ABSTRACT
T-cell clones (TCC) derived from human peripheral blood lymphocytes of a young control, a healthy elderly (SENIEUR) donor, or from CD34(+) hematopoietic progenitor cells were utilised in this study to examine how in vivo and in vitro ageing affects T-cell apoptotic capability. The role of CD25, CD28 and the intracellular proteins, FLICE-inhibitory protein (FLIP), receptor-interacting protein (RIP) and caspase 3 were investigated. We observed an age-related decline in the expression of the IL-2 receptor alpha chain CD25, and absence of the co-stimulatory receptor CD28 on three of the four TCC studied. In young donor- and CD34 cell-derived TCC, but not in SENIEUR donor-derived TCC, we observed an age-related increase in susceptibility of the cells to mFas-L-induced apoptosis, which correlated with the age-related decrease of CD25 expression. Expression levels of full-length RIP and FLIP did not show any correlation to apoptotic susceptibility. However, expression levels of the cleaved form of RIP were greatly reduced in the SENIEUR donor-derived TCC, which together with a trend towards increased caspase 3 activity, could indicate an age-related alteration in utilisation of different apoptotic signalling pathways.
Subject(s)
Aging/immunology , Antigens, CD34/analysis , Apoptosis/immunology , Intracellular Signaling Peptides and Proteins , Lymphocyte Activation/physiology , T-Lymphocytes/immunology , Adult , Aged , Aged, 80 and over , CASP8 and FADD-Like Apoptosis Regulating Protein , CD28 Antigens/metabolism , Carrier Proteins/metabolism , Caspase 3 , Caspases/metabolism , Cells, Cultured , Cellular Senescence/immunology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Humans , Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases , Receptors, Interleukin-2/metabolism , Signal Transduction/immunology , T-Lymphocytes/cytology , T-Lymphocytes/metabolismABSTRACT
CD95-induced apoptosis is an important regulatory mechanism in T cells and this complex signalling pathway is now thought to include the protein kinase RIP. Although, RIP is best known for its role in TNF signalling and NF-kappaB activation, it contains a death domain and it is capable of causing apoptosis upon cleavage. In the present study, the role of RIP in CD95-induced apoptosis and its inter-relationship with the caspase cascade was investigated. Studies were performed on both a RIP-/- T cell line and peripheral T lymphocytes, where RIP was degraded through the addition of geldanamycin. Apoptosis was induced by membrane CD95-L, thought to be the most physiological relevant form of CD95-L. Results showed that RIP-/- cells had a decreased susceptibility to death, thus confirming a role for RIP in CD95-induced apoptosis. Furthermore, it was confirmed that RIP is cleaved upon CD95-L stimulation, a process that can be inhibited by Z-VAD. However, only partial inhibition in peripheral T lymphocytes by Z-VAD was observed, suggesting a potential caspase-independent processing of RIP. Studies performed on the activity of effector caspase 3 and on the initiator caspases 2, 8, and 9 revealed that, in the absence of RIP, the activity of these caspases decreases, indicating that RIP-associated apoptosis is caspase-dependent. Hence, these studies support a caspase-related role for RIP in CD95-induced T apoptosis.
