones could modulate the expression of ECM ligands and receptors by murine TNC or TEC line cultures. When 2-day cultures of the murine TEC lines were treated with PRL or GH, they consistently exhibited an enhancement of fluorescence staining for type IV collagen, laminin, fibronectin, VLA-5 and VLA-6 ŽTable 1., an effect also observed following hormonal treatment of 5-day TNC-derived TEC primary cultures ŽFig. 1.. Although quantitative measurements were not performed, we noticed a higher fluorescence intensity in the hormone-treated cultures as a whole. The numbers of positive cells also appeared to be

increased. The enhancement of VLA-5 and VLA-6 immunolabeling on TEC cultures treated or not with PRL or GH was also confirmed by cytofluorometry, as exemplified in Fig. 2 depicting profiles of the laminin receptor

Fig. 5. Modulation of TECrthymocyte adhesion by PRL, GH, IGF-I Žrespectively in panels a, b and c.: blocking with corresponding antihormone or anti-hormone receptors antibodies. In panel Ža. epithelial cultures remained untreated or were treated with 10y8 M PRL alone, PRL in the presence of anti-PRL mAb diluted 1:100, or PRL after treatment with the anti-PRL receptor mAb diluted 1:100. Controls included untreated TEC cultures, as well as cultures incubated with corresponding antibodies, in the absence of PRL. In panel Žb. TEC were treated with 10y8 M GH alone, GHqanti-GH diluted 1:100, GHqantiIGF-I mAb Ž10 m grml. or GHqanti-IGF-I receptor mAb, also at 10 m grml. Additionally, some cultures were subjected to each antibody in the absence of GH. Similar protocol is seen in panel Žc. as regards the effect of IGF-I. Following these procedures, TEC were washed with PBS and co-cultured with freshly-isolated thymocytes and their adhesion degree to TEC was evaluated. No hormone effects were inhibited when TEC were treated with purified rat Ig, mouse Ig or normal rabbit serum Žnot shown.. Results were expressed as mean"SE of association indexes. Data are representative of three separate experiments. Values of the hormone-treated cultures were statistically different from the corresponding untreated controls Ž p- 0.01.. Blocking effects of each hormone with the corresponding anti-hormone or anti-hormone receptor antibodies were also significant as compared to the hormonal treatment alone Ž p- 0.05.. Panel a: Žwhite area. hormone untreated TEC; Žshaded area. hormone untreated TECqanti-PRL mAb; Ždiagonally striped area. hormone untreated TECqanti-PRL receptor mAb; Žsolid area. PRL treated TEC; Žshaded area with dark surround. PRL treated TECqanti-PRL mAb; Ždiagonally striped area with dark surround. PRL treated TECqanti-PRL receptor mAb. Panel b: Žwhite area. hormone untreated TEC; Žshaded area. hormone untreated TECqanti-GH mAb; Žcross-hatched area. hormone untreated TECqanti-IGF-I mAb; Ždiagonally striped area. hormone untreated TECqanti-IGF-I receptor mAb; Žsolid area. GH treated TEC; Žshaded area with dark surround. GH treated TECqanti-GH mAb; Žcross-hatched with dark surround. GH treated TECqanti-IGF-I mAb; Ždiagonally striped area with dark surround. GH treated TECqanti-IGF-I receptor mAb. Panel c: Žwhite area. hormone untreated TEC; Žshaded area. hormone untreated TECqanti-IGF-I mAb; Ždiagonally striped area. hormone untreated TECqanti-IGF-I receptor mAb; Žsolid area. IGF-I treated TEC; Žshaded area with dark surround. IGF-I treated TECqantiIGF-I mAb; Ždiagonally striped area with dark surround. IGF-I treated TECqanti-IGF-I receptor mAb.

