Expression of protein tyrosine kinases in the murine thymus stroma

ELSEVIER

Immunology

Expression

Letters

50 (1996) 65-69

of protein tyrosine kinases in the murine thymus stroma E.F. Potworowski*,

Institute Armand-Frappier, Received

6 October

Claire Beauchemin

P.O. Box 100. LavaI, QC, Canada, H7N 423

1995; revised 15 December 1995; accepted 14 February

1996

-. Abstract Thymocytes not only receive signals from thymic epithelial cells but can also activate the latter, at least in the medulla. We have previously reported tyrosine phosphorylation of medullary epithelial cell substrates, after co-culture with thymocytes, and identified a number of protein tyrosine kinases in a line of thymic epithelial cells. We report here the in situ localisation by

immunohistochemistry of JAK2 in medullary epithelial cells, of PDGF-R in medullary vascular endothelium. Hassall’s corpuscles, and the weak expression of JAKl and RYK throughout the thymus. Keywords:

Thymus;

Tyrosine

kinases;

PTK;

Epithelium;

There is a growing body of evidence that signal transduction, in the thymus, occurs not only in the direction from stromal cells to thymocytes, but the other way as well. This has been documented in SCID mice, where the scattered medullary epithelial cells responded to TCR + T lymphocytes by expressing the medullary epithelial-specific antigen ER-TRS, and by organizing into a histologically normal adult medulla [1,2]. In a similar experiment, injection of normal bone marrow to SCID mice resulted in the expression of B7 on medullary epithelial cells [3]. Direct demonstration of signal transduction in a lymph0 + stromal direction was provided using medullary epithelial cells (E-5 line) which responded to contact with thymocytes by tyrosine phosphorylation on a protein substrate with an Mw of N 90 kDa [4], and by a thymocyte-triggered, phosphotyrosine-dependent transient upregulation of a surface gp23 [5], a chain of the adhesion receptor molecule for thymocytes [6]. Using the conserved PTK catalytic domain sequence as a primer in PCR, we have identified in E-5 cells 11 known tyrosine kinases, namely JAKI, JAK2, PDGF-R type a, FGF-R, MET, tyro-3, c-abl, src, lyn,

0165-2478/96/$12.00

author.

0 1996 Elsevier

PII SO1 65-2478(96)02520-5

in

Medulla

1. Introduction

* Corresponding

of FGF-R

Science B.V. All rights reserved

bek and RYK. In addition, two new sequences ETECK and THY were identified; they are respectively homologs of Cek-5 and c-yes [7]. In order to further understand activation pathways in the process of thymocyte maturation control, it became pertinent to establish whether some of these tyrosine kinases were expressed in the thymus. In the present work, we localized by immunohistochemistry four of the above tyrosine kinases to distinct stromal elements of the thymus.

2. Materials

and methods

2.1. Antibodies

Rabbit polyclonal antibodies to JAKI, JAK2, FGFR type a, SRC and PDGF-R were obtained from Upstate BiQtechnology Inc., Lake Placid. New York. A rabbit polyclonal anti-serum was raised to the KFQQLVQCLTEFHAALGAYV peptide of RYK according to Hovens et al. [S]. Peroxidase-conjugated swine anti-rabbit Ig was obtained from DAK0 AS, Copenhagen, peroxidase-conjugated rabbit anti-rat IiG/M was from Zymed Laboratories, San Francisco, and an aminoethyl carbazole (AEC) substrate kit (Zymed) was used for revelation. Rat C3Cl2 monoclonal antibody, reacting with E-5 cells and with a

66

E.F. Potworowski,

C. Beauchenzin

/ Inmunologq~

Letters

50 (1996)

65-69

Fig. 1. Anti-JAK2 staining of thymic section. (A) Staining confined to the medulla and occasional cortical cells (40 x ) (B) Medullary epithelial cells showing staining along areas of contact with thymocytes (arrow) (630 x ).

Fig. 2. ER-TR5 staining of thymic section. (A) The antigen distribution is similar to that of JAK-2 (40 x ). (B) Cytoplasmic staining is uniform throughout (630 x ).

subset of medullary epithelial cells and with some elements of the cortex has been described elsewhere [6]. ER-TR5 rat antibody reacting with all medullary epithelial cells [9] was a gift of Prof. W. Van Ewijk.

