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The death domain kinase RIP protects thymocytes from tumor necrosis factor receptor type 2-induced cell death.

Cusson N, Oikemus S, Kilpatrick ED, Cunningham L, Kelliher M - J. Exp. Med. (2002)

Bottom Line: We observed a decrease in rip-/- thymocytes and T cells in both wild-type C57BL/6 and recombination activating gene 1-/- irradiated hosts.However, rip-deficient mice contain few viable CD4+ and CD8+ thymocytes, and rip-/- thymocytes are sensitive to TNF-induced cell death.Taken together, this study implicates RIP and TNFR2 in thymocyte survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Program in Immunology/Virology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Fas and the tumor necrosis factor receptor (TNFR)1 regulate the programmed cell death of lymphocytes. The death domain kinase, receptor interacting protein (rip), is recruited to the TNFR1 upon receptor activation. In vitro, rip-/- fibroblasts are sensitive to TNF-induced cell death due to an impaired nuclear factor kappaB response. Because rip-/- mice die at birth, we were unable to examine the effects of a targeted rip mutation on lymphocyte survival. To address the contribution of RIP to immune homeostasis, we examined lethally irradiated mice reconstituted with rip-/- hematopoietic precursors. We observed a decrease in rip-/- thymocytes and T cells in both wild-type C57BL/6 and recombination activating gene 1-/- irradiated hosts. In contrast, the B cell and myeloid lineages are unaffected by the absence of rip. Thus, the death domain kinase rip is required for T cell development. Unlike Fas-associated death domain, rip does not regulate T cell proliferation, as rip-/- T cells respond to polyclonal activators. However, rip-deficient mice contain few viable CD4+ and CD8+ thymocytes, and rip-/- thymocytes are sensitive to TNF-induced cell death. Surprisingly, the rip-associated thymocyte apoptosis was not rescued by the absence of TNFR1, but appears to be rescued by an absence of TNFR2. Taken together, this study implicates RIP and TNFR2 in thymocyte survival.

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DP thymocyte apoptosis in the absence of rip and tnfr1. Flow cytometric analysis of thymocytes from rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice. (A) Thymocytes from day-6 rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice were stained with PE–anti-CD4, FITC–anti-CD8, and LDS-751. The percent of viable cells are indicated in each quadrant. (B) Thymocyte and spleen cell counts of rip+/+/tnfr1−/− and rip−/−/tnfr1−/− mice. Cell counts were performed in triplicate. Results are expressed ± SEM of at least six animals between 6 to 8 d old. (C) Thymus from control littermates and rip−/−/tnfr1−/− mice was stained with a lineage-specific cocktail containing biotinylated-IgM, -Ter 119, -Gr1, –Mac-1, -PanNK, -CD3, -CD4, and -CD8. Some samples were then stained with FITC-CD44, PE-CD25, and Streptavidin-CyChrome. The Cy− or DN cells were further analyzed according to their expression of CD44 and CD25. The percent of positive cells are indicated in each quadrant. Seven rip−/−/tnfr1−/− mice and seven littermate controls were analyzed. One representative experiment is shown.
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fig6: DP thymocyte apoptosis in the absence of rip and tnfr1. Flow cytometric analysis of thymocytes from rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice. (A) Thymocytes from day-6 rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice were stained with PE–anti-CD4, FITC–anti-CD8, and LDS-751. The percent of viable cells are indicated in each quadrant. (B) Thymocyte and spleen cell counts of rip+/+/tnfr1−/− and rip−/−/tnfr1−/− mice. Cell counts were performed in triplicate. Results are expressed ± SEM of at least six animals between 6 to 8 d old. (C) Thymus from control littermates and rip−/−/tnfr1−/− mice was stained with a lineage-specific cocktail containing biotinylated-IgM, -Ter 119, -Gr1, –Mac-1, -PanNK, -CD3, -CD4, and -CD8. Some samples were then stained with FITC-CD44, PE-CD25, and Streptavidin-CyChrome. The Cy− or DN cells were further analyzed according to their expression of CD44 and CD25. The percent of positive cells are indicated in each quadrant. Seven rip−/−/tnfr1−/− mice and seven littermate controls were analyzed. One representative experiment is shown.

Mentions: To determine whether the rip-associated thymocyte apoptosis was TNFR1-mediated, we examined the CD4/CD8 profiles of the rip+/−/tnfr1−/− and rip−/−/tnfr1−/− neonatal thymus. Dramatic differences in the overall cellularity were observed, suggesting that survival is not mediated through the TNFR1. The rip-deficient thymus consists of 10× fewer thymocytes than rip+/+/tnfr1−/− littermates (Fig. 6 B). Similar to what was observed in the rip−/− thymus, four- to sevenfold increases in the percent of apoptotic cells were also observed in the rip−/−/tnfr1−/− thymocytes by staining with FITC–annexin V/PI or 7-AAD (unpublished data). In addition to the decreased cellularity and increase in apoptotic cells, there were concomitant decreases in the DP thymocyte population (31, 66, 29, and 56% in four age-matched rip−/−/tnfr1−/− mice compared with 84, 83, 86, and 88% in littermate controls). Thus, the absence of RIP resulted in an average 22-fold decrease in the absolute numbers of DP thymocytes.