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VLA-6. With the high sensitivity threshold of this technique, more than 95% of control TEC were labeled and a shift of the histograms towards a higher fluorescence intensity was recorded upon hormone treatments. Interestingly, IGF-I, a major mediator of the physiological effects associated with GH, also promoted an enhancement of ECM ligands and receptors in the three models of cultured epithelial cells. It should be noted that the hormones applied at various concentrations ranging from 10y6 to 10y1 0 M promoted a similar effect on the detection of ECM ligands and receptors, whereas higher dilutions were ineffective Ždata not shown.. In control experiments no fluorescent signal was detected in either untreated or hormone-treated TEC cultures, when unrelated rabbit or rat immunoglobulins were applied as primary antibodies Žsee insert in Fig. 1.. 3.2. Modulation of thymic nurse cell complexes by pituitary hormones In a first series of experiments we showed that the release of thymocytes in 42 h cultures of TNCs was enhanced under treatment with PRL, GH or IGF-1 ŽFig. 3a.. The global statistical analysis of this event showed that the exit of thymocytes from the lymphoepithelial complexes was increased, as compared to controls, as ascertained by lower values of lymphocyte-containing TNC complexes together with an increased in lymphocyte-free epithelial cells in hormone-treated cultures. Moreover, an increase in TNC reconstitution was seen when TNC-derived lymphocyte-free 5-day TEC primary cultures were hormonally-treated and co-cultured with freshly-isolated fetal thymocytes ŽFig. 3b..

the hormones Ždata not shown.. Moreover, incubation of untreated TEC cultures with various anti-hormone or antihormone receptor antibodies did not promote significant decrease in the degree of thymocyte adhesion. These blocking experiments are summarized in Fig. 5. 3.4. InÕolÕement of extracellular matrix on thymocyter TEC interactions modulated by pituitary hormones Since pituitary hormones were able to enhance the presence of extracellular matrix ligands and receptors in TEC cultures and to modulate thymocyterTEC interactions, we wondered whether the two effects were related. We used the model of thymocyte adhesion to the mouse TEC line; treating or not the epithelial cells with a given hormone, in the absence or presence of antibodies able to specifically recognize distinct ECM ligands and receptors. In keeping with data previously reported ŽLannes-Vieira et al., 1993; Lagrota-Candido et al., 1996., we first obˆ served that the anti-type IV collagen, anti-fibronectin antilaminin, anti-VLA-5 and anti-VLA-6 antibodies, when applied to growing TEC prior to the coculturing with thymocytes, significantly prevented their adhesion to the epithelial cells. In further experiments, we demonstrated that the enhancement in thymocyterTEC adhesion promoted by PRL, GH or IGF-I could also be consistently abrogated when TEC were subjected to anti-fibronectin, anti-VLA-5, anti-laminin or anti-VLA6 antibodies before addition of thymocytes these results are summarized in Fig. 6.

3.3. Pituitary hormones enhance adhesion of thymocytes to thymic epithelial cells In a second group of experiments, we evidenced that PRL and GH induced an enhancement of TECrthymocyte adhesion as demonstrated by ELISA and by the association index between the two cell types. Both assays revealed statistically significant differences between hormonetreated and untreated control cultures ŽFig. 4.. The specificity of this effect was confirmed by blocking such hormone-induced enhancement with corresponding antihormone antibodies. Moreover, as regards PRL, not only the anti-PRL but also the anti-PRL receptor mAb were effective in blocking the PRL effect. Finally, the GH-induced enhancement of TECrthymocyte adhesion could also be blocked by anti-IGF-I and anti-IGF-I receptor antibodies. Indeed, these reagents also prevented the enhancing effect promoted by pre-treating TEC with IGF-I. Unrelated rat, rabbit or mouse immunoglobulins did not induce any detectable changes in the patterns of thymocyterTEC adhesion, either in the presence or absence of

Fig. 6. Possible involvement of extracellular matrix ligands and receptors in GH-stimulated TECrthymocyte adhesion. Cultures of TEC remained untreated ŽI. or were treated with 10y8 M GH alone Žcross-hatched., or in the presence of anti-fibronectin Ž a FN., anti-VLA-5, anti-laminin Ž a LN. or anti-VLA6 Žcross-hatched. antibodies. Data were expressed as the means"SE of association indexes. The figure is representative of three separate experiments. Values of the GH-treated cultures were statistically different from the corresponding untreated control Ž p- 0.01.. Blocking effects of GH with anti-extracellular matrix or anti-extracellular matrix receptor antibodies were also significant as compared to the hormonal treatment alone Ž p- 0.05..

