Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

Abstract Neurohypophysial oxytocin (OT) and vasopressin (VP) genes are transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.


INTRODUCTION
A repertoire of neuroendocrine-related genes are expressed within the thymus parenchyme of different species (Martens et al., 1996). Thymic neuroendocrine-related genes are highly conserved throughout evolution of their family and one dominant member is expressed in the thymus network: insulin-like growth factor 2 (IGF2) for the insulin family, neurokinin A (NKA) for tachykinins (Ericsson et al., 1990), and neurotensin (NT ) for neuromedins (Vanneste et al., 1997). Thymic precursors encoded by these genes exert a dual role in T cell differentiation according to their behavior either as cryptocrine/juxtacrine ligands or as neuroendocrine self-antigens (Geenen et al., 1999;Geenen et al., in press). With regard to the neurohypophysial gene family, both oxytocin (OT ) and vasopressin (VP) genes are expressed in thymic epithelial and nurse cells (TEC/TNC) from different species. At the peptide level however, thymic OT concentration is much higher than VP Geenen et al., 1998). During mouse ontogeny, OT and VP expression starts in thymic epithelium on embryonic day E13, while neurohypophysial transcripts are evidenced in the brain only on E15 (submitted for publication). Specific OT and VP binding sites have been evidenced in the rat thymus (Elands et al., 1990), as well as on a murine CD4 2 CD8 2 T cell line (RL12-NP) derived from X rayinduced thymic lymphoma in C57BL/Ka mice . These binding sites function as true receptors and are able to transduce neurohypophysial ligands into phosphoinositide turnover followed by proliferation effects . More specifically, neurohypophysial peptides, and OT in particular, markedly stimulate the phosphorylation of focal adhesion kinases in RL12-NP cells .
Distinct genes (OTR, V1R, V2R and V3R) encode four neurohypophysial peptide receptors which all belong to the superfamily of G-coupled seven-transmembrane proteins. OTR is primarily expressed in uterus myometrium, V1R in vascular smooth muscle and endothelium as well as in liver epithelial cells, V2R in the epithelium of kidney water collecting tubules, and V3R in anterior pituitary corticotroph cells (Gainer and Wray, 1994). OTR, V1R and V3R are coupled to protein G q/11 and activate phospholipase C pathway (Thibonnier et al., 1993), while V2R is coupled to protein G s and activates adenylate cyclase pathway (Barberis and Tribollet, 1996).
This study aimed to determine the nature and distribution of OTR, V1R, V2R and V3R expressed by murine thymic T cell subpopulations and by thymic lymphoma-derived T cell lines RL12-NP and BW5147. In addition, a preliminary insight into the functional role played by the thymic neurohypophysial axis in T cell development was investigated through the use of fetal thymic organ cultures (FTOC) (Jenkinson and Anderson, 1994).

OTR Expression
RT-PCR with OTR specific primers revealed the presence of a 273-bp product in the mouse uterus as positive control. A same sized RT-PCR product was detected in all thymic T cell subsets, and in RL12-NP and BW5147 lines (Fig. 1A). Southern blotting of the PCR product and hybridization with a specific probe confirmed the presence of OTR transcripts in thymocytes (Fig. 1B). Further sequencing of the 237-bp product amplified from RL2-NP cells showed a 97.1% homology with murine OTR sequence. Thymocytes freshly isolated from C57BL/6 mice were also positively stained with a specific Ab to OTR (Fig. 1C).

V3R Expression
RT-PCR with V3R primers identified a 254-bp product in mouse anterior pituitary as positive control. A same sized product was similarly detected in thymic CD4 þ CD8 þ and CD8 þ T cells, but neither in CD4 2 CD8 2 nor in CD4 þ T cells ( Fig. 2A). No amplified product was detected in RL12-NP and BW5147 lines (not shown). Southern blotting and hybridization with a specific probe confirmed V3R expression in CD4 þ CD8 þ and CD8 þ thymic T cells (Fig. 2B).

V1R and V2R Expression
RT-PCR with V1R and V2R primers revealed the presence of 409 and 437-bp products in liver and kidney, respectively, but failed to amplify any product in thymic T cells and T cell lines. Hybridization with specific probes confirmed the absence of V1R and V2R transcripts in all T cell samples (data not shown).

Effects of Neurohypophysial Receptor Antagonists on T Cell Differentiation and Survival in FTOC
From day 0 (corresponding to E14) to day 6 of FTOC, RT-PCR with OT and VP specific forward and reverse primers revealed 211 and 188-bp products, respectively, which hybridized with specific OT and VP oligonucleotide probes after membrane transfer. In FTOC treated with specific OTR and V1R antagonists at 10 26 and 10 29 M, no significant effect could be observed on T cell differentiation. However, the OTR antagonist (10 26 and 10 29 M)-but not the V1R antagonist-induced a significant increase of late apoptosis parameters (Fig. 3).

