Hydrogen peroxide induced down-regulation of CD28 expression of Jurkat cells is associated with a change of site α-specific nuclear factor binding activity and the activation of caspase-3
Introduction
In 1956, Denham Harman first proposed the ‘free radical theory’ of aging, speculating that endogenous ROS were generated in cells and caused cumulative damage (Harman, 1957). Now, many kinds of ROS have been found, including superoxide, H2O2, nitric oxide (NO), and hydroxyl radicals. These various reactive species can either be generated exogenously or produced intracellularly in different ways. They have been recognized to regulate many important cellular events including gene expression (Lo and Cruz, 1995), transcription factor activation (Schreck et al., 1991), and cellular proliferation (Murrell et al., 1990). Such ROS-caused events can influence cellular processes related to the development of age-related diseases.
Among the many consequences of aging, a main factor is the progressive decline in protective immunity. The incidence of malignancies increases with age, and aging people have increased susceptibility to infections and blunted responses to vaccines. Although the molecular mechanisms of immunosenescence are not yet clear, the loss of effective immune activity is largely due to alterations within T cells. With advancing age, substantial changes in both the functional and phenotypic profiles of T cells have been reported and reviewed (Pawelec et al., 2001, Ginaldi et al., 1999). One of the most important changes in the phenotypic CD4+ T cell profiles is the declined expression of CD28 and the appearance of a subgroup of CD28null cells (Caruso et al., 1996, Vallejo et al., 1998). CD28 is a major co-stimulatory molecule in the generation of T cell-mediated immune responses (Ward, 1996, Lenschow et al., 1996). Ligation of CD28 promotes proliferation and cytokine production following antigen engagement (Jenkins et al., 1991). The CD28 gene knockout mice are unable to form germinal centers and are often severely immunocompromised. The CD28null T cells can initiate, but not sustain or proliferate (Boise et al., 1995, Shahinian et al., 1993). CD28 mediated signals are also implicated in promoting cell survival via bcl-xL up-regulation. Given that these observations attest to the critical role of CD28 signal in allowing proliferation and/or promoting T cell survival, the regulation of CD28-expression may have important consequences for the cellular function. Since ROS is a key factor in immunosenescence, we speculate that ROS may play a critical role in the down-regulation of CD28 in aging.
Jurkat E6-1 cells derive from human T lymphocytes; many surface molecules of human T cells are maintained on Jurkat E6-1, including TCR, CD3, CD4, CD28, CD95 etc. The Jurkat cell is also a good model for biologists to study the molecular events in T lymphocytes (Kolanus et al., 1996, Yang et al., 1996). Activation and replicative senescence are two aspects in the study of senescence in vitro. But both neglect the influence of ROS on cultured cells. In this study, Jurkat E6-1 cells were used to study the influence of H2O2 (an important precursor of highly reactive free radicals) on the expression of CD28. During replicative senescence, the progressive loss of CD28-expression was associated with a decrease of site β-binding activity (Vallejo Abbe et al., 1999). In contrast, when cultured in H2O2, the loss of CD28-expression was related to a decrease of site α -binding activity.
The caspases are a novel class of cysteine proteases that play a critical role during apoptosis (Maroto and Perez-Poro, 1997, Li et al., 1997). Among the 10 or more caspases identified so far, caspase-3, a member of the Asp-Glu-Val-Asp-specific caspases, is an attractive candidate for the putative executor of apoptosis because it is commonly activated by numerous apoptotic stimuli, and can also be activated by ROS (Do et al., 2000, Huaruki et al., 2000). Accumulative evidence suggests the presence of the two main caspase cascades. One is triggered by the activation of death receptors that results in the recruitment of the death-inducing signaling complex (DISC) and procaspase-8 (FLICE) as the principal components (Ashkanazi and Dixit, 1998). Activation of caspase-8, in turn, cleaves and activates the downstream caspases such as caspase-6, -7, and -3 (Boldin et al., 1986, Srinivasula et al., 1991, Monney et al., 1998). The other is triggered by cytochrome c released from mitochondria which forms a multi-subunit complex with Apaf-1 and procaspase-9, leading to the activation of caspase-9 (Zou et al., 1999, Susin et al., 1999). Subsequently the activated caspase-9 cleaves to activate caspase-3. In this study, we found that inhibitors of caspase-3, but not those of caspase-8, could counteract the influence of H2O2 on the decline of CD28 expression.
Section snippets
Cell culture and concentration definition of H2O2
Jurkat E6-1 cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of sciences (Shanghai, China). The cells were maintained in antibiotic-free RPMI 1640 medium containing 10% fetal calf serum (FCS), 2 μM glutamine, 50 μM 2-mercaptoethanol, and 25 mM HEPES buffer solution at 37 °C in a humidified atmosphere containing 5% CO2. PHA-l (Sigma) and phorbol 12-myristyl 13-acetate (PMA) (Sigma) are used at final concentrations of 1 μg/ml and 50 ng/ml, respectively. In order to
Oxidative stress: the effect of H2O2
H2O2 was used as one resource of ROS to cultured Jurkat E6-1 cells in this study. The appropriate concentration of H2O2 ([H2O2]) was determined by the Annexin V-FITC and Propidium iodide method. The apoptotic response of Jurkat cells to [H2O2] was assayed after 24 h of culture in medium containing different [H2O2]. The number of apoptotic cells increased with increasing [H2O2], as shown in Fig. 2. Most of the cells did not undergo apoptosis when H2O2 concentration was under 300 μM. Most cells
Discussion
Immunosenescence involves the morphological and functional integrity of all organs, including the cellular and humoral immunological functions. These changes are often associated with important clinical manifestations, such as increased susceptibility to infection and cancer, which are frequently observed in the elderly population. The loss of effective immune activity is largely due to alterations within T cells. Substantial changes in both the functional and phenotypic profiles of T cells
Acknowledgements
This study was supported by a grant from the Japan Institute for the Control of Aging and a grant (2001CB5100009) from The National Program for key Basic Research Projects, Ministry of Science and Technology, China P.R. We thank Yu Hu for her assistance in cell culture. We gratefully acknowledge the technical assistance from Dr Yonghai Li and Mrs Qingfeng Liu.
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