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4. Discussion In the present report we show that PRL and GH are able to modulate Žat least in vitro. the physiology of the thymic epithelium, with consequences on TECrthymocyte membrane interactions. In fact, the molecular basis for such effects were previously provided with the demonstration of specific receptors for such molecules on TEC. As regards PRL receptor for example, it was defined by immunocytochemistry, immunoblotting and northern blotting. Moreover, we showed that they are actually functional, since we could modulate both thymulin production and TEC growth with appropriate agonistic doses of anti-PRL receptor antibodies ŽDardenne et al., 1991.. Also, we demonstrated the expression of specific GH receptors on TEC. Binding displacement and autoradiographic procedures revealed the presence of GH receptor in TEC ŽBan et al., 1990.. This was further confirmed by cytofluorometry ŽDardenne et al., 1994. and more recently the corresponding mRNA was defined by the PCR approach Žmanuscript in preparation.. From a functional point of view, we showed in previous work, that thymic endocrine function was under pituitary hormone control, with enhancement of thymulin secretion promoted by PRL and GH, both in vivo and in vitro ŽDardenne et al., 1989; Goya et al., 1992; Goff et al., 1987; Timsit et al., 1989, 1992.. The enhancement of ECM ligands and receptors in both TEC lines and primary cultures of TNC-derived epithelial cells reported herein clearly reinforces the notion that these two pituitary hormones exert a pleiotropic action upon the thymic microenvironment. Importantly, the in vitro effects described herein are likely to occur in vivo, since pilot experiments using young adult transgenic mice for bovine GH evidenced an increase in the intrathymic contents of ECM components Žour unpublished data.. Using the in vitro model of thymic nurse cells, we evidenced that the thymocyte release from TNC complexes as well as TNC reconstitution were upregulated by both PRL and GH. This issue should be discussed in the context of thymocyte migration. As previously postulated ŽSavino et al., 1993; Villa-Verde et al., 1994., the exit of thymocyte from TNCs as well as the entrance of these cells to reconstitute lymphoepithelial complexes with TNC-derived epithelial cultures, can be considered as an in vitro model of intrathymic T-cell migration. In this respect, one can envision that pituitary hormones can modulate thymocyte migration through an action on the thymic epithelium. In this regard, it is noteworthy that thyroid hormones also appear to modulate this process ŽVilla-Verde et al., 1993.. In addition, we observed that treatment of the mouse TEC line with PRL or GH did promote an enhancement of thymocyte adhesion. This is an apparent paradox with the modulation of thymocyte release from TNC complexes. Nonetheless, these effects more likely reveal the transient nature of the membrane events under analysis, thus allowing a continuous interaction between a migrating thymo-