The death domain kinase RIP protects thymocytes from tumor necrosis factor receptor type 2-induced cell death.

Cusson N, Oikemus S, Kilpatrick ED, Cunningham L, Kelliher M - J. Exp. Med. (2002)

DP thymocyte apoptosis in the absence of rip and tnfr1. Flow cytometric analysis of thymocytes from rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice. (A) Thymocytes from day-6 rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice were stained with PE–anti-CD4, FITC–anti-CD8, and LDS-751. The percent of viable cells are indicated in each quadrant. (B) Thymocyte and spleen cell counts of rip+/+/tnfr1−/− and rip−/−/tnfr1−/− mice. Cell counts were performed in triplicate. Results are expressed ± SEM of at least six animals between 6 to 8 d old. (C) Thymus from control littermates and rip−/−/tnfr1−/− mice was stained with a lineage-specific cocktail containing biotinylated-IgM, -Ter 119, -Gr1, –Mac-1, -PanNK, -CD3, -CD4, and -CD8. Some samples were then stained with FITC-CD44, PE-CD25, and Streptavidin-CyChrome. The Cy− or DN cells were further analyzed according to their expression of CD44 and CD25. The percent of positive cells are indicated in each quadrant. Seven rip−/−/tnfr1−/− mice and seven littermate controls were analyzed. One representative experiment is shown.
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fig6: DP thymocyte apoptosis in the absence of rip and tnfr1. Flow cytometric analysis of thymocytes from rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice. (A) Thymocytes from day-6 rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice were stained with PE–anti-CD4, FITC–anti-CD8, and LDS-751. The percent of viable cells are indicated in each quadrant. (B) Thymocyte and spleen cell counts of rip+/+/tnfr1−/− and rip−/−/tnfr1−/− mice. Cell counts were performed in triplicate. Results are expressed ± SEM of at least six animals between 6 to 8 d old. (C) Thymus from control littermates and rip−/−/tnfr1−/− mice was stained with a lineage-specific cocktail containing biotinylated-IgM, -Ter 119, -Gr1, –Mac-1, -PanNK, -CD3, -CD4, and -CD8. Some samples were then stained with FITC-CD44, PE-CD25, and Streptavidin-CyChrome. The Cy− or DN cells were further analyzed according to their expression of CD44 and CD25. The percent of positive cells are indicated in each quadrant. Seven rip−/−/tnfr1−/− mice and seven littermate controls were analyzed. One representative experiment is shown.
Mentions: To determine whether the rip-associated thymocyte apoptosis was TNFR1-mediated, we examined the CD4/CD8 profiles of the rip+/−/tnfr1−/− and rip−/−/tnfr1−/− neonatal thymus. Dramatic differences in the overall cellularity were observed, suggesting that survival is not mediated through the TNFR1. The rip-deficient thymus consists of 10× fewer thymocytes than rip+/+/tnfr1−/− littermates (Fig. 6 B). Similar to what was observed in the rip−/− thymus, four- to sevenfold increases in the percent of apoptotic cells were also observed in the rip−/−/tnfr1−/− thymocytes by staining with FITC–annexin V/PI or 7-AAD (unpublished data). In addition to the decreased cellularity and increase in apoptotic cells, there were concomitant decreases in the DP thymocyte population (31, 66, 29, and 56% in four age-matched rip−/−/tnfr1−/− mice compared with 84, 83, 86, and 88% in littermate controls). Thus, the absence of RIP resulted in an average 22-fold decrease in the absolute numbers of DP thymocytes.

Bottom Line: We observed a decrease in rip-/- thymocytes and T cells in both wild-type C57BL/6 and recombination activating gene 1-/- irradiated hosts.However, rip-deficient mice contain few viable CD4+ and CD8+ thymocytes, and rip-/- thymocytes are sensitive to TNF-induced cell death.Taken together, this study implicates RIP and TNFR2 in thymocyte survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Program in Immunology/Virology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Fas and the tumor necrosis factor receptor (TNFR)1 regulate the programmed cell death of lymphocytes. The death domain kinase, receptor interacting protein (rip), is recruited to the TNFR1 upon receptor activation. In vitro, rip-/- fibroblasts are sensitive to TNF-induced cell death due to an impaired nuclear factor kappaB response. Because rip-/- mice die at birth, we were unable to examine the effects of a targeted rip mutation on lymphocyte survival. To address the contribution of RIP to immune homeostasis, we examined lethally irradiated mice reconstituted with rip-/- hematopoietic precursors. We observed a decrease in rip-/- thymocytes and T cells in both wild-type C57BL/6 and recombination activating gene 1-/- irradiated hosts. In contrast, the B cell and myeloid lineages are unaffected by the absence of rip. Thus, the death domain kinase rip is required for T cell development. Unlike Fas-associated death domain, rip does not regulate T cell proliferation, as rip-/- T cells respond to polyclonal activators. However, rip-deficient mice contain few viable CD4+ and CD8+ thymocytes, and rip-/- thymocytes are sensitive to TNF-induced cell death. Surprisingly, the rip-associated thymocyte apoptosis was not rescued by the absence of TNFR1, but appears to be rescued by an absence of TNFR2. Taken together, this study implicates RIP and TNFR2 in thymocyte survival.

Show MeSH
Related in: MedlinePlus