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Deficiency of the cyclin kinase inhibitor p21(WAF-1/CIP-1) promotes apoptosis of activated/memory T cells and inhibits spontaneous systemic autoimmunity.

Lawson BR, Baccala R, Song J, Croft M, Kono DH, Theofilopoulos AN - J. Exp. Med. (2004)

Bottom Line: Absence of p21 resulted in enhanced Fas/FasL-mediated activation-induced T cell death, increased activation of procaspases 8 and 3, and loss of mitochondrial transmembrane potential.Increased apoptosis was also associated with p53 up-regulation and a modest shift in the ratio of Bax/Bcl-2 toward the proapoptotic Bax.Thus, modulation of the cell cycle pathway may be a novel approach to reduce apoptosis-resistant pathogenic lymphocytes and to ameliorate systemic autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
A characteristic feature of systemic lupus erythematosus is the accumulation of activated/memory T and B cells. These G0/G1-arrested cells express high levels of cyclin-dependent kinase inhibitors such as p21, are resistant to proliferation and apoptosis, and produce large amounts of proinflammatory cytokines. Herein, we show that ablation of p21 in lupus-prone mice allows these cells to reenter the cell cycle and undergo apoptosis, leading to autoimmune disease reduction. Absence of p21 resulted in enhanced Fas/FasL-mediated activation-induced T cell death, increased activation of procaspases 8 and 3, and loss of mitochondrial transmembrane potential. Increased apoptosis was also associated with p53 up-regulation and a modest shift in the ratio of Bax/Bcl-2 toward the proapoptotic Bax. Proliferation and apoptosis of B cells were also increased in p21-/- lupus mice. Thus, modulation of the cell cycle pathway may be a novel approach to reduce apoptosis-resistant pathogenic lymphocytes and to ameliorate systemic autoimmunity.

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Enhanced proliferation and apoptosis of p21−/− T cells from male BXSB mice. (a) Increased proliferation of p21−/− T cells after in vitro cross-linking of antigen receptors. LN cells (n = 3 mice/group) were stimulated with increasing concentrations of plate-bound anti-CD3 (0.1–20 μg/ml) plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at 72 h (mean ± SEM cpm). (shaded bars) BXSB p21−/− mice. (unshaded bars) p21+/+ BXSB mice. (b) Kinetics of in vitro proliferation. LN cells (n = 3 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at the indicated time points. Results are representative of two independent experiments (n = 3 mice/group). (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (c) Increased AICD of p21−/− T cells. T cell were stimulated with 0.5 μg/ml of soluble anti-CD3 for 48 h followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 (n = 3 mice/group). Percent of apoptotic (Annexin V+PI−) CD4+ T cells was determined by FACS®. Similar results were obtained with CD8+ T cells. (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (d) Increased proliferation of wild-type BXSB T cells treated with p21 AS oligonucleotides. LN cells (n = 4 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies in the presence of either p21 AS or control oligonucleotides (all AS oligonucleotides at 400 nM) and assessed for [3H]thymidine incorporation (mean ± SEM cpm). (e) Increased AICD of wild-type BXSB T cells treated with p21 AS oligonucleotides. Wild-type BXSB T cells were stimulated with 0.5 μg/ml of soluble anti-CD3 followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 in the constant presence of p21 AS or control oligonucleotides (400 nM; n = 3 mice/group). (▪) AS oligo no. 1. (□) AS oligo no. 2. (▴) Control oligonucleotide. (e, inset) Western blot analysis of wild-type BXSB T cells treated with either p21 AS or control oligonucleotides. T cells were stimulated with 10 μg/ml anti-CD3 and 5 μg/ml CD28 antibodies in the presence of AS or control oligonucleotides (400 nm). Whole cell lysates were analyzed by Western blot using anti-p21 and antiactin antibodies. *, P < 0.05 by Student's t test.
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fig3: Enhanced proliferation and apoptosis of p21−/− T cells from male BXSB mice. (a) Increased proliferation of p21−/− T cells after in vitro cross-linking of antigen receptors. LN cells (n = 3 mice/group) were stimulated with increasing concentrations of plate-bound anti-CD3 (0.1–20 μg/ml) plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at 72 h (mean ± SEM cpm). (shaded bars) BXSB p21−/− mice. (unshaded bars) p21+/+ BXSB mice. (b) Kinetics of in vitro proliferation. LN cells (n = 3 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at the indicated time points. Results are representative of two independent experiments (n = 3 mice/group). (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (c) Increased AICD of p21−/− T cells. T cell were stimulated with 0.5 μg/ml of soluble anti-CD3 for 48 h followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 (n = 3 mice/group). Percent of apoptotic (Annexin V+PI−) CD4+ T cells was determined by FACS®. Similar results were obtained with CD8+ T cells. (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (d) Increased proliferation of wild-type BXSB T cells treated with p21 AS oligonucleotides. LN cells (n = 4 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies in the presence of either p21 AS or control oligonucleotides (all AS oligonucleotides at 400 nM) and assessed for [3H]thymidine incorporation (mean ± SEM cpm). (e) Increased AICD of wild-type BXSB T cells treated with p21 AS oligonucleotides. Wild-type BXSB T cells were stimulated with 0.5 μg/ml of soluble anti-CD3 followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 in the constant presence of p21 AS or control oligonucleotides (400 nM; n = 3 mice/group). (▪) AS oligo no. 1. (□) AS oligo no. 2. (▴) Control oligonucleotide. (e, inset) Western blot analysis of wild-type BXSB T cells treated with either p21 AS or control oligonucleotides. T cells were stimulated with 10 μg/ml anti-CD3 and 5 μg/ml CD28 antibodies in the presence of AS or control oligonucleotides (400 nm). Whole cell lysates were analyzed by Western blot using anti-p21 and antiactin antibodies. *, P < 0.05 by Student's t test.