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cyte and the sessile microenvironment. In this respect, it is actually possible that de-adhesion mechanisms may also be modulated by pituitary hormones. One candidate molecule is tenascin. This ECM component, whose expression in adult life is restricted to very few organs, exhibits de-adhesion properties ŽChiquet-Ehrismann, 1995. and is constitutively expressed intrathymically ŽOcklind et al., 1993; Freitas et al., 1995.. Moreover, we recently observed that in a different model, the murine acute Chagas’ disease, there is a simultaneous increase Žboth in vivo and in vitro. of adhesive ECM proteins such as fibronectin and laminin, as well as of tenascin ŽSavino et al., 1989; Cotta-de-Almeida, 1996.. Experiments are know being scheduled to approach this hypothesis. In any case, the ECM-related effect of PRL and GH does not seem to be thymus-restricted since ECM-mediated mature T-cell adhesion and migration can also be under pituitary hormone influence. It was shown that recombinant human GH stimulates in vitro migration of resting and activated human T-cells from peripheral blood and T-lymphocyte engraftment in the thymus of SCID mice via b 1 and b 2 integrins ŽTaub et al., 1994.. It is also relevant that the enhancing effect of GH on TECrthymocyte interactions could also be obtained with IGF-I. We showed that the GH-induced enhancement of thymocyte adhesion could be significantly abrogated by treating TEC with anti-IGF-I or anti-IGF-I receptor mAbs. This finding clearly favors the hypothesis of an autocrine IGF-IrIGF-I receptor circuit mediating the actions of GH upon the thymic epithelium. This notion is supported by the fact that the increase in thymulin secretion induced by GH could also be prevented by anti-IGF-I and anti-IGF-I receptor mAbs ŽTimsit et al., 1992.. Furthermore, this hypothesis is in keeping with our recent data Žunpublished., showing by immunocytochemistry, cytofluorometry and immunoblotting, the expression of IGF-I receptors by cultured TEC. The effect of pituitary hormones upon intrathymic cell–cell heterotypic interactions should also be discussed in conjunction with the PRLrGHrIGF-I-induced increase in ECM ligands and receptors. The rationale is the previous demonstration that similar TECrthymocyte interactions are at least partially mediated by extracellular matrix ligands and receptors ŽSavino et al., 1993; Lannes-Vieira et al., 1993; Villa-Verde et al., 1994; Lagrota-Candido et ˆ al., 1996.. In fact, the hypothesis by which the modulation of TECrthymocyte interactions by pituitary hormones is secondary to the upregulation of ECM ligands and receptors by TEC is supported by the experiments showing that the enhancement of thymocyte adhesion to hormonallytreated TEC could be blocked with anti-ECM or anti-ECM receptor antibodies. Additionally, it is worthwhile to mention that IGF-I has been shown to enhance ECM extrathymically, namely in the human mesangial cells from renal glomeruli ŽPricci et al., 1996.. Nevertheless, since thymocyterTEC adhesion was not totally prevented by

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those reagents, one cannot discard the possible involvement of other adhesion molecules. In this respect, blocking experiments using antibodies able to recognize ICAM-1 and LFA-3 adhesion molecules, both expressed by the thymic epithelium ŽNonoyama et al., 1989; Vollger et al., 1987., may provide useful information. On the other hand, the possibility that, in the experiments reported herein, hormones are directly affecting the thymocytes led to interact with cultured TEC, seems unlikely, since only TEC were treated, being largely washed before being co-cultured with thymocytes. In any case, although in the present and previous studies we described pituitary hormone effects on the thymic epithelium, that may ultimately modulate the physiology of thymocytes, we should not discard the alternative pathway by which these hormones could exert a direct influence upon differentiating thymocytes. This possibility is supported by the demonstration of GH and PRL receptors in both murine and human thymocytes ŽBan et al., 1990; Gagnerault et al., 1993; Dardenne et al., 1994.. Lastly, our data should be discussed in the light of recent findings showing the intrathymic production of PRL, GH as well as IGF-I. It has been shown that thymocytes, express mRNA coding for PRL as well as the peptide itself ŽPelegrini et al., 1992; Wu et al., 1996a.. More recently, expression of GH by TEC was ascertained by immunocytochemistry and in situ hybridization ŽMaggiano et al., 1994; Wu et al., 1996b.. Additionally, the production of IGF-I by the thymic epithelium has been demonstrated ŽGeenen et al., 1993; our unpublished data.. These findings raise the hypothesis that, besides the endocrine influences of pituitary hormones on TECrthymocyte interactions, an autocrinerparacrine circuitry mediated by the same hormones may be involved.

Acknowledgements We thank Mr. Alvaro L. Bertho for cytofluorometric analysis and Mrs. D. Broneer for English review. This work was supported by CAPES, CNPq and PADCTrCNPq ŽBrazil. and INSERM ŽFrance..

