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INSERM U-344, Endocrinologie Moleculaire (V.d.M.-C., J.-C.S., M.-C. P.-V.), and Universite Paris V, CNRS URA 1461, Hopital Necker (V.d.M.-C., M.-C.G., M.D.), Paris, France; the Laboratory on Thymus Research, Department Of Immunology, Foundation Oswaldo Cruz (W.S.), Rio De Janeiro, Brazil; and the Department of Medicine, Ludwig Maximilians Universität (C.J.S.), Munich, Germany
Address all correspondence and requests for reprints to: Dr. Marie-Catherine Postel-Vinay, INSERM U-344, Endocrinologie Moléculaire, Faculté Necker Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France. E-mail: postel-vinay{at}necker.fr
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Introduction |
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Intrathymic T cell differentiation is driven by the thymic microenvironment, a tridimensional network composed of distinct cell types, such as thymic epithelial cells (TEC), macrophages, and dendritic cells, as well as extracellular matrix elements (3, 4). The thymic epithelium, the major component of the thymic microenvironment, influences differentiating thymocytes by the secretion of various polypeptides, including thymic hormones and cytokines and also by cell-cell contacts: interactions involving major histocompatibility complex gene products expressed by thymic epithelial cells with the T cell receptor, and interactions through classical adhesion molecules (3, 4). Lastly, TEC can bind to and interact with maturing thymocytes by means of extracellular matrix ligands and receptors (5).
Numerous recent studies demonstrate that distinct intrathymic cellular interactions are under neuroendocrine control (6); in particular, GH has been shown to modulate thymus physiology. The secretion of thymulin, a chemically defined thymic hormone produced by TEC, is up-regulated by GH in different species, including mouse (7), dog (8), and man (9, 10). GH also increases TEC proliferation in vitro as well as the expression of extracellular matrix ligands and receptors with consequent modulation of extracellular matrix-mediated TEC/thymocyte interactions (9, 11). In vivo experiments evidenced changes in thymocyte differentiation under GH influence. It was shown that GH injections in aging rats increased total thymocyte number and the percentage of CD3-bearing cells (12, 13). Accordingly, we showed enhanced concanavalin A mitogenic response as well as interleukin-6 production by thymocytes from GH-treated aging animals (7). Moreover, in dwarf mice, long term treatment with GH restored the thymic hypoplasia and decreased number of CD4+CD8+ thymocytes observed in these animals (14). Lastly, recombinant human GH (hGH) was shown to stimulate human peripheral blood lymphocyte engraftment and migration of T cells into the thymus of SCID (severe combined immunodeficiency) mice (15).
It has been difficult to identify GH receptors (GHRs) in thymic cells, probably because of the low receptor number. Initial studies revealed GH binding sites in murine TEC (16). More recently, the presence of GHR was demonstrated in murine thymocyte subsets by means of cytofluorometry (17). Concerning the human thymus, in situ hybridization studies revealed a positive signal for the GHR messenger RNA (mRNA) throughout the cortex as well as the medullary TEC (18).
In addition to GHR expression, intrathymic expression of GH was suggested with the detection of the specific mRNA by in situ hybridization and of the protein by immunocytochemistry (19, 20).
In the present work, using RT-PCR and immunoradiometric assay, we show the gene expression and the production of hGH in human TEC and thymocytes in culture. Moreover, GHRs are identified at distinct stages of thymocyte differentiation by cytofluorometry and in cultured TEC by immunohistochemistry.
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Materials and Methods |
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Culture of TEC
Surgical discarded thymic tissues were obtained from children
undergoing cardiac surgery (n = 12), aged from 5 days to 3 yr. The
thymic capsula was removed, and the lobules were transfered to a
conical tube containing RPMI 1640, supplemented with 10%
heat-inactivated FCS, 10 mM HEPES, 1 mM sodium
pyruvate, 1% nonessential amino acids, 5 x 10-5
M 2-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml
streptomycin (all from Life Technologies, Cergy-Pontoise, France).
Tissue was minced, and the supernatant containing thymocytes was
collected and subjected to centrifugation (1800 rpm for 20 min) on
Ficoll-Hypaque density gradient. To obtain fibroblast-free TEC primary
culture, small thymic explants were cultured in 75-cm3
flasks in D-valine-containing Eagle’s MEM supplemented
with 2 mmol/L L-glutamine, 10 mmol HEPES, 100 U/ml
penicillin, 100 mg/ml streptomycin, and 10% FCS, as previously
described for culture of cortical TEC lines from fetal and postnatal
human thymi (23). The purity of epithelial cells in culture was
ascertained by anticytokeratin immunostaining and was determined to be
over 96%.
