Expression of NGF receptors in normal and pathological human thymus
Introduction
The thymus is the primary lymphoid organ where competent and self-tolerant T-cells differentiate (Kruisbeek, 1993; Ritter and Boyd, 1993). This process requires cell to cell interactions and the production of soluble factors by thymocytes or non-lymphoid stromal cells (Boyd et al., 1993; Kruisbeek et al., 1980; Ritter and Boyd, 1993). The stromal population consists in epithelial cells, fibroblasts, macrophages and dendritic interdigitated cells. Three types of epithelial cells, the subcapsular, cortical and medullary cells, have been individualised on the basis of their phenotype and ontogeny (Le Lievre and Le Douarin, 1975; Lobach and Haynes, 1987). Ablation of areas of chick embryos cephalic neural crest results in thymic aplasia/hypoplasia (Bockman and Kirby, 1984).
Nerve Growth Factor (NGF) has been characterised as a trophic factor for the development and maintenance of neural crest-derived sensory and sympathetic neurons (Levi-Montalcini, 1987). NGF is the prototype of a family of target-derived neurotrophic factors that also includes Brain Derived Neurotrophic factor (BDNF), Neurotrophin-3 (NT3) and Neurotrophin-4/5 (NT-4/5) (Lindsay et al., 1994). These neurotrophins (NT) share a common low-affinity p75NGFR receptor (Chao and Hempstead, 1996) that may modulate the interaction of a defined NT to its specific receptor (Rodriguez-Tebar et al., 1992; Clary and Reichardt, 1994). High affinity binding sites require the presence of tyrosine kinase receptors of the trk family (Barbacid, 1994). NGF is the preferred ligand for Trk, also named TrkA (Kaplan et al., 1991a, Kaplan et al., 1991b; Klein et al., 1991a). BDNF and NT4/5 are the preferred ligands for TrkB (Klein et al., 1991b, Klein et al., 1992) while TrkC is activated by a unique ligand, NT-3 (Lamballe et al., 1991). Crosstalks between TrkA and NT-3 (Cordon-Cardo et al., 1991) or NT-4/5 (Berkemeier et al., 1991; Ip et al., 1992) occur at higher ligand concentration (Ip et al., 1993). In some cellular systems, TrkA by itself is sufficient to form high-affinity binding sites through homodimerisation (Klein et al., 1991a; Jing et al., 1992) while p75NGFR potentiates NGF elicited TrkA activation in PC12 cell line (Hempstead et al., 1991).
NGF supports sympathetic innervation of lymphoid tissues (Carlson et al., 1995) but triggers several biological response on immune cells (Otten et al., 1994). NGF is considered as a regulatory cytokine for lymphoid cells differentiation and survival (Ehrhard et al., 1993, Ehrhard et al., 1994; Torcia et al., 1996).
The expression of NTs and their receptors has been recently investigated in the rodent thymus (Laurenzi et al., 1994; Lomen Hoerth and Shooter, 1995; Maroder et al., 1996). Isolation of mononuclear cells from stromal cells allowed the detection of NT-3 and NT-4 transcripts in the thymocytes fraction and of all NT in the corresponding stroma (Laurenzi et al., 1994). While Loemen Hoerth and Shooter (Lomen Hoerth and Shooter, 1995) found a predominant expression of NT receptors transcripts, p75NGFR, trkA and trkB in the thymus stroma, Laurenzi et al. (Laurenzi et al., 1994) found a predominant expression of trkA transcripts in thymus mononuclear cells and of p75NGFR and trkB transcripts in stromal cells. Alternatively, no trkA transcript but trkB transcripts have been detected in mice thymocytes (Maroder et al., 1996). Therefore, a precise anatomical identification of NGF receptors expression would lead to a better understanding of the physiological targets of NGF in thymus development and pathology.
In human, this identification has primarily focused on p75NGFR (Pescarmona et al., 1993). Subcapsular and medullar epithelial cells were found p75NGFR immunoreactive (IR) suggesting a biological role for NGF on neural crest-derived epithelial cells (Pescarmona et al., 1993). An immunohistochemical detection of TrkA was also performed in some thymuses among a wide range of human tissues (Shibayama and Koizumi, 1996). Both thymocytes and epithelial cells were found TrkA-IR. Our preliminary results on three adult thymuses indicated a more restricted distribution of TrkA or p75NGFR receptors on specific stromal cells in the normal human thymus (Labouyrie et al., 1997) analogous to those observed in the pigeon thymus (Ciriaco et al., 1996).
These differences prompted us to investigate the expression of both p75NGFR and TrkA receptors in normal human thymuses, at foetal and adult stages and in pathological conditions including thymic hyperplasia, thymoma and thymic carcinoma. Our results indicate that p75NGFR and/or TrkA are specifically expressed by epithelial or interdigitated reticular cells (IRC) but not by thymocytes. A switch in the normal TrkA positive–p75NGFR negative phenotype to a TrkA negative–p75NGFR positive phenotype was found in histologically aggressive epithelial cells tumours suggesting that NGF and its receptors are involved in the thymus stroma organogenesis and proliferation.
Section snippets
Tissues
Fresh thymuses were divided into one snap-frozen part in liquid nitrogen-cooled isopentane and one part fixed in 10% buffered formalin or Bouin's liquid. Foetal thymuses (n=3) were collected from the files of the Foetopathology Unit of the University Hospital according to the guidelines of the Ethic Committee. Normal human thymuses (n=3) were removed during cardiac surgery (n=2) or collected from autopsy (n=1). Pathological thymuses (n=15) were obtained by surgery. Combined clinical and
Detection of trkAI transcripts in normal and pathological thymuses
We performed RT-PCR on RNA extracted from normal thymuses from two foetuses (weeks 23 and 26, menstrual age), two children (8 months and 8 yr) and one adult (46 yr). We also studied pathological thymuses with thymic hyperplasias (n=3), thymomas (n=6) and thymic carcinoma (n=1). To confirm the specificity of amplification products visualised on ethidium bromide stained gels (Fig. 1), all RT-PCR bands from one adult thymus, one thymic hyperplasia, one thymic thymoma and one carcinoma and from the
Discussion
Our study extends to the molecular level and to human pathology the identification of TrkA receptors in the human thymus previously performed by immunohistochemistry in the pigeon (Ciriaco et al., 1996). Using RT-PCR and sequencing, we found TrkA transcripts in the human thymus both in normal and in pathological conditions. Only the short and peripheral trkAI transcripts were detected in the thymus while trkAII transcripts, that contains the 18 bp alternatively spliced exon (Barker et al., 1993
Acknowledgements
This work was supported by grants from the Association pour la Recherche contre le Cancer (ARC) and the Region Aquitaine. We are grateful to J. Ferrer and C. Bartoli for molecular analysis, M. Turmo and B. Garcia for protein analysis. We acknowledge Dr. L.F. Reichardt for the gift of the rtrkA antibody.
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