DISCUSSION
As suggested by previous studies (Torres and Johnson, 1988;Elands et al., 1990;Martens et al., 1992), T lymphocytes express neurohypophysial peptide receptors, but the precise identity of receptor subtypes and positive T cells still had to be fully characterized. The present study addressed this question by investigating the nature of neurohypophysial receptor expressed by highly purified thymic T cells and by thymic lymphomaderived T cell lines RL12-NP and BW5147. As clearly shown, OTR is transcribed in all MACS-purified thymic T cell subsets (CD4 2 CD8 2 , CD4 þ CD8 þ , CD4 þ and CD8 þ ), as well as in RL12-NP and BW5147 T cell lines. OTR transcription leads to membrane expression of the OTR protein in freshly isolated thymocytes. These data confirm at the molecular level those previously obtained by autoradiography and by classic radio-ligand binding analyses (Elands et al., 1990;Martens et al., 1992).
RT-PCR with V3R primers identified in CD4 þ CD8 þ and CD8 þ cells a product with the same size as the anterior pituitary positive control. Southern blotting and hybridization with a specific probe confirmed that V3R is expressed by these T cell subpopulations. This result indicates that V3R might be the other neurohypophysial receptor expressed by T cells as already suggested in a previous study . Our observation also explains the positive hybridization with V3R cDNA in northern blots from human thymus extracts (Sugimoto et al., 1994). Nevertheless, we cannot definitively exclude that other cell types might also express V3R in the thymus cellular network. V1R expression could not be documented in any of the T cell types investigated. Since vascular endothelial cells express this receptor (Barberis and Tribollet, 1996;Zingg, 1996), this negative result indirectly confirms that our purification procedure is not contaminated by endothelial cells. V2R expression was neither observed in any type of T cells and this concords with our experience about the absence of adenylate cyclase activation by neurohypophysial-related peptides in immature or mature T cells. Previously, a radiobinding study had suggested that, in the immune system, only macrophages express the V2R type of neurohypophysial receptor (Block et al., 1981).
The functional role of thymic neurohypophysial-related peptides must be discussed according to the behavior of the final peptide product processed in TEC following OT and VP transcription. This study characterizes the parameters involved in the signaling between neurohypophysial ligands and receptors, and these may be currently defined as follows. At the level of the neurohypophysial ligands, both OT and VP peptides are synthesized in thymic epithelial cells (TEC), with a dominance of OT. At the level of the receptors, OTR is expressed at all main stages of T cell differentiation, while V3R expression is restricted to CD4 þ CD8 þ and CD8 þ T cells. Thus, a different pattern of neurohypophysial receptor expression occurs along with the process of T cell differentiation. Similarly to what has been shown for IGF type 2 receptors (CD222) (Mason, 2000), it may be expected that OTR and V3R also will be soon identified as new cluster differentiation (CD) markers. Ancient studies have reported that neurohypophysial ligands increase thymocyte growth (Whitfield et al., 1969;Martens et al., 1992) and the level of glucose oxydation in the thymus (Goren et al., 1984). More recently, the binding of neurohypophysial ligands, particularly OT, was shown to increase the level of inositol triphosphate and to phosphorylate focal adhesion-related kinases (FAK) in immature T cells . The implication of these kinases in T cell differentiation has also been described by other authors (Kanazawa et al., 1996). In this study, the FTOC model was used to gain a first insight into the role of the thymic neurohypophysial axis in T cell development. Endogenous OT and VP transcription was controlled throughout FTOC duration. FTOC treatment with a specific OTR antagonist (10 26 and 10 29 M) induced a significant increase in late apoptosis parameters, but did not modify the percentage of T cell subsets. A V1R antagonist did not exert any significant influence, in accordance with the absence of V1R expression by thymic T cell subsets. These data further confirm that an intrathymic OT/OTR signaling is implicated in the general process of T cell development and survival. Since this pathway also controls FAK phosphorylation in CD4 2 CD8 2 RL12-NP cells, thymic OT/OTR signaling could play a significant role in the promotion of immunological synapses between TEC and thymocytes during T cell differentiation.
Finally, it should be stressed that the investigation of neurohypophysial receptor expression by immature CD4 2 CD8 2 T cells lines derived from murine thymic lymphomas showed that OTR is the only neurohypophysial receptor expressed by those cells. With regard to the mitogenic and FAK-promoting activity of thymic OT, as well as proapoptotic properties of the OTR antagonist, this finding suggests that the use of OTR antagonist might be considered in the future therapy of T cell lymphomas/leukemia's.

Staining and Immunomagnetic Separation (MACS)
Thymic lobes were surgically removed from adult female C57BL/6 mice; they were cut in small pieces, filtered and washed in DPBS buffer. Thymocyte suspension was incubated with a mAb cocktail prepared for negative selection of CD4 and CD8 T cells (StemCell Technologies). After washing in DPBS buffer, an anti-biotin tetrameric Ab complex mix (StemCell Technologies) was added. The last incubation was performed with a magnetic colloid (StemCell Technologies). Separation was realized with a MACS column (Miltenyi Biotec).

Total RNA Extraction
Total RNA from murine uterus, liver, kidney and anterior pituitary was extracted with TriPure Isolation Reagent (Boerhinger Mannheim). Total RNA from thymocytes, cell lines and fetal thymic lobes (at different days of culture) was extracted with RNeasy Mini Kit (Qiagen). They were treated (15 min, 378C) with DNase I (5 IU/reaction) in presence of RNase inhibitor (2 IU/ml). Reaction was ended by enzyme inactivation (8 min, 858C). Then, 0.1vol NaAc (3 M) and 2.5vol ethanol (100%) were added and RNA was precipitated (12 h, 2 208C). After two washings with ethanol (70%), RNA was suspended in milli-Q quality water and quantified with Ribogreen RNA Quantitation Kit (Molecular Probes).

Immunocytochemistry
Thymocytes were freshly isolated from C57BL/6 mice, transferred on slides by cytospin centrifugation and fixed acccording to Robert et al. (1991). OTR polyclonal Ab (1:50) was used as first Ab, and FITC goat anti-mouse Ig (Jackson, 1:50) was used in second step.

Statistics
Results were expressed as mean (m)^standard deviation (sd) of values after treatment vs basal conditions. Normality was tested by Kolmogorov-Smirnov test. Differences between groups were estimated by ANOVA and analyzed by multi-comparative Student-Newman -Keuls test.