References Anderson, G., Moore, N.C., Owen, J.J.T., Jenkinson, E.J., 1996. Cellular interactions in thymocyte development. Annu. Rev. Immunol. 14, 73–99. Andrews, P., Boyd, R.L., 1985. The murine thymic nurse cell: An isolated thymic microenvironment. Eur. J. Immunol. 15, 36–41. Ban, E., Gagnerault, M.C., Jammes, H., Postel-Vinay, M.C., Haour, F., Dardenne, M., 1990. Specific binding sites for growth hormone in cultured mouse thymic epithelial cells. Life Sci. 48, 2141–2148. Berrih, S., Savino, W., Cohen, S., 1985. Extracellular matrix of the human thymus. An immunofluorescence study on frozen sections and cultured thymic epithelial cells. J. Histochem. Cytochem. 33, 655–664.

Boyd, R.L., Tucek, C.L., Godfrey, D.I., Izon, D.J., Wilson, T.J., Davidson, N.J., Bean, A.G.B., Ladyman, H.M., Ritter, M.A., Hugo, P., 1993. The thymic microenvironment. Immunol. Today 14, 445–459. Chiquet-Ehrismann, R., 1995. Tenascins, a growing family of extracellular matrix proteins. Experientia 51, 853–862. Cirne-Lima, E.O., van Ewijk, W., Savino, W., 1993. A mouse thymic epithelial cell line bears simultaneously cortical and medullary cytokeratin defined phenotypes. In Vitro Cell. Develop. Biol. 29A, 443– 445. Cotta-de-Almeida, V., 1996. The thymus in the immunopathology of experimental Chagas’ disease. Evidence for a disturbance in the intrathymic T-cell migration and differentiation, Ph.D. Thesis on Cellular and Molecular Biology, Foundation Oswaldo Cruz, pp. 1– 158. Dardenne, M., Savino, W., Gagnerault, M.C., Itoh, T., Bach, J.F., 1989. Neuroendocrine control of thymic hormonal production. I. Prolactin stimulates in vivo and in vitro the production of thymulin by human and murine thymic epithelial cells. Endocrinology 125, 3–12. Dardenne, M., Kelly, P.A., Bach, J.F., Savino, W., 1991. Identification and functional activity of prolactin receptors in thymic epithelial cells. Proc. Natl. Acad. Sci. USA 88, 9700–9704. Dardenne, M., Savino, W., 1994. Neuroendocrine control of thymus physiology by peptidic hormones and neuropeptides. Immunol. Today 15, 518–523. Dardenne, M., Leite-de-Moraes, M.C., Kelly, P.A., Gagnerault, M.-C., 1994. Prolactin receptor expression in human hematopoietic tissues analysed by flow cytometry. Endocrinology 134, 2108–2114. Defresne, M.P., 1986. Le microenvironment thymique et les cellules ´ pre-leucemiques au cours du developpement des lymphomes induits ´ ´ chez la souris. Role des cellules ‘nurses’ thymiques, Ph.D. thesis, ´ Liege ` University, Belgium. Fabris, N., Mocheggiani, E., Mariotti, S., Pacini, F., Pinchera, A., 1986. Thyroid function modulates thymic endocrine activity. J. Clin. Endocrinol. Metabol. 62, 474–478. Freitas, C.S., Lyra, J.S.P.O., Dalmau, S.R., Savino, W., 1995. In vivo and in vitro expression of tenascin by human thymic microenvironmental cells. Develop. Immunol. 4, 139–147. Gagnerault, M.-C., Touraine, P., Savino, W., Kelly, P., Dardenne, M., 1993. Expression of prolactin receptor on murine lymphoid cells in normal and autoimmune conditions. J. Immunol. 151, 1–9. Geenen, V., Achour, I., Robert, F., Vandersmissen, E., Sodoyez, J., Defresne, M.P., Boniver, J., Lefebvre, P.J., Franchimont, P., 1993. Evidence that insulin-like growth factor 2 ŽIGF-2. is the dominant thymic peptide of the insulin superfamily. Thymus 21, 115–124. Goff, B.L., Roth, J.A., Arp, L.H., Incefy, G.S., 1987. Growth hormone treatment stimulates thymulin production in aged dogs. Clin. Exp. Immunol 68, 580–587. Goya, R.G., Gagnerault, M.-C., Leite-de-Moraes, M.C., Savino, W., Dardenne, M., 1992. In vivo effects of growth hormone on thymus function in aging mice. Brain Behav. Immun. 6, 341–350. Itoh, T., Kasahara, S., Mori, T., 1982. A thymic epithelial cell line, IT-45R1, induces the differentiation of prethymic progenitor cells into post-thymic cells through direct contact. Thymus 4, 69–75. Lagrota-Candido, J.M., Vanderlei, F.H. Jr., Villa-Verde, D.M.S., Savino, ˆ W., 1996. Exracellular matrix components of the mouse thymic microenvironment. V. Effects of interferon-g upon thymocyterthymic epithelial cell interactions mediated by extracellular matrix ligands and receptors. Cell. Immunol. 170, 235–244. Lannes-Vieira, J., Dardenne, M., Savino, W., 1991. Extracellular matrix components of the mouse thymus microenvironment. I Ontogenetic studies and modulation by glucocorticoid hormones. J. Histochem. Cytochem. 39, 1539–1546. Lannes-Vieira, J., Chammas, R., Villa-Verde, D.M., Vannier-dos-Santos, M.A., Mello-Coelho, V.M., Souza, S.J., Brentani, R.R., Savino, W., 1993. Thymic epithelial cells express laminin receptors that may modulate interactions with thymocytes. Int. Immunol. 5, 1421–1430.