The two human TEC lines, obtained by an explant technique and limiting dilution cloning, were provided by Dr. M. L. Toribio (Universidad Autonoma de Madrid, Madrid, Spain). One was obtained from a fetal thymus, and the other was derived from explants of a postnatal organ (23). All cultures were maintained using D-valine containing RPMI 1460 medium at 37 C in 5% CO2. The viability of the cells was more than 95%, as evaluated by trypan blue staining.
RNA extraction from thymic cell preparations
TEC in primary culture (11–14 days) or TEC lines were treated
with trypsin/EDTA (Life Technologies) and centrifuged at 4 C (3000 rpm,
10 min) before washing in PBS. Thymocyte suspensions were centrifuged
as described above. The pellets were resuspended in 1 ml RPMI for cell
counting. Total RNA was prepared using Trizol reagent (Life
Technologies) as an improvement to the single step RNA isolation
originally described by Chomczynski and Sacchi (24).
RT-PCR
Various quantities of thymocyte- or TEC-derived total RNA were
denatured for 10 min at 68 C and cooled at 4 C. RT was performed in
solution containing 20 mM Tris-HCl (pH 8.3), 50
mM KCl, 5 mM MgCl2, 10
mM dithiothreitol, 1 mM deoxy-NTP, 1 U/ml
RNasin, and 10 U/ml Moloney murine leukemia virus reverse transcriptase
(Life Technologies) at 37 C for 1 h. The RT reaction was finished
by heating specimens at 96 C for 5 min and cooling at 4 C.
Oligonucleotide sequences of the different primers used are presented
in Table 1
. Ten microliters of RT
products were used in PCR mixture, with 25 pmol of each primer, sense
and antisense, and 1 U Taq polymerase (Perkin-Elmer/Cetus,
Norwalk, CT), diluted in 20 mM Tris-HCl (pH 8.3), 50
mM KCl, 2 mM MgCl2, and 200
mM deoxy-NTP in a final volume of 50 or 30 µl for
thymocytes or TEC, respectively. The PCR profile for GH complementary
DNA (cDNA) amplification consisted of denaturation at 94 C for 5 min
followed by 30 cycles at 94 C for 30 sec, 60 C for 1 min, and 72 C for
1 min. For GHR cDNA amplification, 36 cycles were performed at 94 C for
30 sec, 56 C for 1 min, and 72 C for 1 min and 15 sec. Both cDNA
amplifications were completed by heating at 72 C for 10 min. Amplified
PCR products were electrophoresed in 1% ethidium bromide-agarose gel
and visualized under a UV transluminator. RT-PCR in the absence of RNA
served as a negative control, whereas RNA prepared from human
hypophysis and liver were used as positive controls for GH and GHR gene
expression, respectively.
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Supernatants of thymocyte- or TEC-derived cultures were concentrated 20-fold using Centripep-30 Amicon filters (Grace, Epernon, France). The samples were analyzed by a commercial immunoradiometric assay (Kit 125I hGH U Coatria, BioMerieux, Lyon, France) according to instructions of the manufacturer. This technique has been used for the measurement of urinary GH (25), and the detection limit has been evaluated to be 0.5 pg/ml. Results are expressed as picograms per ml concentrated medium.
Immunocytochemistry
Cultures of epithelial cells were submitted to an indirect
immunofluorescence technique (26). Briefly, cells were fixed with
absolute ethanol for 5 min, incubated with PBS-0.2% BSA for 1 h,
and further washed with PBS. Material was then subjected to
biotinylated anti-GHR antibody (mAb 10B8, diluted 1:10) for 1 h,
washed with PBS, and subjected to streptavidin-FITC (diluted 1:100) for
1 h. After an additional PBS washing, samples were mounted and
analyzed under a Leitz Ortoplan fluorescence microscope (Leitz,
Rockleigh, NJ). To ascertain the epithelial nature of the primary TEC
cultures, anticytokeratin serum (diluted 1:20) was used; it was
revealed with the FITC-labeled goat antirabbit IgG serum.
Flow cytometric analysis and cell sorting
Triple fluorescence immunostaining of thymocytes was performed
by incubating 1 x 106 cells in PBS supplemented with
2% FCS and 0.1% sodium azide in the presence of biotinylated, PE- or
FITC-conjugated specific antibodies or isotype controls at appropriate
concentrations. After PBS washing, cells were incubated with
streptavidin-Cy-Chrome and postfixed in 1% formaldehyde. Incubations
were performed in a final volume of 20 µl in 96-well microtiter
plates for 20 min at 4 C. In some experiments, dual immunostaining was
carried out using two different anti-GHR monoclonal antibodies;
biotinylated 263 mAb was revealed by streptavidin-PE, whereas 10B8 mAb
was directly coupled to FITC. Cell acquisition was performed in a
FACScan flow cytometer, and data were analyzed using Lysis II software
(Becton Dickinson, Grenoble, France). Forward light scatter gates were
set to exclude dead cells and debris.
