A dual altered peptide ligand down-regulates myasthenogenic T cell responses and reverses experimental autoimmune myasthenia gravis via up-regulation of Fas-FasL-mediated apoptosis

Abstract

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent, antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogues of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to down-regulate in vitro and in vivo MG-associated autoreactive responses. The aims of this study were to investigate the possible role of Fas-FasL-mediated apoptosis in the down-regulatory mechanism of the dual APL. We demonstrate here the effect of the dual APL on expression of key molecules involved in the Fas-FasL pathway, in a p195-212-specific T cell line, in mice immunized with Torpedo acetylcholine receptor and in mice afflicted with EAMG (induced with the latter). In vitro and in vivo results show that the dual APL up-regulated expression of Fas and FasL on the CD4 cells. Expression of the pro-apoptotic molecules, caspase 8 and caspase 3, was significantly up-regulated, while anti-apoptotic cFLIP and Bcl-2 were down-regulated upon treatment with the dual APL. The dual APL also increased phosphorylation of the mitogen-activated protein kinases, c-Jun-NH2-terminal kinase and p-38, known to play a role in the regulation of FasL expression. Further, in the T cell line incubated with the dual APL as well as in mice of the SJL inbred strain immunized with the myasthenogenic peptide and treated concomitantly with the dual APL, the percentage of apoptotic cells increased. Results strongly indicate that up-regulation of apoptosis via the Fas-FasL pathway is one of the mechanisms by which the dual APL reverses EAMG manifestations in C57BL/6 mice.

Figure 1

The dual altered peptide ligand (APL) up-regulates expression of Fas and FasL on p195–212-specific T cells. Cells (10⁶ per ml) of the line were stimulated with p195–212 (5 µg per ml) with or without the dual or reversed dual APL (500 µg per ml) for 24 hr. Cells were double-stained for CD45Rb and Fas or FasL and analysed by fluorescence activated cell sorter (FACS). The results presented are after reduction of the background staining obtained with the matched isotype controls. Numbers in the upper levels correspond to CD45Rb^high cell population and those below correspond to CD45Rb^1ow cells. Shown is one of two experiments performed.

Figure 2

Effect of the dual altered peptide ligand (APL) on the expression of apoptotic molecules by a p195–212-specific T cell line. T cells (5 × 10⁶) of the line were stimulated with p195–212 with or without the dual or the reversed dual APL for 24 hr. Total RNA was extracted, and real time polymerase chain reaction (PCR) was performed for caspase 8, caspase 3, cFLIP and Bcl-2 as described in Materials and methods. Results are expressed as the mean percentage of the gene expression of triplicate samples ± SD values. *P < 0·05, **P < 0·0005, when gene expression in the presence of the dual APL was compared with that in the absence of the dual APL. Shown is one of two experiments performed.

Figure 3

The dual altered peptide ligand (APL) up-regulates phosphorylation of pJNK and p-38 in p195–212-specific T cells. T cells (5 × 10⁶) of the line were stimulated with p195–212 with or without the dual or the reversed dual APL for 24 hr. Whole-cell lysates were prepared, separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane that was later blotted with anti-p-JNK or anti-p-38 or their respective totals. Results are expressed as densitometry units (measured with National Institutes of Health image software). Shown is one of two experiments performed.

Figure 4

The dual altered peptide ligand (APL) up-regulates annexin V positive cells in a p195–212-specific cell line. Cells (10⁶ per ml) of the line were stimulated with p195–212 (5 µg per ml) with or without the dual or reversed dual APL (500 µg per ml) for 24 hr. Cells were double-stained for annexin V-fluorescein isothiocyanate (FITC) and PI-PE. The results are presented after reduction of background obtained with unstained cells. Percentage of annexin V positive and PI negative cells are depicted in the lower right quadrant. Shown is one of two experiments performed.

Figure 5

The dual altered peptide ligand (APL) up-regulates annexin V positive cells in lymph node (LN) derived CD4⁺ cells of SJL inbred mice immunized with p195–212. SJL mice were either immunized with p195–212 [10 µg per mouse in complete Freund's adjuvant (CFA)] and concomitantly administered subcutaneously (s.c.) with the dual APL [200 µg per mouse in phosphate-buffered saline (PBS)] or immunized with p195–212 or PBS alone. Ten days after immunization, spleen cells (a) and popliteal LN cells (b) were harvested. Cells were stained for CD4, annexin V and PI and analysed by fluorescence activated cell sorter (FACS).(a, bi) Staining for PI and annexin-V. Percentages in the upper left and right quadrants represent PI-stained necrotic cells while that in the lower right quadrant indicates annexin V positive apoptotic cells. (a, bii–iv) Percentage of CD4⁺ cells out of the gated annexin-V positive and PI negative cells. Shown is one of three experiments performed. (a, bv) Results are also expressed as the mean percentage of CD4 cells positive for annexin in the three experiments performed ± SD values. Percentage of apoptotic CD4⁺ cells of PBS immunized mice was considered as 100%. *P < 0·05 when compared with the group immunized with the peptide alone.