V. de Mello-Coelho et al.r Journal of Neuroimmunology 76 (1997) 39–49 Le, P.T., Tuck, D.T., Dinarello, C.A., Haynes, B.F., Singer, K.H., 1988. Human thymic epithelial cells produce IL-1. J. Immunol. 138, 2520– 2525. Maggiano, N., Piantelli, M., Ricci, R., Larocca, L., Capelli, A., Ranelletti, F.O., 1994. Detection of growth hormone-producing cells in human thymus by immunohistochemistry and non-radioactive in situ hybridization. J. Histochem. Cytochem. 42, 1349–1354. Nonoyama, S., Nakayama, M., Shiohara, T., Yata, J., 1989. Only dull CD3q thymocytes bind to thymic epithelial cells. The binding is elicited by both CD2rLFA-3 and LFA-1rICAM-1 interactions. Eur. J. Immunol. 19, 1631–1635. Ocklind, G., Talts, J., Fassler, R., Mattsson, A., Ekblom, P., 1993. Expression of tenascin in developing and adult mouse lymphoid organs. J. Histochem. Cytochem. 41, 1163–1169. Patel, D.D., Haynes, B.F., 1993. Cell adhesion molecules involved in intrathymic T-cell development. Semin. Immunol. 5, 283–292. Pelegrini, I., Lebrun, J.J., Ali, S., Kelly, P.A., 1992. Expression of prolactin and its receptor on human lymphoid organs. Cell. Mol. Endocrinol. 6, 1023–1029. Pricci, F., Pugliese, G., Romano, G., Romeo, G., Locuratolo, N., Pugliese, F., Merie, ´ P., Galli, G., Casini, A., Rotella, C.M., Di Mario, U., 1996. Insulin-like growth factors I and II stimulate extracellular matrix production in human glomerular mesangial cells. Comparison with transforming growth factor-beta. Endocrinology 137, 879–885. Savino, W., Wolf, B., Aratan-Spire, S., Dardenne, M., 1984. Thymic hormone containing cells. IV. Fluctuations in the thyroid hormone levels in vivo can modulate the secretion of thymulin by epithelial cells of young mouse thymus. Clin. Exp. Immunol. 55, 629–635. Savino, W., Dardenne, M., 1984. Thymic hormone containing cells. VI. Immunohistologic evidence for the simultaneous presence of thymulin, thymopoietin and thymosin a-1 in normal and pathological human thymuses. Eur. J. Immunol. 14, 987–991. Savino, W., Itoh, T., Imhof, B.A., Dardenne, M., 1986. Immunohistochemical studies on the phenotype of murine and human thymic stromal cell lines. Thymus 8, 245–256. Savino, W., Bartoccioni, E., Homo-Delarche, F., Gagnerault, M.C., Itoh, T., Dardenne, M., 1988. Thymic hormone containing cells. IX. Steroids in vitro modulate thymulin secretion by human and murine thymic epithelial cells. J. Steroid Biochem. 29, 135–140. Savino, W., Leite-de-Moraes, M.C., Hontebeyrie-Joskowicz, M., Dardenne, M., 1989. Studies on the thymus in Chagas’ disease. I. Changes in the thymic microenvironment in mice acutely infected with Trypanosoma cruzi. Eur. J. Immunol. 19, 1727–1733.