For analysis of CD34+ CD2+ triple negative (TN) cells, thymocytes were first depleted of CD3-expressing cells by incubation with biotinylated anti-CD3 mAb (Caltag) followed by one cycle of streptavidin microbeads (Miltenyi, Tebu, Le Perray-en-Yvelines, France). The remaining cells were incubated with PE-labeled anti-CD2 mAb and FITC-conjugated anti-CD3, -CD4, -CD8, and -CD19 mAbs (Immunotech). CD2+ CD3- CD4- CD8- CD19- cells were then sorted with a FACS Vantage cell sorter (Becton Dickinson). Reanalysis of sorted cells indicated a purity of 98% or more.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
was detected in freshly isolated thymocytes (lanes 1–4) and in primary
culture of TEC (lane 5), where the signal was weaker. A PCR product of
similar size (390 bp) was amplified with RNA extracted from human
pituitary (lane 6). No signal was detected for controls (RT-PCR in the
absence of RNA; lanes 7 and 8).
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).
Immunoreactive hGH was detected in the media of seven different
thymocyte cultures with large variations in the hormone concentration.
The high value (264 pg/ml) found in one thymocyte culture medium was
confirmed by assaying serial dilutions of the medium. In addition,
three media of primary TEC cultures contained immunoreactive hGH.
However, no GH gene expression was found in fetal and postnatal TEC
lines, and no GH-immunoreactive material was detected in the culture
media of the cells (not shown).
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shows the PCR products revealed in 1%
agarose gel by ethidium bromide staining. A band of the expected size
of 330 bp was visualized in thymocytes (lane 1), TEC (lane 2), as well
as fetal (lane 3) and postnatal (lane 4) TEC lines. The negative
controls represent amplification of the reverse transcribed products
lacking RNA, and the positive control represents the amplification of 4
µg total RNA from human liver after reverse transcription (lane
5).
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). The signal for GHR was very weak at
the cell surface. A strong labeling was visualized in the perinuclear
region of the cells (Fig. 3, b–d). As expected, control IgG did not
generate significant fluorescent signal (Fig. 3a). Double labeling with
anticytokeratin or anti-GHR mAb was also performed by
immunocytochemistry on fixed primary TEC or on fetal or postnatal TEC
lines. Over 96% of the cells were positive to cytokeratin detection
(not shown).
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). Comparable results (not shown) were
obtained through dual immunostaining using two different anti-GHR
monoclonal antibodies, allowing the use of either of the two antibodies
to study GHR expression.
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and Table 3
). However, although higher
frequencies of GHR-positive cells were found within this immature
population, GHR density on cell membrane appeared similar in the
distinct CD4/CD8-defined subsets.
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. Moreover, as CD2 has been recently
shown not to be restricted to T cell lineage, but is also expressed on
thymic B cells bearing the CD19+ marker (27),
CD19+ cells were also separated by sorting. Sorted
thymocytes expressing the CD2+ CD3-
CD4- CD8- CD19- phenotype were
then examined for their expression of CD34, a marker for intrathymic T
cell progenitors (28) and GHR. Over 90% of cells with the
CD34+ CD2+ phenotype expressed GHR at the cell
membrane, as shown in Fig. 5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intrathymic production of GH has originally been reported on the basis of immunocytochemical and in situ hybridization data (19, 29), which revealed positive signals in cortical epithelial cells and in septal, phenotypically undefined cells, but not in thymocytes. However, immunoreactive and biologically active GH was shown to be produced by isolated human and rat thymocytes (30, 31). In another study, a GH-specific RT-PCR product was revealed in total thymus extracts (but not in distinct cell types), and in situ hybridization signal was found in epithelial cells and in some thymocytes (20). Our data represent the first demonstration of the presence of GH mRNA in human thymocytes and TEC, with the secretion of a GH-like peptide by both cell types. However, the subpopulation of GH-producing cells in human thymus remains to be identified.
The fact that the two TEC lines presented neither detectable hormone production nor GH gene expression remains intriguing. It is unlikely that nonepithelial contaminants in TEC primary cultures are responsible for GH expression because 1) very few contaminant cells are present in the cultures; and 2) previous findings, using immunocytochemistry, have demonstrated subseptal TEC positive for anti-GH labeling (19). A more likely explanation is loss of the ability of immortalized TEC to constitutively produce GH.
