Clin. exp. Immunol. (1992) 89, 158-163
Intracellular free Ca2 + fluxes and responses to phorbol ester in T lymphocytes from healthy elderly subjects J. R. NAYLOR, S. R. JAMES & L. K. TREJDOSIEWICZ Department of Clinical Medicine, St James's University Hospital, University of Leeds, Leeds, UK
(Acceptedfor publication 20 January 1992)
SUMMARY A group of healthy elderly subjects (a 75 years) was selected by the strict criteria of the SENIEUR protocol, and compared with healthy young (< 35 years) volunteers. Mitogenic responses of peripheral blood mononuclear cells to phytohaemagglutinin and anti-CD3 were significantly reduced in the elderly (P < 0 0002), thereby confirming that even though in perfect health, elderly individuals show impaired cell-mediated immunity. However, no abnormality of intracellular free Ca2+ fluxes could be detected in purified T cells from the elderly subjects when stimulated with anti-CD3 antibody. Nevertheless, both the proliferative responses of purified T cells to phorbol ester and calcium ionophore (Ionomycin) and the phorbol ester-induced inhibition of the Ca2+ response were defective in the elderly subjects (P < 0003 and P < 0-0002, respectively). These data suggest that signal transduction and the generation of second messengers proceed normally in T cells from the elderly, but downstream events mediated by activation of protein kinase C are dysfunctional.
Keywords Mitogenic responses intracellular free calcium phorbol ester protein kinase C
INTRODUCTION All available evidence suggests that ageing is accompanied by a progressive dysfunction of cell-mediated immunity (reviewed in A serum immunoglobulin concentrations are [1]). Although oncentrations ar
[1]).edinlthough serumanimmunoglobuint
appears to be limited to T lymphocyte-dependent antigens [3], implying no defect in B lymphocyte function per se. There is no clear hypothesis to explain why cell-mediated immunity declines with age. Thymic atrophy alone is unlikely to account for immunesenescence, as thymic weight and volume change little between the ages of 5 and 84 years [4]. Immunesenescence could be due to a T cell dysfunction, or due to associated extrinsic factors, such as deficient accessory cell function, a changing balance of helper and suppressor cells or excessive suppression by monocyte-derived factors (e.g. by prostaglandin E2). Several recent studies have focused on IL-2 secretion and membrane expression ofthe IL-2 receptor (IL-2R) [5-7]. These reports suggest that age-related T cell proliferative abnormality is due to defects in IL-2 secretion, expression of high affinity IL-2R and transcription of IL-2R mRNA [7]. As evidence relating to immunesenescence points to a defect or defects preceding IL-2 production and up-regulation of IL-2R, it is possible that such defects arise as a defect in the membrane transduction of mitogenic signals. This might be due
to the subnormal generation of inositol trisphosphate (IP3) and diacylglycerol (DG) second messengers, the respective mediators of intracellular calcium fluxes and activation of protein PC 89.Teei vdnei upr fti kns
8,9.Teeievdnensuprofts
.iaeC hypothesis from murine models, showing that mitogen-evoked fluxes in intracellular free Ca2+ concentration ([Ca2+]i) decline in splenocytes with age [10]. However, a recent study in humans has suggested that [Ca2 +] fluxes remain normal in T cells in the . . elel [1 Thus, ipsb tha seondfessenger producbt PC
a e tion aisimpairedwbutrtat.th eves. tream anormalitiesi In order to test these hypotheses, we have studied a carefully
selected group of healthy elderly subjects, in order to investigate T cell mitogen-induced [Ca.2 +1, fluxes and the effects of pharmacological activation of PKC by means of phorbol ester [12]. Here
report th ahough [Caer ron tohorbolester wer
flxe remainedunimaied, T b d
SUBJECTS AND METHODS
Subjects All subjects were living in the community and were selected by the rigorous criteria of the SENIEUR protocol [13] to exclude sub-clinical or other disease as a confounding variable. In addition to the protocol criteria, subjects were tested on at least two occasions 1 month apart to ensure all were in a steady state. Subjects were excluded if they had taken any medication,
Correspondence: L. K. Trejdosiewicz, Department of Clinical Medicine, C.S.B. Level 7, St James's University Hospital, Leeds LS9 7TF, UK.
