Mohammad A. Pahlavani

Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System and Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78284

Received 10/4/98 Accepted 10/12/98

4. AGE-RELATED CHANGES IN SIGNAL TRANSDUCTION IN T CELL

4.1 Calcium signaling and second messenger generation

Activation of T cells results in a transient increase in intracellular free calcium ion concentrations [Ca²⁺]_i. The rise in [Ca²⁺]_i results from the release of intracellular stores and also from the influx of extracellular Ca²⁺. Several studies have been focused on the effect of age on either total levels of intracellular calcium by using a calcium probe (fluorochrome indo-1) or the influx of extracellular Ca²⁺ by radiolabeled calcium (⁴⁵Ca²⁺). These studies which are listed in table 1 indicate that the induction of calcium signal generation is altered with age in mice and humans. For example, an early study showed that the basal level of intracellular calcium was slightly higher in T cells from old mice than T cells from young mice and that the induction of intracellular calcium level by mitogen did not change with age (113). Another report demonstrated that resting T cells from old mice had more uptake of Ca²⁺ than the resting T cells from young mice. However, the induction of Ca²⁺ uptake was less for T cells from old mice than T cells from young mice (115). In one study, it was shown that the induction of the intracellular calcium level by anti-CD3 but not by PHA, was lower in T cells from old mice compared to T cells from young mice (118).

In contrast to the reported studies in mice, the data in humans on effect of age on calcium signaling in T cells have not been consistent. For example, two studies indicated no age-related changes in the uptake of Ca²⁺ in resting or PHA-stimulated peripheral blood lymphocytes in humans (132,136). However, another report showed that the induction of [Ca²⁺]_i by anti-CD3 and by PHA decreased in T cells from elderly humans subjects compared to T cells from young subjects (133-135). One question that has been addressed is whether the calcium signal generation is faulty in all T cell populations from old experimental subjects, or whether only certain subsets of T cell exhibit less response to calcium mobilization and others have a response similar to young subjects. In a study in mice, it was shown that among the T cell populations, the ionomycin-resistant T cell subsets were predominantly of the memory phenotype (Pgp-1^hig) and these cells were hyporesposive with respect to the helper and cytotoxic function in both young and old animals (118).

A common feature of the antigen receptor-mediated signaling is the activation of PLC-g, resulting in the hydrolysis of phosphoinositide lipids and the production of IP3 and DAG. Production of these second messengers in turn leads to the increase in the intracellular free calcium ion concentrations [Ca²⁺]_i and the activation of PKC, respectively. Therefore, it has been argued that the age-related changes in calcium signal mobilization might result from alterations in production of IP3. The initial report on the effect of age on IP3 generation indicated that the basal level of IP3 was slightly higher in splenic T cells from old mice compared to the level in young mice. However, Con A induction of IP3 generation was lower in splenic T cell from old mice than young mice (113). In contrast, another study showed no age-related difference in the basal or the induced level of IP3 in T cells from mice (115).

Table 1 lists studies on the effect of age on the generation of the second messenger DAG and the activation of PKC. The initial report showed that the basal level of total PKC activity was slightly higher in T cells from old mice, whereas the membrane and the cytoplasmic distribution of PKC were similar in T cells from both old and young mice (113). In addition, PKC translocation did not change with age when cells were stimulated with phorbol myristate acetate (PMA). However, when cells were stimulated with Con A the induction of PKC translocation was lower (50% ) in T cells from old mice than T cells from young mice (113). This study suggested that the age-related decline in PKC might reflect changes in generation of DAG. One study reported four-fold decrease in membrane-associated PKC activity in T cells from old mice compared to T cells from young mice (122). A study in humans has shown that the basal level of PKC in peripheral blood lymphocytes from elderly subjects was similar to the levels in peripheral blood lymphocytes from young subjects. However, PHA induction of PKC was significantly less in peripheral blood lymphocytes from elderly subjects compared to the level in young subjects (136). Recently, it was reported that the expression of PKC a-isomers but not b-isomers decreased in peripheral blood T cells from elderly human subjects compared to the peripheral blood T cells from young subjects (139). This study suggested that selective alteration in PKC isoenzymes in T cells during aging might contribute to alterations in intracellular signaling events.

4.2 Protein tyrosine phosphorylation and Ras/MAPK activation

Because of the ubiquitous role of protein phosphorylation in the initiation of physiological signals, it has been postulated that changes in the phosphorylation of the key proteins with age might be the primary cause of defect in T cell function. The studies listed in table 1 show that phosphorylation of various cellular proteins is impaired in T cells from humans (138,142,144) and rodents (123,124,130). In one study, it was shown that T cells from elderly humans were more susceptible to herbimycin A (a PTK inhibitor) which inhibits signal transduction. However, no age-related changes in tyrosine phosphorylation of endogenous proteins were found when cells were stimulated with pervandate (a PTK activator). The increase in sensitivity of T cells from elderly humans to PTK inhibitors was associated with a decrease in the inducibility of tyrosine phosphorylation of the endogenous protein substrate (138). This study suggested that alterations in upstream signaling events might be the underlying cause of the decline in tyrosine phosphorylation with age.

