HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/11/1538 02-0869fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by SUZUKI, H. Articles by NAKASHIMA, I. Search for Related Content PubMed PubMed Citation Articles by SUZUKI, H. Articles by NAKASHIMA, I.

Involvement of MKK6 in TCRß^intCD69^lo: a target population for apoptotic cell death in thymocytes¹

HARUHIKO SUZUKI², JIANGHONG WU, KHALED HOSSAIN, TATSUYA OHHATA, JUN DU^*, ANWARUL A. AKHAND, AKEMI HAYAKAWA, HIROSHI KIMURA, MASATOSHI HAGIWARA and IZUMI NAKASHIMA

Department of Immunology and Department of Equipment Center for Research and Education, Nagoya University Graduate School of Medicine, Aichi, Japan;
^* Department of Medical Technology, Nagoya University School of Health Sciences, Aichi, Japan; and
Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

²Correspondence: Department of Immunology, Nagoya University Graduate School of Medicine, Aichi 466-8550, Japan. E-mail: k46200a{at}nucc.cc.nagoya-u.ac.jp

SPECIFC AIMS

The aim of this study is to elucidate the role of MKK6-p38 pathway in physiological development and selection process of thymocytes.

PRINCIPAL FINDINGS

1. TCRß^int/hiCD69^lo population is a real target for apoptotic cell death in thymocytes
To analyze events of cellular apoptosis in specific subpopulations of thymocytes in real time, we used a system for detecting caspase activity in live cells and searched for specific populations that have high frequencies of apoptosis. In the staining of TCRß and CD69, most thymocytes were found to be TCRß^loCD69^-/lo and some cells expressed intermediate-high levels of TCRß (Fig. 1 A). The expression level of CD69 in TCRß^int/hi cells was either higher than or equivalent to that in TCRß^lo cells, indicating that both the TCRß^int and TCRß^hi cells could be divided into at least two subpopulations of CD69^hi (G1, G2) and CD69^lo (G3, G4) (Fig. 1A ). We used a PhiPhiLux^TM system to detect real-time activation of caspase in live cells. PhiPhiLux incorporated into a cell is specifically cleaved by caspase-3 or caspase-7, and the cleaved products give fluorescence that can be detected by flow cytometric analysis. The use of this system combined with staining for cell surface antigens enabled detection of caspase-activity in particular populations of thymocytes. When we examined caspase activity in each population (G1-G4), determined by TCRß and CD69 expression levels, we found that TCRß^intCD69^lo and TCRß^hiCD69^lo cells included large percentages of caspase-active cells, but no other populations showed high percentages of caspase-active cells (Fig. 1B ). We also examined DNA fragmentation in cells collected by cell sorting and found that the TCRß^int-hiCD69^lo population (G3+G4) included a significantly higher percentage of cells with fragmented DNA than did other populations such as the TCRß^int-hiCD69^hi (G1+G2) or TCRß^loCD69^-/lo (Fig. 1C ). These results indicate that TCRß^int/hiCD69^lo cells are target populations for apoptotic cell death in thymocytes.

View larger version (50K): [in this window] [in a new window]   Figure 1. High percentage of apoptotic thymocytes in the TCRßint/hiCD69lo population. A) Thymocytes from C57BL/6 mice were analyzed by flow cytometry. Thymocytes from C57BL/6 mice were stained with PE-conjugated anti-TCRß antibody and biotin-conjugated anti-CD69 antibody, visualized by streptavidin-APC. The profile of TCRß and CD69 expression is shown. The areas of four gates (G1: TCRßintCD69hi, G2: TCRßhiCD69hi, G3: TCRßintCD69lo, G4: TCRßhiCD69lo) and percentages of cells in each area are also shown. B) Cells were treated using a PhiPhiLuxTM apoptosis detection kit and stained with PE-conjugated anti-TCRß antibody and biotin-conjugated anti-CD69 antibody, visualized by streptavidin-APC. Cells were analyzed for PhiPhiLux fluorescence (FL1) and their profiles are shown for each subpopulation in the region gates (G1-G4) shown in panel A). Percentages of cells with high caspase activity are also shown. C) Thymocytes were stained with FITC-conjugated anti-CD69 and PE-conjugated anti-TCRß antibodies; TCRßloCD69-/lo cells, TCRßint/hiCD69hi cells (G1+G2), and TCRßint/hiCD69lo cells (G3+G4) were collected by cell sorting. DNA fragmentation in each sorted sample was analyzed by PI staining and flow cytometry. Percentages of cells with fragmented DNA are shown.

