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Defect in ERK2 and p54^JNK Activation in Aging Mouse Splenocytes

^a Center for Gerontological Research, MCP-Hahnemann University, Philadelphia, Pennsylvania
^b Department of Microbiology and Immunology, MCP-Hahnemann University, Philadelphia, Pennsylvania
^c Cell and Molecular Biology Program, Institute for Biomedical Sciences, School of Medicine, University of Chile, Santiago

Felipe Sierra, Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096 E-mail: sierraf{at}mlhs.org.

Decision Editor: Edward J. Masoro, PhD

	Abstract

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We have previously reported on a defect in both extracellular signal-regulated protein kinase (ERK) and c-jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) activation in splenocytes obtained from old rats. In order to investigate whether these effects are conserved across species, we have now used mouse splenocytes to measure the effect of aging on the activation of the same two MAPK families: ERK and JNK. Our results demonstrate that, as in rats, both MAPK signal transduction pathways are affected by aging in mice, indicating the existence of a further defect located downstream of the receptor-proximal events. Whereas ERK1 and p46^JNK activation were not significantly modified, the kinetics of both ERK2 and p54^JNK activation and inactivation were affected in splenocytes from old animals. Specifically, by analyzing the kinetics of activation and inactivation of these enzymes, we found a nearly 50% decrease in the fold of activation of both ERK2 and p54^JNK. These defects result in an overall diminution of enzyme activities without changes in the steady-state levels of relevant proteins. The impaired activity of these two MAPK pathways is likely to play a role in the reduced expression of interleukin-2 and diminished lymphoproliferation observed in old animals.

T-CELL activation and proliferation require both the T-antigen receptor and costimulatory molecules. The early events after T-cell stimulation lead to the activation of several protein tyrosine kinases (PTKs) of the src family, such as Lck and Fyn, as well as the zeta-chain-associated protein kinase ZAP-70 ⁽¹⁾. These events result in the recruitment and phosphorylation of anchoring proteins, which indirectly couple to phospholipase C (PLC) activation, resulting in increased levels of intracellular calcium and activation of protein kinase C (PKC) ⁽²⁾. The activation of PTKs also leads to the recruitment of adaptor proteins such as Shc, growth factor receptor-bound protein 2 (Grb2), and son of sevenless (Sos) to the cytoplasmic membrane. Sos, a guanine exchange factor, induces Ras activation resulting in the activation (via Raf-1 and MEK) of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (ERK) ⁽³⁾. It has been described that activation of the ERK pathway is required for interleukin-2 (IL-2) gene induction, and IL-2 is the major growth factor in T cells ⁽⁴⁾.

Evidence for the requirement of ERK activity for IL-2 gene expression comes from experiments in Jurkat cells, where overexpression of ERK isoforms leads to increased IL-2 gene expression, whereas overexpression of a dominant-negative mutant of Raf-1 results in diminished IL-2 expression ⁽⁵⁾. Furthermore, it has been shown that inhibition of the ERK pathway in T-cell clones challenged with antigen also results in a decrease in IL-2 mRNA levels ⁽⁶⁾.

Activation of T lymphocytes also requires the activity of another member of the serine/threonine MAPK family, c-jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) ⁽¹⁾. Whereas ERK activation only requires the T-cell receptor (TCR) stimulus, JNK activation is synergistic upon costimulation of both the TCR and CD28 receptors ⁽⁷⁾. The JNKs are encoded by at least three separate genes, each of which can give rise to several isoforms by alternative splicing. JNK3 is specifically expressed in brain, and each one of the other genes, JNK1 and JNK2, may originate isoforms of 54 and 46 kDa ⁽⁸⁾. They are activated preferentially by cellular stress, but also by growth factors and G protein-coupled receptor agonists ⁽⁹⁾. The JNK pathway is distinct but parallel to the pathway responsible for activation of ERK ^(10)(11). It is clear that the activation of both MAPK pathways is required for maximal IL-2 gene expression.

The immune response is compromised in aged individuals, and this occurs at several different levels ^{(12)(13)(14)(15)(16)(17)(18)(19)}. The most consistent change with increasing age is a decrease in the proliferative response of T cells in response to a variety of stimuli ^(12)(13). Most but not all researchers also find a decrease in IL-2 production (reviewed in ^{(1)(14)(16)(17)(18)(19)}). Miller has argued that the disagreements in the literature are possibly the result of differences in the method of activation used ⁽¹⁹⁾. A series of studies suggest that age-related disturbances in signal transduction pathways might explain the diminished IL-2 gene expression. It has been shown that tyrosine phosphorylation of Shc, Fyn, and ZAP-70 are all diminished in anti-CD3-stimulated T cells from old mice ⁽²⁰⁾ and humans ⁽²¹⁾. In contrast, in ConA-stimulated T cells from rats, an age-related defect was observed only on the activation of Lck and ZAP-70, but not Fyn ⁽²²⁾. These results suggest the existence of a defect in the early steps of T-cell activation. Additional studies performed in aged murine and human T cells have shown an age-related impairment in the activation of both Ras and Raf-1 ^(23)(24), again suggesting age-related impairments located upstream of these molecules within the pathway. Kinetic studies show that the age-related impairments represent reductions in both the level and duration of MAPK activation ^{(25)(26)(27)(28)(29)(30)}.