Members of the Src (Lck and Fyn) and Syk (ZAP-70) family of PTKs play a critical role in TCR-mediated signal transduction. table 1 list the studies on the effect of age on the activation of Lck, Fyn, and ZAP-70. The induction of Lck (144) and ZAP-70 (142) activity has been shown to decrease with age in humans. One study reported that Fyn activity but not Lck activity by anti-CD3< was less in T cells from elderly subjects than T cells from young subjects (140). Similarly, a recent study in mice showed that the induction of Fyn and ZAP-70 activity decreased with age in T cells (121). More recently, our laboratory reported that the kinase activities (autophsphorylation) associated with Lck and ZAP-70 but not Fyn were significantly less (by 56% and 76%, respectively) in T cells from old rats compared to T cells from young rats (130). Furthermore, our study showed that the decrease in Lck and ZAP-70 activities with age was not due to changes in their corresponding protein levels.

Stimulation of T cells through TCR/CD3 complex results in sequential phosphorylation and activation of a number of signaling molecules that eventually lead to the activation of the Ras/MAPKs signaling cascade. MAPKs in turn phosphorylate and activate a variety of regulatory proteins and transcription factors involved in regulation of cytokine genes (e.g., IL-2). Because the age-related decline in T cell function (i.e., IL-2 expression and proliferation) has been well documented and because activation of MAPK is essential for the induction of cytokine gene expression, it has been hypothesized that the age-related decline in T cell function might occur as a result of a decrease in MAPK activation. Several studies (table 1) have provided evidence in support of the view that the induction of MAPK activity decreases with age. For example, a study in humans has shown that MAPK (ERK1 and ERK2) and MEK activities decreased with age when cells were stimulated with anti-CD3, PHA, PHA plus PMA, or PMA plus ionomycin (141). In addition, it has recently been reported that the induction of Raf-1, MAPK (ERK2), and JNK activities decreased with age in humans (143). Similarly, a study in mice has shown that the induction of MAPK and MEK activity in T cells decreased with age (121,128,129). In this study, MAPK and MEK activity was assessed by monitoring the phosphorylation of the MAPK and MEK substrate in T cell lysates by the mobility shift assay (129). This study showed that a shift in mobility (phosphorylation) of one of the ERK2 substrate (the ribosomal S6 protein kinase pp90^rsk) was less in the anti-CD3 stimulated T cells from old mice compared to anti-CD3 stimulated T cells from young mice. In addition, this study indicated that the age-related decline in MAPK/MEK activation was not due to changes in the proportion of naive/virgin or memory T cell subsets because MAPK/MEK activity was equally diminished in both naive and memory helper T cells from old mice (128).

Our laboratory has recently investigated the effect of age on signal transduction in T cells from rats. Figure 3 shows data in which the induction of MAPK, JNK, and Ras activation was assessed in T cells from young (6 month) and old (24 months) F344 rats. Aging had no effect on the basal level of MAPK or JNK activity or the protein levels of these regulatory proteins. However, Con A induction of MAPK activity but not JNK activity was significantly less (by 65%) in T cells isolated from old rats compared with T cells isolated from young rats. Furthermore, we found that the age-related decline in MAPK activity was correlated with a decrease in phosphorylation of p44^MAPK protein (130).

Figure 3. Effect of age on the induction of MAPK, JNK, and Ras in T cells from F344 rats. The splenic T cells from young (6 months) and old (24 months) rats were stimulated with Con A for 5 to 10 min. Protein was isolated and the activity associated with the immunoprecipitated p44 and p42 MAPK, p46 JNK or p21ras was measured. Data were taken from Pahlavani et al. (130). The values (*) for the old rats are significantly different from the values for the young rats at p<0.001.

Why is MAPK activation reduced in aging T cells? Based on the current model (figure 1), the age-related decrease in MAPK activation could occur by at least two distinct mechanisms. First, the decrease in the activity of MAPK may be due to the upregulation of the MAPK phosphatase (MPK-1). That is, similar levels of MAPK activity are present in the activated T cells from young and old animals; but in response to stimulation, the activity of MPK-1 that is involved in dephosphorylation of MAPK increases in the T cells from old animals. Second, the decrease MAPK activity with age could arise from reduced activity of the proximal signaling molecules such as MEK or Ras. In another word, less MAPK activity is observed in T cells of old animals because less MEK or Ras activity is present in these cells. Our laboratory has addressed the question of whether Ras activation alters with age and if the changes are correlated with an alteration in the expression of p21^ras protein. The Ras activity was assessed by measuring the accumulation of GTP and GDP-bound p21^ras in the immunoprecipitated p21^ras protein in the unstimulated and Con A stimulated T cells. As shown in figure 3, the percentage of GTP-p21^ras observed in the Con A-stimulated T cells from old rats was significantly less (53%) than the level observed in T cells isolated from young rats. The age-related decrease in Ras activation was not associated with changes in the p21^ras protein level (130). This study suggests that aging alters the activation of Ras/MAPK cascade that leads to cytokine gene expression and T cell function.