Mentions: Previous in vitro analyses with p21−/− T cells from nonautoimmune mice showed increased proliferation compared with wild-type T cells (36, 37). Similarly, BXSB p21−/− T cells showed enhanced proliferation compared with wild-type T cells (P < 0.05) after engagement of CD3 and CD28, with no apparent alteration of the TCR-induced activation threshold (Fig. 3 a). Increased proliferation was not attributed to enhanced TCR expression, which was equivalent between the two types of T cells before and after stimulation (unpublished data). These findings were further supported by time-kinetic analysis showing equal proliferation of p21−/− and wild-type T cells up to 24 h, but significantly greater proliferation of p21−/− T cells by 72 h (Fig. 3 b). Interestingly, p21−/− T cell proliferation declined faster than control T cells, reaching baseline levels by ∼120 h after stimulation compared with >150 h for the p21+/+ cells, suggesting increased apoptosis by the p21-deficient cells.


Deficiency of the cyclin kinase inhibitor p21(WAF-1/CIP-1) promotes apoptosis of activated/memory T cells and inhibits spontaneous systemic autoimmunity.

Lawson BR, Baccala R, Song J, Croft M, Kono DH, Theofilopoulos AN - J. Exp. Med. (2004)

Enhanced proliferation and apoptosis of p21−/− T cells from male BXSB mice. (a) Increased proliferation of p21−/− T cells after in vitro cross-linking of antigen receptors. LN cells (n = 3 mice/group) were stimulated with increasing concentrations of plate-bound anti-CD3 (0.1–20 μg/ml) plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at 72 h (mean ± SEM cpm). (shaded bars) BXSB p21−/− mice. (unshaded bars) p21+/+ BXSB mice. (b) Kinetics of in vitro proliferation. LN cells (n = 3 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at the indicated time points. Results are representative of two independent experiments (n = 3 mice/group). (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (c) Increased AICD of p21−/− T cells. T cell were stimulated with 0.5 μg/ml of soluble anti-CD3 for 48 h followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 (n = 3 mice/group). Percent of apoptotic (Annexin V+PI−) CD4+ T cells was determined by FACS®. Similar results were obtained with CD8+ T cells. (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (d) Increased proliferation of wild-type BXSB T cells treated with p21 AS oligonucleotides. LN cells (n = 4 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies in the presence of either p21 AS or control oligonucleotides (all AS oligonucleotides at 400 nM) and assessed for [3H]thymidine incorporation (mean ± SEM cpm). (e) Increased AICD of wild-type BXSB T cells treated with p21 AS oligonucleotides. Wild-type BXSB T cells were stimulated with 0.5 μg/ml of soluble anti-CD3 followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 in the constant presence of p21 AS or control oligonucleotides (400 nM; n = 3 mice/group). (▪) AS oligo no. 1. (□) AS oligo no. 2. (▴) Control oligonucleotide. (e, inset) Western blot analysis of wild-type BXSB T cells treated with either p21 AS or control oligonucleotides. T cells were stimulated with 10 μg/ml anti-CD3 and 5 μg/ml CD28 antibodies in the presence of AS or control oligonucleotides (400 nm). Whole cell lysates were analyzed by Western blot using anti-p21 and antiactin antibodies. *, P < 0.05 by Student's t test.
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fig3: Enhanced proliferation and apoptosis of p21−/− T cells from male BXSB mice. (a) Increased proliferation of p21−/− T cells after in vitro cross-linking of antigen receptors. LN cells (n = 3 mice/group) were stimulated with increasing concentrations of plate-bound anti-CD3 (0.1–20 μg/ml) plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at 72 h (mean ± SEM cpm). (shaded bars) BXSB p21−/− mice. (unshaded bars) p21+/+ BXSB mice. (b) Kinetics of in vitro proliferation. LN cells (n = 3 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies and assessed for [3H]thymidine incorporation at the indicated time points. Results are representative of two independent experiments (n = 3 