The early events in MAPK signal transduction can be circumvented through the use of phorbol 12-myristate 13-acetate (PMA) plus the Ca⁺² ionophore (CaI) A23187, which together act as effective comitogens for some species of lymphocytes ⁽³¹⁾. It has been shown, however, that the age-related defect in immunoproliferation is still apparent when cells are stimulated with PMA plus CaI ⁽³²⁾, and this suggests that a further defect in signaling might be located downstream of the TCR-proximal events. It is important to note, however, that although activation of total splenocytes by means of the TCR/CD3 complex results in specific activation of T lymphocytes, the use of PMA plus CaI is likely to activate other cell populations as well, including B cells ⁽³³⁾. Indeed, it has been reported that stimulation of human B cells with either CaI or PMA alone can induce Tyr phosphorylation, as well as the enzymatic activation of ERK ⁽³⁴⁾. Similarly, JNK activity is increased after both CD40 and B-cell receptor ligation ⁽³⁵⁾. Using mouse splenocytes as a model, we have used PMA plus CaI to further investigate the existence of age-related defects in signal transduction pathways located downstream of the receptor-proximal defects already described. Studies on the effect of age on PMA plus CaI-induced activation of T cells have generally used proliferation as an end point. We chose to assess the changes directly in ERK and JNK MAP kinase activities in response to PMA plus CaI stimulation. In rats, we have observed an age-related decrease in these activities ⁽²⁸⁾. We extend these results to the mouse model, and further we show here that the kinetics of activation of both pathways is affected by aging in mouse splenocytes. In addition, our results indicate that not all isoforms of these enzymes are equally affected by the aging process.

	Methods

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Mice
C57BL/6 male mice were used for all experiments. They were purchased from the National Institute on Aging colony of the Charles River Laboratory, and upon arrival, they were housed in our specific pathogen-free facility for at least 2 weeks before use. The mice received food and water ad libitum. The mean life span of male C57BL/6 mice is approximately 24 months. In this study, young or old refers to ages of 4–6 months or 24–26 months, respectively. The young animals are therefore sexually mature at the time of assay.

In all experiments, aliquots of the cells were used to assess proliferative capacity in response to 4 µg/ml ConA. All pools of splenocytes from old animals demonstrated a diminished proliferation in this assay, ranging between 35% and 62% of the level observed in matched samples from young animals (data not shown). This is in accordance with data from the literature ^(36)(37)(38).

Spleen Lymphocyte Isolation
Animals were sacrificed by cervical dislocation. The spleens from three animals, either young or old, were pooled and homogenized in a Dounce homogenizer in Roswell Park Memorial Institute (RPMI) 1640 (BioWhittaker, Walkersville, MD). The cell suspension was then carefully overlaid on Histopaque 1083 (Sigma, St. Louis, MO), centrifuged at 800 x g for 20 minutes at room temperature, and the splenic mononuclear cells were carefully collected from the media/Ficoll interphase and resuspended in RPMI 1640.

Cell Stimulation and Whole Cell Extract Preparation
After one wash in RPMI, the splenocytes were resuspended at 5 x 10⁶ cells/ml in RPMI without serum, and cells were allowed to equilibrate at 37°C, 5% CO₂ for 1–2 hours. Before stimulation, 5% fetal bovine serum was added, and the cells were stimulated with PMA (50 ng/ml) plus the CaI A23187 (1 µg/ml) for the indicated times.

For whole cell extracts (WCE) to be prepared, harvested cells were suspended in lysis buffer as described by Li and colleagues ⁽²⁸⁾, shaken at 4°C for 30 minutes, and cleared by centrifugation. The supernatant (WCE) was collected and protein concentration was measured by the Bradford assay as commercialized by BioRad (Hercules, CA).

