Massive lymphocyte apoptosis in the thymus of functionally deficient TrkB mice

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Abstract

The occurrence of TrkB in the murine thymus (15-day and 3-month old) was investigated by Northern blot, Western blot and immunohistochemistry. Furthermore, the thymus of 15-day-old mice carrying a non-functional mutation on trkB was analyzed. Both trkB mRNA and 145 kDa TrkB protein were detected. In addition, isolated lymphocytes and stromal cells also expressed this protein. The thymus of homozygous functionally TrkB-deficient animals showed structural and ultrastructural changes consistent with massive death of cortical lymphocytes, confirmed with TUNEL. Present results suggest a role for TrkB in maintaining the survival or preventing massive death of lymphocytes in the mammalian thymus.

Introduction

Neurotrophins (NTs) are a family of growth factors primarily known for their role in promoting and maintaining the survival of discrete populations of developing and mature neurons (see Fariñas, 1999, Huang and Reichart, 2001). However, they are now regarded as growth factors with a wide spectrum of functions (Tessarollo, 1998), including regulation of the immune system (for references, see Aloe et al., 1999, Garcı́a-Suárez et al., 2000a). The biological effects of the NTs are mediated by a group of tyrosine-kinase receptors codified by the trk family of proto-oncogenes, denominated TrkA, TrkB and TrkC. Full-length and truncated (i.e. lacking the tyrosine-kinase domain) isoforms have been identified for TrkB and TrkC, whereas all isoforms of TrkA contain the catalytic domain (Lewin and Barde, 1996). The full-length isoform of TrkB, 145 kDa, serves as a signal transducing receptor for brain-derived growth factor (BDNF) and neurotrophin 4/5 (NT-4/5; Lewin and Barde, 1996) and it is widely distributed in both nervous and non-nervous tissues Shibayama and Koizumi, 1996, Yamamoto et al., 1996.

TrkB, at the mRNA or protein level, has been found in lymphoid organs and immunocompetent cells Laurenzi et al., 1994, Lomen-Hoerth and Shooter, 1995, Aloe et al., 1999, D'Onofrio et al., 2000. The rat thymus contains mRNA for both full-length and truncated TrkB Laurenzi et al., 1994, Lomen-Hoerth and Shooter, 1995, although the thymic cells expressing them have not been fully characterized. In fact, TrkB mRNA has been found in immature cortical thymocytes Maroder et al., 1996, Besser and Wank, 1999 but also in stromal cells Laurenzi et al., 1994, Lomen-Hoerth and Shooter, 1995, especially ED1+ macrophages of the cortico-medullary junction (Garcı́a-Suárez et al., 1998). On the other hand, the mRNAs for BDNF and NT4/5 have been detected in the thymus Timmusk et al., 1993, Laurenzi et al., 1994, Maroder et al., 1996, thus suggesting an autocrine and/or paracrine loop of the BDNF/NT-4/5/TrkB system within this organ.

Data about the presence of TrkB in the mouse thymus are scarce. Therefore, the first goal of the present study was to analyze the expression (Northern blot and Western blot) and distribution (light immunohistochemistry) of TrkB in the murine thymus at different ages. In addition, in order to clarify its function in the thymus, we have studied in detail the structure and ultrastructure of this organ in mice lacking functional TrkB (Klein et al., 1993). Because preliminary studies demonstrated increased lymphocyte death in animals deficient in functional TrkB, the TUNEL technique to detect apoptosis was also carried out.

Section snippets

Animals and tissues

The thymus of C57B1/6 mice of two ages (15 days n=14 and 3 months n=11) was removed under deep chloral hydrate anesthesia (350 mg/kg, i.p.). In five animals per age group, the two lobes of the organ were separated: one was embedded in a cryoprotectant medium, snap-frozen in liquid nitrogen and stored at −80° until use; the other was fixed in Bouin's fixative for 12 h and routinely processed for paraffin embedding. The thymuses and the brains of three animals per age group were used to isolate

Expression of TrkB mRNA

The expression of trkB in the thymus and brain of mouse was examined by determination of steady state levels of mRNA. All experiments were performed in triplicate. Fig. 1A shows the autoradiographs of trkB and β-actin detected by Northern blot. Relative levels of trkB mRNA expression in both tissues at the two examined ages examined demonstrate a decrease in the expression of trkB mRNA of the thymus from young to adult animals, whereas the level of expression remain stable in the brain (Fig. 1B)

Discussion

During the last decade, evidence has been accumulated for a role of neurotrophins in regulating the immune system (see Otten and Gadient, 1995, Aloe et al., 1999, Ruberti et al., 2000). Thus, both neurotrophins and their signal transducing receptors were localized in both lymphoid tissues and immunocompetent cells Laurenzi et al., 1994, Lomen-Hoerth and Shooter, 1995, Torcia et al., 1996, Hannestad et al., 1997, Besser and Wank, 1999, Garcı́a-Suárez et al., 1998, Garcı́a-Suárez et al., 2000b,

Acknowledgements

The authors thank Ms. M. Pérez-Pérez for the critical reading of the manuscript, and Mr. D.F Monjil for the technical assistance This study was supported in part by a. This study was supported in part by a grant from Spanish DGICYT (CC-99-SAF-0119-C0202) Fo J.A.V.

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    These authors contributed equally to this study.

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