mice/group). (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (c) Increased AICD of p21−/− T cells. T cell were stimulated with 0.5 μg/ml of soluble anti-CD3 for 48 h followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 (n = 3 mice/group). Percent of apoptotic (Annexin V+PI−) CD4+ T cells was determined by FACS®. Similar results were obtained with CD8+ T cells. (•) BXSB p21−/− mice. (○) BXSB p21+/+ mice. (d) Increased proliferation of wild-type BXSB T cells treated with p21 AS oligonucleotides. LN cells (n = 4 mice/group) were stimulated with 10 μg/ml of plate-bound anti-CD3 plus 5 μg/ml of soluble anti-CD28 antibodies in the presence of either p21 AS or control oligonucleotides (all AS oligonucleotides at 400 nM) and assessed for [3H]thymidine incorporation (mean ± SEM cpm). (e) Increased AICD of wild-type BXSB T cells treated with p21 AS oligonucleotides. Wild-type BXSB T cells were stimulated with 0.5 μg/ml of soluble anti-CD3 followed by TCR religation with 10 μg/ml of plate-bound anti-CD3 in the constant presence of p21 AS or control oligonucleotides (400 nM; n = 3 mice/group). (▪) AS oligo no. 1. (□) AS oligo no. 2. (▴) Control oligonucleotide. (e, inset) Western blot analysis of wild-type BXSB T cells treated with either p21 AS or control oligonucleotides. T cells were stimulated with 10 μg/ml anti-CD3 and 5 μg/ml CD28 antibodies in the presence of AS or control oligonucleotides (400 nm). Whole cell lysates were analyzed by Western blot using anti-p21 and antiactin antibodies. *, P < 0.05 by Student's t test.
Mentions: Previous in vitro analyses with p21−/− T cells from nonautoimmune mice showed increased proliferation compared with wild-type T cells (36, 37). Similarly, BXSB p21−/− T cells showed enhanced proliferation compared with wild-type T cells (P < 0.05) after engagement of CD3 and CD28, with no apparent alteration of the TCR-induced activation threshold (Fig. 3 a). Increased proliferation was not attributed to enhanced TCR expression, which was equivalent between the two types of T cells before and after stimulation (unpublished data). These findings were further supported by time-kinetic analysis showing equal proliferation of p21−/− and wild-type T cells up to 24 h, but significantly greater proliferation of p21−/− T cells by 72 h (Fig. 3 b). Interestingly, p21−/− T cell proliferation declined faster than control T cells, reaching baseline levels by ∼120 h after stimulation compared with >150 h for the p21+/+ cells, suggesting increased apoptosis by the p21-deficient cells.

Bottom Line: Absence of p21 resulted in enhanced Fas/FasL-mediated activation-induced T cell death, increased activation of procaspases 8 and 3, and loss of mitochondrial transmembrane potential.Increased apoptosis was also associated with p53 up-regulation and a modest shift in the ratio of Bax/Bcl-2 toward the proapoptotic Bax.Thus, modulation of the cell cycle pathway may be a novel approach to reduce apoptosis-resistant pathogenic lymphocytes and to ameliorate systemic autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
A characteristic feature of systemic lupus erythematosus is the accumulation of activated/memory T and B cells. These G0/G1-arrested cells express high levels of cyclin-dependent kinase inhibitors such as p21, are resistant to proliferation and apoptosis, and produce large amounts of proinflammatory cytokines. Herein, we show that ablation of p21 in lupus-prone mice allows these cells to reenter the cell cycle and undergo apoptosis, leading to autoimmune disease reduction. Absence of p21 resulted in enhanced Fas/FasL-mediated activation-induced T cell death, increased activation of procaspases 8 and 3, and loss of mitochondrial transmembrane potential. Increased apoptosis was also associated with p53 up-regulation and a modest shift in the ratio of Bax/Bcl-2 toward the proapoptotic Bax. Proliferation and apoptosis of B cells were also increased in p21-/- lupus mice. Thus, modulation of the cell cycle pathway may be a novel approach to reduce apoptosis-resistant pathogenic lymphocytes and to ameliorate systemic autoimmunity.

Show MeSH
Related in: MedlinePlus