Western Blot Analysis
Aliquots (30 µg) of WCE from each time point were used for a Western blot analysis. Samples were denatured by boiling in sodium dodecyl sulfate (SDS) loading buffer, reduced with 5% ß-mercaptoethanol, and fractionated in 10% or 12.5% SDS-polyacrylamide gel electrophoresis mini gels. Proteins were then transferred to nitrocellulose filters (BA 85, Schleicher & Schuell, Keene, NH); the membranes were blocked with bovine serum albumin (BSA) buffer (1% BSA, 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature and probed with horseradish peroxidase conjugated antiphosphotyrosine monoclonal antibody (Transduction Laboratories, Lexington, KY) for 1 hour at room temperature. After the samples were washed three times for 15 minutes in TNT buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20), detection was performed by using the enhanced chemiluminescence (ECL) system from Amersham (Arlington Heights, IL), according to the instructions supplied by the manufacturer.

To measure steady-state levels of kinases, we used 30 µg of WCE of unstimulated mouse splenocytes prepared from four individual animals of each age. The protocol was analogous to the previous one, except that after the transfer, the membrane was blocked three times for 15 minutes with 5% nonfat dry milk (Carnation, Nestlé USA, Solon, OH) prepared in a buffer of 25 mM Tris, pH 7.5, 1 mM ethylenediamine tetra-acetic acid, and 140 mM NaCl (TEN buffer). Membranes were then probed with specific primary antibodies (Transduction Laboratories) overnight at 4°C, followed by three washes of 20 minutes each in 3% nonfat milk in TEN. The blot was then exposed to peroxidase-conjugated goat antimouse IgG secondary antibody for approximately 1.5 hours at room temperature and then washed extensively in TEN buffer. Detection was performed by using ECL as before. Before reprobing with a new antibody, the blot was stripped by shaking for 30 minutes at 50°C in a solution containing 62 mM Tris (pH 6.7), 2% SDS, and 100 mM ß-mercaptoethanol. Then the membrane was extensively washed in TEN buffer and reprobed with another antibody. As a normalization control, we probed the membrane with antiactin antibody (ICN; Biomedical Research Products, Costa Mesa, CA).

Western blot analysis with the antiactive ERK and antiactive JNK antibodies was performed exactly as suggested by the manufacturer (Promega, Madison, WI).

"In gel" Kinase Assays
Ten or 30 µg of WCE were used for the ERK and JNK kinase assays, respectively, according to the procedures described previously ⁽²⁸⁾.

Data Acquisition and Analysis
Films from appropriately short exposure times were scanned and quantitated using the ImageQuaNT software analysis program (Molecular Dynamics, Sunnyvale, CA). The values represent the mean ± standard error obtained from three independent pools of splenocytes. The nonparametric Kruskal–Wallis test was used in the analysis of nonrelated samples (i.e., young versus old), whereas the Friedman and Quade test was used for related samples (i.e., time course experiments). For statistical significance, we considered as significant a value of p < .05.

	Results

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Spleen Cells Show Diminished Proliferation and an Impairment in Tyrosine Phosphorylation
In this study, we measured ERK and JNK activities in total unfractionated mouse splenocytes. These represent a complex population containing approximately 25–30% T cells, 60–65% B cells, and approximtely 5% macrophages and natural killer cells. In order to characterize the impairment in proliferation previously described in cells from aging subjects, we stimulated spleen cells obtained from young or old mice. In these experiments, we observed that spleen cells from older mice demonstrate an approximately 47% (p < .05) lower proliferative potential than cells obtained from young animals. Furthermore, and in agreement with previous studies, we observed an age-related decline in total tyrosine phosphorylation ⁽³³⁾. Fig. 1 shows the kinetics of tyrosine phosphorylation of proteins in the 40- to 60-kDa range of molecular weight in pools of mouse splenocytes treated for various times with PMA plus A23187. As shown in this figure, stimulation of cells obtained from young animals leads to a rapid and strong induction of phosphorylation of two proteins in the apparent molecular weight range of 42–44 kDa. This event is diminished in cells obtained from old animals. The apparent molecular weight of these species suggests that they might correspond to ERK2 and ERK1.

View larger version (64K): [in this window] [in a new window]   Figure 1. Protein tyrosine phosphorylation in response to phorbol 12-myristate 13-acetate plus calcium ionophore induction is diminished in splenocytes from old mice. Pools of splenocytes isolated from three young and three old C57BL/6 mice were induced with phorbol 12-myristate 13-acetate (50 ng/ml) plus A23187 (1 µg/ml) for the times indicated. Aliquots (30 µg) of the whole cell extract were electrophoresed on a 12.5% sodium dodecyl sulfate polyacrylamide gel and transferred to nitrocellulose membranes. The stimulation time (in minutes) is indicated. A: Immunodetection was performed with horseradish peroxidase conjugated anti-P-Tyr antibody. Binding was detected by an enhanced chemiluminescence system. B: Both extracellular signal-regulated protein kinase (ERK) isoforms (ERK1 and ERK2) were detected by Western blot analysis with anti-pan-ERK antibody, followed by horseradish peroxidase-conjugated secondary antibody.

