B 220 is Expressed on ApoptotEc Thymocytes Indsuced lvy X-Krradiation

CD45 is cell surface g}ycoprotein and expressed on all haematopoietic cells except mature erythrocytes and platelcts, Eight iseforms of CD45 are generated by alternative splicing ef exons 4-6. B220 includiing all three exons is expressed specifically on pan-B cell Iineage. Recently, it vvas reported that B220 wtts expressed on apoptotic T cells induced by staphylococcal enteretoxin B (SEB). In the present study, we inyestigated the exprcssion of B220 on murine thymocytes aftcr whele-body X-irradiation. We used the forward Iight scattering of flow cytometry as a parameter of ceil size, and defined two pepulations; FSChigh (normal cell size) and FSCtow (correspond to apoptotic cell in size) fraction. B220' cells in FSChigh fraction reached a maximum value (3S%) at 18 hr after irradiation. In FSCTOW fraction, 40-60% cells wvere positive for B220 at any tirne points. These results suggest that B220 is expressed on thymocytes in the pre-apoptotic stage, because B220 was expressed on not only FSCiO" cells but also FSChigh cells.-KEy woRDs: apoptosis, B220, mouse, thymocyte,X-irradiation. '-'""'' '''-"--J, Vet, Med. Sci. 61(4): 337-341,1999 CD45 is a rnajor protein tyrosine phosphatase (PTPase) expressing on all hematopoietic cells except mature erythrocytes and platelets [16]. It is reported that this cell surface antigen has a critical rele in signal transduction concerning actiyation or apoptosis of lymphocytes via T cell receptor (TCR) or B cell recepter (BCR; surface IgM) [11, 17], CD45 occurs in multiple isoforms with variable involvement of exons 46 due to alternative splicing [16], Molecular weights of these isoforms are distributed between 180 and 22e kDa [131. The 220 kDa isoform contains these all three exons and have been known to be preferentially expressed on B cells. Thus, this B cell-specific isoform is called "B220". It is known that CD45RA, including exon 4 of the three exons, is expresscd on naivelresting T cells and medullary thymocytes [7, 191. 0n the other hand, CD45RO (the iow molecular weight isoform), lacking exons 4-6, is expressed on memorylactivated T cells and cortical thymocytes [6, 8]. Generally, it is regarded that B220 was not appeared on thymocytes or mature T cells. There are some reports, however, concerning the expression of B220 on the apoptotic T cells induced by anti-CD3 monoclonal antibodies (mAb), concanavalin A (Con A), staphylococcal enterotoxin B (SEB) {15, 181 or Interleukin-2 (IL-2) [9], On the other hand, there is no report about B220 expression on the apoptotic T cells induced by radiation, a potent inducer of apoptosis. In this study, we analyzed the cell size, DNA contents and cell surface antigens including B220 of thymocytes obtained froiT} DBAI mice at various time after the 6.8 Gy whole-body irradiation. MATERIALS AND METHODS Animals: DBAI mice wcre originally provided by Dr. J, Hayakawa (Kanazawa University, Kanazawa, Japan) and have been bred at our laboratory since 1995. Mice were kept in a room at a temperature 22 ± 1eC and in a relative humidity of 50 ± 5%, and the room was lighted fer 12 hrl day, Mice were provided commercial NMF diet (Oriental Yeast Co,, Ltd., Tokyo, Japan) and acidified water (pH 3) ad libitum. In the present study, each point in the figure is the mean ± standard deviation of three to seven mice. irradiation: Fernale 6-week-old mice were exposed to 6.8 Gy of whole-body X-irradiation at a dose rate of O.46Gyt min with X-ray irradiator (Radiofiex-350; Rigaku Denki Co,, Ltd., Tokyo, Japan) operated at 25e kV and 12,5 rriA with of e.3 rnm Cu and O.5 mm AI filtration. Mice were subsequently killed at O (control), 3, 6, 9, 12, 18, 24, 36, 48, 72 and 168 hr after the irradiation, and then thymi were excised and weighted. Thymocyte suspensions were preparcd by gently teasing the thymus with forceps in Hank's balanced salt solution containing 1% fetal bovine serum (HBSS-FBS), The cell suspensions were washed once with HBSS-FBS and the number of cells was counted with a Neubauer haemocytometer, The viability of cells was determined by Trypan blue exclusion. Antibodies and flow cytemetry: Two-color flowcytometric analysis was performed with a Cyto ACE-150 (Japan Spectrosc6pic Co,, Ltd., Tokyo, Japan). The data were analyzed with the Cyto ACE system program version 3,04 (Japan Spectroscopic Co,, Ltd,), The thymocytes were stained with PE-corijugated anti-Thy-1 (5a-8, Cedarnane) ancl FITC-coajugated anti-B220 (RA3-6B2, Caltag Laboratories) monoclonal antibodies (mAb), The cells (1 × 106) were incubated for 3e min on ice in 1OO Ld HBSS-FBS containing the appropriate dilution of antibedies, and washed once with the same buffer. DiVA labeling with PI: Apoptotic thymocyZes were counted by fiow cytometry after propidium iodide (PI) staining. DNA content of thymocytes was anaayzed as described by Nicoletii et al. IIO]. Briefly, thymocytes (1 × 1e6) were suspended in O.5 ml of PBS (-) and then O,5 mt Japanese Society of Veterinary Science NII-Electronic Library Service apaneseSociety f eterinary cience 338 S. OKA, K. KUBO, S. MATSUYAMA AND Y. TAKAMORI of O.1% Pelyoxyethylene-10-octylphenyl ether (Triton X100 ; Wako Pure Chemical Industries, Ltd,, Osaka, Japan) and 1 ml of 1 mglml RNase A (Sigma, St. Louse, Mo, U,S.A.) were added to the bell suspension. After incubation for 20 min at 37eC, the samples were mixed with 2 ml of PBS containing PI (S pgXmt, Sigma) and kept at room temperature for 15 min in the dark before the flow cytometric analysis. Detection of DIVA f}'agmentation on agarose gels: Analysis of DNA fragmentation was performed as described by Pilarski et al. [12] with a slight medificatien, Thymocytes (1 × 106) were lysed in 20 pt of 1O mM EDTA, 50 mM Tris-HCI (pH 8.0) containing O.5% (wlv) sodiumN-lauroylsarcosinate. The samples were digestecl by addition of 1 ptl RNase solution (10 rnglmt) at 500C for 30 min. One pl of proteinase K solution (10 mg/ml) was then added and the solution was incubated at 500C for 1 hr. The samples were electrophoreised in 2% agarose gel at 12,5 V/ cm for 30 rnin. Statistical nalysis: Statistical significance of the experimental data was examined by student's t-test.