158
Defective phorbol ester responses in the elderly
159
prescribed or otherwise, during the month of screening or between screening and study. 'Aged' donors were all > 75 years, and the 'young' donor control group was selected from laboratory and hospital staff volunteers aged
Intracellular free Ca2 + fluxes and responses to phorbol ester in T lymphocytes from healthy elderly subjects J. R. NAYLOR, S. R. JAMES & L. K. TREJDOSIEWICZ Department of Clinical Medicine, St James's University Hospital, University of Leeds, Leeds, UK
(Acceptedfor publication 20 January 1992)
SUMMARY A group of healthy elderly subjects (a 75 years) was selected by the strict criteria of the SENIEUR protocol, and compared with healthy young (< 35 years) volunteers. Mitogenic responses of peripheral blood mononuclear cells to phytohaemagglutinin and anti-CD3 were significantly reduced in the elderly (P < 0 0002), thereby confirming that even though in perfect health, elderly individuals show impaired cell-mediated immunity. However, no abnormality of intracellular free Ca2+ fluxes could be detected in purified T cells from the elderly subjects when stimulated with anti-CD3 antibody. Nevertheless, both the proliferative responses of purified T cells to phorbol ester and calcium ionophore (Ionomycin) and the phorbol ester-induced inhibition of the Ca2+ response were defective in the elderly subjects (P < 0003 and P < 0-0002, respectively). These data suggest that signal transduction and the generation of second messengers proceed normally in T cells from the elderly, but downstream events mediated by activation of protein kinase C are dysfunctional.
Keywords Mitogenic responses intracellular free calcium phorbol ester protein kinase C
INTRODUCTION All available evidence suggests that ageing is accompanied by a progressive dysfunction of cell-mediated immunity (reviewed in A serum immunoglobulin concentrations are [1]). Although oncentrations ar
[1]).edinlthough serumanimmunoglobuint
appears to be limited to T lymphocyte-dependent antigens [3], implying no defect in B lymphocyte function per se. There is no clear hypothesis to explain why cell-mediated immunity declines with age. Thymic atrophy alone is unlikely to account for immunesenescence, as thymic weight and volume change little between the ages of 5 and 84 years [4]. Immunesenescence could be due to a T cell dysfunction, or due to associated extrinsic factors, such as deficient accessory cell function, a changing balance of helper and suppressor cells or excessive suppression by monocyte-derived factors (e.g. by prostaglandin E2). Several recent studies have focused on IL-2 secretion and membrane expression ofthe IL-2 receptor (IL-2R) [5-7]. These reports suggest that age-related T cell proliferative abnormality is due to defects in IL-2 secretion, expression of high affinity IL-2R and transcription of IL-2R mRNA [7]. As evidence relating to immunesenescence points to a defect or defects preceding IL-2 production and up-regulation of IL-2R, it is possible that such defects arise as a defect in the membrane transduction of mitogenic signals. This might be due
to the subnormal generation of inositol trisphosphate (IP3) and diacylglycerol (DG) second messengers, the respective mediators of intracellular calcium fluxes and activation of protein PC 89.Teei vdnei upr fti kns
8,9.Teeievdnensuprofts
.iaeC hypothesis from murine models, showing that mitogen-evoked fluxes in intracellular free Ca2+ concentration ([Ca2+]i) decline in splenocytes with age [10]. However, a recent study in humans has suggested that [Ca2 +] fluxes remain normal in T cells in the . . elel [1 Thus, ipsb tha seondfessenger producbt PC
a e tion aisimpairedwbutrtat.th eves. tream anormalitiesi In order to test these hypotheses, we have studied a carefully
selected group of healthy elderly subjects, in order to investigate T cell mitogen-induced [Ca.2 +1, fluxes and the effects of pharmacological activation of PKC by means of phorbol ester [12]. Here
report th ahough [Caer ron tohorbolester wer
flxe remainedunimaied, T b d
SUBJECTS AND METHODS
Subjects All subjects were living in the community and were selected by the rigorous criteria of the SENIEUR protocol [13] to exclude sub-clinical or other disease as a confounding variable. In addition to the protocol criteria, subjects were tested on at least two occasions 1 month apart to ensure all were in a steady state. Subjects were excluded if they had taken any medication,
Correspondence: L. K. Trejdosiewicz, Department of Clinical Medicine, C.S.B. Level 7, St James's University Hospital, Leeds LS9 7TF, UK.
158
Defective phorbol ester responses in the elderly
159
prescribed or otherwise, during the month of screening or between screening and study. 'Aged' donors were all > 75 years, and the 'young' donor control group was selected from laboratory and hospital staff volunteers aged