2.2, Irnmunoperoxidase Frozen

thymus

staining

sections

(5 /lrn) were air-dried,

briefly

fixed with acetone at room temperature, and reacted in a humid chamber with primary antibodies for 30 min, rinsed in PBS-Tween (0.05% Tween 20, pH 7.8) and incubated for an additional 30 min with the appropriate peroxidase conjugate. The reaction was revealed by a 1.5min incubation with AEC. The preparations were mounted in buffered glycerol and sealed.

E.F. Potworowski.

Fig. 3. C3C12 staining (B) Cortical capillaries

C. Beauclwnlin

1 Irrmum~log~

of thymic sections. (A) Medullary ‘islands’ with a strongly in longitudinal and transverse section showing positively

Fig. 4. Anti-PDGF-R staining of thymic section. (A) Staining is found medulla (40 x ). (B) Enlargement of the area above the upper left corner Cortical cell (possibly dendritic) (630 x ).

3. Results The anti-JAKl serum gave a very weak but even reaction throughout the entire thymus; while the reaction was clearly cytoplasmic, there was no apparent distinction between the various cellular elements. Anti-JAK2 stained all medullary epithelial cells, as well as a few isolated cells in the cortex (Fig. 1). The medullary cells stained by anti-JAK2 were distributed

Letters

staining staining

67

50 (I 996) 65-69

central cell, surrounded by a more diliusc endothelial cells (630 x ).

mainly in the endothelium of some blood of the inset, showing negative and positive

area (40 x ).

vessels. particularly in the blood vessels (630 x ). (C)

throughout the entire medulla and corresponded to those that reacted with ER-TR5 antibody (Fig. 2). The anti-JAK2 staining however was less even than that of ER-TR5. and some areas of the medulla stained more intensely than others. The reaction with anti-JAK2 seemed to be strongest in areas where epithelial cells are in contact with lymphocytes (Fig. I b). JAK2 + cells included those stained by C3C12 (Fig. 3). The latter stains only some epithelial cells dis-

68

E.F. Potworowski. C. Beauchemin /Immunology Letters 50 (1996) 65-69

Fig. 5. Anti-FGF-R staining of thymic sections. (A) Staining confined to the medulla (40 x ). (B) Hassall’s corpuscles showing outer layer of stained epithelial cells and concentric layers of acellular material. The center is negative (630 x ).

tributed in characteristic medullary islands with a highly stained central core and diffuse staining in the periphery. C3C12 + cell islands tended to be localised in the outer medulla. Cortical cells stained by antiJAK2 were few, evenly distributed and did not form a reticulum, whereas cortical cells stained by C3C12 were elongated and appeared to be associated with capillaries (Fig. 3b). Anti-PDGF-R type A antibody stained strongly vascular endothelium of some but not all small blood vessels, mostly in the medulla (Fig. 4A,B); it stained some isolated cells with cytoplasmic prolongations (possibly dendritic cells) mostly in the medulla but also to a smaller extent in the cortex (Fig. 4C). The reaction to anti-FGF-R anti-serum was confined to the medulla (Fig. 5A), where it stained the endothelium of some blood vessels, Hassall’s corpuscles and associated epithelial cells (Fig. 5B). The reaction of anti-RYK serum with medullary and cortical epithelial cells was extremely weak compared to that obtained in kidney, where the reaction was strongest in glomeruli and in the major and minor calyces (not shown). The anti-SRC serum failed to react with thymic tissue, even when used undiluted.

4. Discussion Of the PTKs detected in the thymus, JAK2 was of particular relevance. It is a widely distributed receptor tyrosine kinase of the Janus family. It is associated

with, and can be activated by the engagement of receptors for prolactin [lo], IL-3 [ll], growth hormone and IFN-), [ 12,131. It is autophosphorylated and, in addition, induces the phosphorylation of a p91 (or stat91), a transcriptional activator. IFN-y is known to up-regulate the expression of a number of thymic epithelial membrane proteins, including CDRl [14] in the cortex and gp23 (unpublished), class I and class II MHC, and TSA-1 in the E-5 medullary epithelial cells [15]. It is of interest that single positive medullary CD4 + HSA’“Qa2+, thymocytes believed to be the most mature CD4+ subset in the thymus, secrete IFN-), [16]. It is quite likely that CD4 + HSA’“Qa2+ thymocytes could have a paracrine activation effect on medullary epithelial cells, via IFN-1;. RYK has not been as thoroughly documented in terms of activation. It is a receptor tyrosine kinase which is expressed with different intensity in several organs; it was shown by immunoprecipitation to react strongly with the kidney and marginally with thymus [8] which corroborates our results. The reactivity of some blood vessels to anti-PDGF-R a has been documented previously. It was found in some capillaries, and in larger vessels, and has been associated with endothelial cells [ 171 and arterial smooth muscle [18]. It is particularly evident during angiogenesis [ 191 FGF-R localisation was studied most thoroughly in the rat. It was found in a variety of tissues during embryogenesis, particularly in mesoderm- and neuroectoderm-derived tissues [20]. In the adult, it was found primarily in cells of the central nervous system