2. Reduced number of apoptotic cells in the TCRß^intCD69^lo population of MKK6-deficient thymocytes
To analyze a role of the MKK6-p38 pathway in thymocyte development, we generated MKK6-deficient mice. When we examined caspase activity in TCRß^int/hiCD69^lo cells from MKK6^+/+ or MKK6^-/- mice, we found that TCRß^intCD69^lo cells from MKK6^-/- mice included a significantly lower percentage of caspase-active cells than did those from MKK6^+/+ mice, whereas no difference was found between caspase activities in TCRß^hiCD69^lo cells from MKK6^+/+ mice and those from MKK6^-/- mice (Fig. 2 A, B). We collected TCRß^intCD69^lo (G3) and TCRß^hiCD69^lo (G4) cells from MKK6^+/+ or MKK6^-/- mice by cell sorting and analyzed DNA fragmentation in each population. Similarly, TCRß^intCD69^lo (G3) cells from MKK6^-/- mice included lower percentage of cells with DNA fragmentation than did TCRß^intCD69^lo cells in MKK6^+/+ mice (Fig. 2C, D ).

View larger version (57K): [in this window] [in a new window]   Figure 2. Reduced number of apoptotic cells in the TCRßintCD69lo population of MKK6-deficient mice. A) Thymocytes from MKK6+/+ or MKK6-/- mice of the same littermate were treated with a PhiPhiLuxTM apoptosis detection kit, then stained with PE-conjugated anti-TCRß antibody and biotin-conjugated anti-CD69 antibody, visualized by streptavidin-APC. PhiPhiLux fluorescence profiles are shown for each subpopulation shown in Fig. 1A . Percentages of cells with high caspase-activity are shown. B) Percentages of PhiPhiLux-positive cells in indicated subpopulations are shown as averages and standard deviations obtained from the analysis of three mice in each group. The mean absolute numbers of thymocytes were 12.5 x 107 in MKK6+/+ mice and 11.8 x 107 in MKK6-/- mice. *Significantly different by statistical analysis (P=0.006, Student’s t test). C) Thymocytes from MKK6+/+ or MKK6-/- mice were stained with FITC-conjugated anti-CD69 and PE-conjugated anti-TCRß antibodies, and TCRßintCD69int cells (G3) and TCRßhiCD69lo cells (G4) were collected by cell sorting. DNA fragmentation in each sorted sample was analyzed by PI staining and flow cytometry. Percentages of cells with fragmented DNA are shown in each panel. D) Percentages of cells with fragmented DNA in indicated subpopulations are shown as averages and standard deviations obtained from 3 independent analyses in each group. #Significantly different by statistical analysis (P=0.024, Student’s t test).

CONCLUSIONS AND SIGNIFICANCE

By using a method to detect activation of caspase, we extensively analyzed ongoing apoptosis in thymocyte subpopulations of different differentiation steps. The caspase-active cells were predominantly abundant in TCRß^int/hiCD69^lo thymocytes. The results of our study indicate that caspase activity is increased during physiological apoptosis in thymocytes, and increased activity of caspase is a marker for ongoing apoptosis in thymocytes. We examined DNA fragmentation in cells of each population collected by cell sorting and found that the TCRß^int/hiCD69^lo population contained a higher percentage of DNA-fragmented cells than did other populations, indicating that apoptotic cell death occurs at this developmental stage. We demonstrated for the first time that the target subpopulation for ongoing apoptotic cell death is in the CD69^lo population, not in the CD69^hi population. Our results suggested that the TCRß^int/hiCD69^lo subpopulation might be essentially distinguished from TCRß^int/hiCD69^hi cells by the percentage of cells undergoing apoptotic cell death.

We generated MKK6-deficient mice in order to clarify the roles of the p38 pathway in thymocyte development and, for the first time, proved the involvement of the MKK6-p38 pathway in particular developmental stages of thymocytes in a physiological condition. In the analysis of caspase activity, we found a statistically significant difference between MKK6-deficient mice and age-matched control mice in TCRß^intCD69^lo cells but not in TCRß^hiCD69^lo cells. This suggests that thymocytes undergoing apoptotic cell death may be classified into two subpopulations according to dependency on MKK6 in the process of apoptosis: one of mostly TCRß^intCD69^lo cells in a relatively immature MKK6-dependent stage and the other of mostly TCRß^hiCD69^lo cells in a relatively mature MKK6-independent stage.

In this study, we identified a real target population for apoptotic cell death in thymocytes as TCRß^int/hiCD69^lo and elucidated the role of MKK6 in a restricted subpopulation among them. MKK6 is crucially and selectively involved in thymocyte development at the stage of TCRß^intCD69^lo in which physiological negative selection possibly takes place.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of the proposed course of thymocyte development and selection. Immature thymocytes of TCRßloCD69lo phenotype develop to mature TCRßhi thymocytes via TCRßint stage. TCRßint/hi thymocytes express either a low or high level of CD69 and only CD69lo cells become a target population for apoptosis. This apoptosis possibly includes negative selection of thymocytes and is MKK6 dependent in TCRßintCD69lo cells, but MKK6 independent in TCRßhiCD69lo cells. Pathways indicated with dashed arrows have not been proven.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0869fje; doi: 10.1096/fj.02-0869fje

This Article Full Text (PDF) All Versions of this Article: 17/11/1538 02-0869fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by SUZUKI, H. Articles by NAKASHIMA, I. Search for Related Content PubMed PubMed Citation Articles by SUZUKI, H. Articles by NAKASHIMA, I.