Figure 6

The dual altered peptide ligand (APL) up-regulates Fas and FasL expression in mice immunized with Torpedo acetylcholine receptor (TAChR). C57BL/6 mice were either immunized with TAChR [10 µg per mouse in complete Freund's adjuvant (CFA)] and concomitantly administered subcutaneously (s.c.) with the dual APL/reversed dual APL [200 µg per mouse in phosphate-buffered saline (PBS)] or immunized with TAChR alone. Ten days after immunization, popliteal lymph node (LN) cells were harvested. LN cells were stained for CD4, CD25, and Fas or FasL and analysed by fluorescence activated cell sorter (FACS). The results presented are after reduction of the background staining obtained with the matched isotype controls. Shown is one of three experiments performed.

Figure 7

Effect of the dual altered peptide ligand (APL) on the expression of apoptotic markers in lymph node (LN) cells of C57BL/6 mice immunized with Torpedo acetylcholine receptor (TAChR). C57BL/6 mice were either immunized with TAChR [10 µg per mouse in complete Freund's adjuvant (CFA)] and concomitantly administered subcutaneously (s.c.) with the dual APL/reversed dual APL [200 µg per mouse in phosphate-buffered saline (PBS)] or immunized with TAChR alone. Ten days after immunization, popliteal LN cells were harvested. (a) Total RNA was extracted from LN cells and real time polymerase chain reaction (PCR) was performed for caspase 8, caspase 3, cFLIP and Bcl-2 as described in Materials and methods. Results are expressed as the mean percentage of the gene expression of triplicates ± SD values. *P < 0·05, **P < 0·001 when gene expression in the presence of the dual APL was compared with that in the absence of the dual APL. (b) Lysates were prepared from LN cells, separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane that was later blotted with anti-p-c-Jun-NH2-terminal kinase (JNK) or anti-p-38 or their respective totals. Results are expressed as densitometry units (measured with National Institutes of Health image software). Shown is one of two experiments performed.

Figure 8

Effect of the dual altered peptide ligand (APL) on the expression of Fas, FasL by lymph node (LN) cells of C57BL/6 mice with experimental autoimmune myasthenia gravis (EAMG). (a) EAMG was induced in C57BL/6 mice by immunization and boost with Torpedo acetylcholine receptor (TAChR). Mice were treated for 4 weeks with either phosphate-buffered saline (PBS) (vehicle) or the dual APL (see Materials and methods). Mice were graded before and after treatment as follows: grade 3, severe muscle weakness, paralysis, dehydration, moribund; grade 2, weakness at rest; grade 1, moderate muscular weakness after exercise; and grade 0, no definite muscular weakness after exercise. (b) At the end of treatment, popliteal LN cells were harvested, stained for CD4, CD25, and Fas or FasL and analysed by fluorescence activated cell sorter (FACS). The results are from pooled samples (LN cells) of mice with EAMG that were treated with the dual APL or of diseased mice that were administered with PBS (as shown in a). Results presented are after reduction of the background staining obtained with the matched isotype controls. Shown is one of two experiments performed.

Figure 9

Effect of the dual altered peptide ligand (APL) on the expression of apoptotic markers by lymph node (LN) cells of C57BL/6 mice with experimental autoimmune myasthenia gravis (EAMG). EAMG was induced in C57BL/6 mice by immunization with Torpedo acetylcholine receptor (TAChR). After 4 weeks of treatment with either phosphate-buffered saline (PBS) (vehicle) or the dual APL, popliteal LN cells were harvested of four mice of each of the treatment groups. Total RNA was extracted from lymph node cells and real time polymerase chain reaction (PCR) was performed for caspase 8 and cFLIP expression as described in Materials and methods. Results are expressed as mean percentage gene expression of triplicates ± SD values. *P < 0·001, **P < 0·005 when gene expression of the dual APL-treated mice was compared with that of PBS-administered mice.