The physiological relevance of the locally produced GH needs to be further studied. In addition to actions of pituitary-derived GH, aspects of thymus physiology, including thymulin production (6), and some thymocyte/microenvironmental interactions (11) as well as progress of thymocyte differentiation may be under the control of thymus-derived GH. In a recent work, Sabharwal and Varma showed that GH is produced by human thymic cells and is able to stimulate the proliferation of cultured thymocytes. The authors also provided evidence favoring an indirect GH effect, mediated by locally produced insulin-like growth factor I (IGF-I) (31). It has also been reported that exogenous GH is able to stimulate the production of thymulin and IGF-I as well as the proliferation of TEC (6). All of these results suggest that locally synthesized GH and IGF-I could play important roles as growth factors for thymocytes and TEC.
The biological effects of GH are mediated by receptors located in target cell membranes. The presence of GH-binding sites on cultured murine TEC was shown initially (16). Using RT-PCR followed by Southern analysis, GHR mRNA was identified in rat thymus, and in situ hybridization experiments revealed the presence of GHR mRNA on human medullary microenvironmental cells and thymocytes (18). More recently, using cytofluorometry, the expression of GHRs was demonstrated on murine thymocytes (17). Nonetheless, the expression of GHRs in human thymocytes had not been reported. We demonstrate that GHRs are expressed in thymocytes, and that the expression is restricted to a small percentage of cells, which is in contrast with the large expression of PRL receptors in most thymocytes (32). Despite comparable effects of PRL and GH on the thymic microenvironment (6, 11), direct targeting of these two hormones on the lymphoid compartment of the organ might be differential.
Human thymocytes have been shown to differentiate from immature TN CD3- CD4- CD8- precursors to mature single positive CD4+ or CD8+ populations expressing high levels of CD3. Our data demonstrate that GHR are mainly present in these immature CD3- CD4- CD8- TN precursors. However, TN thymocytes that lack the markers CD3, CD4, and CD8 constitute a heterogeneous population of precursors with various capacities for differentiation (33). Convincing evidence has now been obtained that the most immature thymocytes able to differentiate in CD3+ cells express the pluripotent marker stem cell CD34 (28, 34). CD34+ TN cells can be further subdivided according to phenotype. The finding that CD34+ coexpress CD2, an early T cell marker, suggests further that these cells are T cell precursors, although the expression of CD2 has been recently shown on some thymic mature B cells (defined by the CD19 marker). Thus, using these three markers and cell sorting, we defined the phenotype of immature thymic precursors expressing GHR and showed, on CD3-depleted cells, that GHR+ cells were mainly restricted to a subpopulation of TN cells expressing CD34 and CD2, but not CD19. These findings are in agreement with the expression of GHR in immature murine thymic cell subsets (17) and also with in vivo data observed in dwarf mice; the animals presented a deficiency in T cell progenitors in the thymus, and GH treatment resulted in the expansion of CD4+/CD8+ double positive cells (14). Taking into account the comitogenic activity of GH on concanavalin A-treated mouse thymocytes (35), it is conceivable that in man, GH may play a role in the continuous expansion of cells that have not yet been subjected to the process of positive and negative selection of their repertoire. It is of interest to recall the dual effector theory for GH, which was proposed by Green for preadipocytes but can be applied to other cell types (36): GH directly affects the differentiation of cell precursors and indirectly, through IGF-I, has a mitogenic effect on young differentiated cells.
By immunocytochemistry, using biotinylated monoclonal antireceptor antibody, the presence of GHRs is shown in TEC in primary culture and also in two human cell lines. The subcellular distribution of the GHRs is in accordance with the findings of previous studies in other cells; the receptors are more numerous in intracellular compartments than at the cell surface (37).
Constant interactions exist between TEC and differentiating thymocytes,
with well known effects of TEC on intrathymic T cell
migration/differentiation. Considering that GH can be produced by TEC
and thymocytes, and that both cell types express GHRs, a paracrine
GH-mediated lymphoepithelial cross-talk is conceivable (38). Indeed,
thymocyte-derived cytokines, such as interferon-
, can modulate TEC
proliferation and extracellular matrix secretion with consequent
changes in the thymocyte adhesion pattern to epithelial cells (39, 40).
Likewise, GH affects various functions of thymocytes and TEC,
supporting the hypothesis of a bidirectional GH-mediated cross-talk
between the two cell types. It was recently shown that GH is able to
regulate the expression of several cytokines in bovine thymic stromal
cells (41).
In conclusion, our results support a paracrine/autocrine mode of action for GH in the human thymus.
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Acknowledgments |
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Footnotes |
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Received March 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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modulates thymocyte/thymic
epithelial cell interactions via extracellular matrix ligands and
receptors. Cell Immunol 170:235–244[CrossRef][Medline]
, IL-1ß and IL-6 expression by growth hormone
and prolactin in bovine thymic stromal cells. Mol Cell Endocrinol 128:117–127[CrossRef][Medline]
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