Ubiquitin–proteasome pathway is compromised in CD45RO+ and CD45RA+ T lymphocyte subsets during aging

doi:10.1016/S0531-5565(01)00203-0

Experimental Gerontology

Volume 37, Issues 2–3, 3 January 2002, Pages 359-367

https://doi.org/10.1016/S0531-5565(01)00203-0 Get rights and content

Abstract

Recent reports from our laboratory have demonstrated that CD45RO⁺ and CD45RA⁺ T lymphocytes from the elderly are compromised in their response to activation-induced IL-2 receptor expression, IκB-α degradation, as well as nuclear translocation of NFkB. To understand the basis of this activation-induced dysfunction in the elderly, we have examined the role of the ubiquitin–proteasome pathway. Our results demonstrate that both CD45RO⁺ and CD45RA⁺ T lymphocytes from the elderly show significant reduction in the constitutive 26S proteasome-associated chymotryptic activity, when compared to those in the young. Additionally, anti-CD3–CD28 treatment induced enhancement of proteasome-associated enzymatic activity in cells from the young, but not in cells from the elderly. Lowered proteasome-associated activity and its effect on reduced immune responses in the elderly could be mimicked by experiments which involved pretreatment of T cells from young donors with a proteasome specific inhibitor, lactacystin. These data demonstrate that IL-2 receptor induction is clearly compromised in T cells from the young when proteasomes are inhibited by pretreatment with lactacystin. An examination of ubiquitin specific hydrolase activity, demonstrated a decrease in activated CD45RA⁺ and CD45RO⁺ T cell subsets from the elderly when compared to young.

These results suggest that lowered proteasome-associated enzymatic activity in combination with compromised de-ubiquitinating activity may be responsible for lowered activation-induced NFkB and NFkB-mediated gene expression in elderly subjects.

Introduction

Most measures of T lymphocyte function following antigenic exposure, such as cytokine-receptor expression, cytokine production, cytotoxicity and proliferation, show age-associated impairments, in both humans and inbred strains of mice (Trebilcock and Ponnappan, 1998, Nagel et al., 1988, Miller, 1996). One of the characteristic phenotypic alterations that accompanies aging in the T cell compartment, is a shift towards an increase in the number of CD45RO⁺ T cells with a concomitant decrease in CD45RA⁺ T cells, in both murine and human populations (De Paoli et al., 1988, Flurkey et al., 1992).

Previous studies from our laboratory focusing on transcription factor NFkB in T cell subsets during aging have demonstrated age-dependent decrease in the function of both CD45RA⁺ and CD45RO⁺ T cells in the elderly (Trebilcock and Ponnappan, 1998). This decrease manifested as lowered nuclear induction of the transcription factor NFkB, NFkB regulated expression of IL-2 receptor and CD3-induced proliferation in these subsets. As transcription factor NFkB is an important regulator of immune response genes (Ghosh et al., 1998), and the induced nuclear levels of this factor are regulated by the 26S proteasome, alterations in the induction and regulation of this transcription factor may well be attributed to the functional fine-tuning of the 26S proteasome activity (May and Ghosh, 1998). Indeed, our studies on T cells during aging have clearly demonstrated lowered proteasomal chymotryptic activity (Ponnappan et al., 1999). A similar decline in proteolytic activity associated with the proteasome has been demonstrated in other cell types such as rat liver, human dermal fibroblasts and skeletal muscles (Shibatani et al., 1996, Ly et al., 2000, Lee et al., 1999). Therefore, to elucidate the basis for the decline in NFkB induction and NFkB-mediated responses in the T cell subsets in the elderly, we have extended our analyses to 26S proteasome, focusing on the chymotryptic activity responsible for the induction of transcription factor NFkB, and vital in mediating immune response gene expression.

Results presented in this manuscript on the analyses of the 26S proteasome-associated enzymatic activity clearly demonstrate that both CD45RA⁺ and CD45RO⁺ T cell subsets manifest a decline in chymotryptic activity. A similar age-related decline in activity could also be discerned with respect to the de-ubiquitinating activity associated with activated T cells, while the constitutive de-ubiquitinating activity remained similar in T cell subsets from the young and the elderly. This is in keeping with our previously reported findings in T cells during aging (Ponnappan et al., 1999). A cursory examination of the alpha and beta subunits of the 20S proteasome core demonstrated limited alterations in the levels of 20S proteasome protein in T cells. Taken together, these data suggest that altered proteasome-associated enzymatic activity in combination with decreased de-ubiquitinating activity may underlie the observed decline in immune function associated with aging in T cell subsets.

Section snippets

Reagents

Antibodies to CD45RO and CD45RA used for purification of memory and naive T cell subsets along with antibodies used for flow cytometry including fluoroscein (FITC) labeled antibodies to CD45RA and CD45RO, phycoerythrin (PE) labeled anti-CD3, and FITC- and PE-labeled isotype controls were obtained from Sigma Chemical Co. (St Louis, MO). Anti-CD3 and anti-CD28 used for activation were from Zymed (San Francisco, CA). Anti-IgG coupled to horseradish peroxidase was obtained from BD-Transduction

Proteasome associated chymotryptic activity in CD45RA⁺ and CD45RO⁺ T lymphocyte subsets is decreased in the elderly

To understand the basis of decline in the nuclear induction of NFkB in T cell subsets from the elderly, we assayed 26S proteasome associated chymotryptic activity. Chymotryptic activity detected by the cleavage of fluorogenic substrate specific for the activity was carried out using proteasome-enriched cell lysates of T cell subsets obtained from five pairs of young and elderly donors. In-gel overlay assays were performed to ensure the fidelity of the enriched fractions for proteasome. Results

Discussion

These studies help to elucidate the effect of aging on the ubiquitin–proteasome pathway on immune function in the elderly. Results presented here for the first time demonstrate that alongside defects in the 26S proteasome associated enzymatic activity, defects can also be discerned in cellular de-ubiquitinating activity in the T cell compartment during aging. This is particularly significant, as defects in de-ubiquitination can directly impact on the levels of ubiquitinated proteins. Presence

Acknowledgements

This material is based upon the work supported by grants provided by NIH/NIA RO1 AG13081, MO1RR14288 and in part by the use of facilities at the VA Medical Center, Little Rock, Arkansas. This work would not have been possible without the expert technical assistance of Dr Mingzhong Zheng and Mrs Virginia Fitzhugh, and assistance provided by the General Clinical Research Center, in sample collection. Thanks are due to Dr S. Ponnappan for critical reading of the manuscript.

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Ubiquitin–proteasome pathway is compromised in CD45RO+ and CD45RA+ T lymphocyte subsets during aging

Abstract

Introduction

Section snippets

Reagents

Proteasome associated chymotryptic activity in CD45RA+ and CD45RO+ T lymphocyte subsets is decreased in the elderly

Discussion

Acknowledgements

Exp. Gerontol.

Clin. Immunol. Immunopathol.

J. Biol. Chem.

Trends Neurosci.

Immunol. Today

Neuron

Cell. Immunol.

Mech. Ageing Dev.

Structure and functions of the proteasomes

Annu. Rev. Biochem.

Lactacystin and clasto-lactacystin β-lactone modify multiple proteasome subunits and inhibit intracellular protein degradation and major histo-compatibility complex class I antigen presentation

J. Biol. Chem.

Ubiquitin–proteasome pathway is compromised in CD45RO⁺ and CD45RA⁺ T lymphocyte subsets during aging

Proteasome associated chymotryptic activity in CD45RA⁺ and CD45RO⁺ T lymphocyte subsets is decreased in the elderly