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Savino, W., Villa-Verde, D.M.S., Lannes-Vieira, J., 1993. Extracellular matrix proteins in intrathymic T-cell migration and differentiation?. Immunol. Today 14, 158–161. Savino, W., Dardenne, M., Carnaud, C., 1996. Conveyor belt hypothesis for intrathymic cell migration: Possible relationship with extracellular matrix. Immunol. Today 27, 97–98. Taub, D.D., Tsarfaty, G., Lloyd, A.R., Durum, S.K., Murphy, W.J., 1994. Growth hormone promotes human T-cell adhesion and migration to both human and murine matrix proteins in vitro and directly promotes xenogeneic engraftment. J. Clin. Invest. 94, 293–300. Timsit, J., Safieh, B., Gagnerault, M.-C., Savino, W., Lobetzki, J., Bach, J.F., Dardenne, M., 1989. Augmentation des taux circulants de thymuline au cours de l’hyperprolactinemie et de l’acromegalie. C.R. Acad. Sci. Paris 310 ŽSerie III., 7–13. Timsit, J., Savino, W., Safieh, B., Chanson, P., Gagnerault, M.-C., Bach, J.F., Dardenne, M., 1992. Effects of growth hormone and insulin-like growth factor 1 in thymic hormonal function in man. J. Clin. Endocrinol. Metab. 75, 183–188. van Ewijk, W., 1991. T-cell differentiation is influenced by thymic microenvironments. Ann. Rev. Immunol. 9, 591–615. Villa-Verde, D.M.S., Mello-Coelho, V., Farias-de-Oliveira, D., Dardenne, M., Savino, W., 1993. Pleiotropic influence of triiodothyronine on thymus physiology. Endocrinology 133, 867–875. Villa-Verde, D.M.S., Chammas, R., Lagrota-Candido, J.M., Brentani, ˆ R.R., Savino, W., 1994. Exracellular matrix components of the mose thymic microenvironment. IV. Thymic nurse cells express extracellular matrix ligands and receptors. Eur. J. Immunol. 24, 659–664. Vollger, L.W., Tuck, D.T., Springer, T.A., Haynes, B.F., Singer, K.H., 1987. Thymocyte binding to human thymic epithelial cells is inhibited by monoclonal antibodies to CD2 and LFA-3. J. Immunol. 138, 358–363. Wekerle, H., Ketelsen, U.P., Ernst, M., 1980. Thymic nurse cells. Lymphoepithelial cell complexes in murine thymuses: Morphological and serological characterization. J. Exp. Med. 151, 925–943. Wu, H., Devi, R., Mamarkey, W.B., 1996a. Localization of prolactin messenger ribonucleic acid in the human immune system. Endocrinology 137, 349–353. Wu, H., Devi, R., Mamarkey, W.B., 1996b. Localization of growth hormone messenger ribonucleic acid in the human immune system: A clinical research center study. J. Clin. Endocrinol. Metabol. 81, 1278–1282.