E.F. Potwjorowski. C. Beauckemin /Immunology Letters 50 (1996) 65-69

[21]. While, to the best of our knowledge, its presence in the thymus has not been previously reported, it is not entirely surprising to find yet another molecule shared between the brain and the thymus. The question arises as to why E-5 cells expressed sequences for all the PTKs described in the present work [7], whereas the in situ expression of these proteins was selectively distributed among a variety of stromal elements. One possibility is the different sensitivities of the detection techniques used, PCR being by far more sensitive than immunohistochemistry. It is also conceivable that E-5, being a transformed cell line, has reverted to a less differentiated stage, and expresses a wider range of tyrosine kinases. If such were the case, antibodies to PTKs could become a useful tool to monitor the differentiation and the activation state of thymic epithelial cells.

Acknowledgements

This work was partIy funded by a grant of the Medical Research Council of Canada.

References [l] Shores, E.W., van Ewijk, W. and Singer, Immunol. 21, 1657. [2] Surh. C.D., Ernst, B. and Sprent. 611.

A. (1991).

Eur.

J. (1992) J. Exp. Med.

J. 176,

69

Degermann, S., Surh. C.D., Glimcher. L.11.. Sprenl, 1. and Lo, D. (1994) J. Immunol. 152. 3254. C.. Amarante-Mendes, G. and Potworowski. E.F. 141 Couture, (1992) Eur. J. Immunol. 22. 2579. E.F., Chamberland. C‘.. Beauchemin. C. and VI Potworowski, Marceau, N. (1994) Cell. Immunol. 153. 256. [61Couture, C.. Patel. P.C. and Potworowski. t1.1:. (1990) Eur. J. Immunol. 20. 2769. E.F. [71 HCbert. B., Bergeron, J.. Tijssen, I’. and Potuorowski. (1994) Gene 143. 257. AC.. Ilarpur, A.G., PI Havens. C.M.. Stacker. S.A., Andre\. Ziemiecki, A. and Wilks, A.F. (1992) Proc. Uatl. Acad. Sci. USA 89, 11818. [91 van Vliet, E.. Melis, M. and van Flwijk. W. t 1Y84) Eur. J. Immunol. 14. 524. 1101 Rui. H.. Kirken, R.A. and Farrar. W.L. ( lYY4) J. Biol. Chem. 269, 5364. [Ill Silvennoinen. 0.. Witthuhn, B.A., Quelle. F.W.. Cleveland. J.L., Yi. T. and Ihle, J.N. (1993) Proc. Natl. Acad. Sci. USA 90, 8429. v21 Silvennoinen. 0.. Ihle. JN., Schlessinger. J. and Levy, DE. (1993) Nature 366. 583. [I31 Shual. K.. Ziemiecki, A., Wilks, A.1:.. Harpur. A.G.. Sadowski, H.B., Gilman, M.Z. and Darnell. J.E. ( IYY3) hature 366, 580. u41 Small, M.. van Ewijk, W.. Gown. A.M. and Rouse. R.V. (1989) Immunology 68, 371. D.I. and Marrack, P.C. [I51 Hugo, P.. Kappler, J.W.. Godfre). (1994) J. Immunol. 152, 1023. [I61 Bendelac, A. and Schwartz, R.H. (lY91) Nature 353, 68. P. and Frackelton. A.R. Jr. [I71 Beitz. J.G.. Kim, I.S., Calabresi. (1992) Experientia (Suppl.) 61. 85. M.. Rahm, M.. Claesson-W&h. L.. Sejersen, T., U81 Sjolund. Heldin. C.H. and Thyberg, J. (1990) Growth Factors 3. 191. S. and Ohlsson. R. [I91 Holmgren. L.. Glaser, A., Pfeiffer-Ohlsson. (1991) Development 113. 749. PO1 Wanaka, A., Milbrandt. J. and Johnson. E.M. Jr. (1991) Development 1 Il. 955. Pll Wanaka. A.. Johnson, E.M. Jr. and Milbrandt. J. (1990) Neuron 5. 267. 131