View larger version (36K): [in this window] [in a new window]   Figure 2. Extracellular signal-regulated protein kinase (ERK) activation in response to phorbol 12-myristate 13-acetate plus calcium ionophore induction is diminished in splenocytes from old mice. A: Aliquots of whole cell extract (10 µg) from the same experiment shown in Fig. 1 were used to measure ERK activity by the in gel kinase assay. B: ERK activity was measured by Western blot analysis of the same membrane, using an anti-active ERK antibody. In both cases, the results are representative of three independent experiments. The phosphorylated forms of ERK are indicated (P-ERK1 and P-ERK2). C: Activation of ERK2 but not ERK1 is impaired in aging splenocytes. Pools of splenocytes (three animals/pool of each age) were induced as in Fig. 1 for the indicated times. Aliquots of whole cell extract (10 µg) were used to measure ERK activity by the in gel kinase assay. The bands corresponding to the 44-kDa ERK1 and the 42-kDa ERK2 were quantitated from three independent experiments. Values are mean ± standard deviation, and values have been standardized against the basal level of activity at time zero; •, young; , old. Similar results were obtained if the raw data (not standardized) are plotted (data not shown).

Whereas the data presented, and that in the literature, indicate that ERK activity is rapidly induced in lymphocytes, we next tested the kinetics of disappearance of ERK activity for periods up to 4 hours. The results (Fig. 2) indicate that the induction of both ERK activities in response to PMA plus CaI is quite persistent and easily detectable above basal levels for at least 4 hours in animals of both age groups. In contrast, T-cell stimulation with either anti-CD3 or anti-CD3 plus PMA leads to ERK activity that starts to decline by 10–15 minutes in both humans and mice ^(23)(38). Our results further indicate that age does not significantly affect the degree of activation of ERK1 in splenocytes exposed to PMA plus CaI. In contrast, compared with that of younger counterparts, ERK2 activity is significantly diminished in splenocytes from old animals at all times examined, with an average diminution of 49 ± 3% (p < .01). Furthermore, as in the case of ERK1, ERK2 activity in young animals reaches a maximum at 15 minutes and then declines slowly. It should be noted that in these cells, ERK2 activity is more predominant than ERK1 (Fig. 2 and Fig. 2).

JNK Activities Are Also Differentially Impaired During Aging
Because JNK, but not ERK, activity requires the synergistic effects of both PMA and A23187, or, in vivo, the costimulation of both the TCR and CD28 pathways ⁽⁷⁾, we wanted to explore whether JNK activation is also affected by age. To test this possibility, we measured JNK activity both by in gel kinase assay and antiactive JNK antibodies. Fig. 3 shows a representative example of the kinetics of JNK activation, as measured by Western blot using antiactive JNK antibodies. The results indicate that, like ERK activity, JNK activity is induced in response to PMA plus A23187, and this activity is diminished during aging. Similar results were obtained using the in gel kinase assay for JNK. Therefore, our results suggest that the defect we observe in JNK activation most likely represents a reduction in signal transduction within the pathway. Fig. 3 indicates the equal loading of the gel used in Fig. 3, as demonstrated by the similar intensity of the p46^JNK protein signal.

View larger version (40K): [in this window] [in a new window]   Figure 3. The c-jun N-terminal protein kinase (JNK) activation in response to phorbol 12-myristate 13-acetate plus calcium ionophore induction is diminished in splenocytes from old mice. A: JNK activity was measured by Western blot analysis of the same membranes used in Fig. 2, using anti-active JNK antibody. The phosphorylated forms of JNK are indicated (p46 and p54). B: The same membrane was probed with anti-JNK1 antibody, followed by horseradish peroxidase-conjugated secondary antibody. C: Activation of p54JNK but not p46JNK is impaired in aging splenocytes. JNK activity was measured in triplicate by in gel kinase assays (three animals/pool of each age). The bands corresponding to the 54-kDa p54JNK and the 46-kDa p46JNK were quantitated. Values are mean ± standard deviation, and they have been standardized against the basal level of activity at time zero; •, young; , old. Similar results were obtained if the raw data (not standardized) are plotted (data not shown).

The Steady-State Level of the Kinases Is Not Affected by Aging
It is generally believed that the activity of MAPK signal transduction pathways is controlled primarily at the level of phosphorylation and dephosphorylation ⁽³⁹⁾. However, it is clear that changes in the abundance of these proteins could also affect the activity of the pathways ⁽⁴⁰⁾. Therefore, we have used Western blot analysis to establish whether or not the steady-state level of different MAPK is affected by age in mouse splenocytes. For this, we have used total cellular extracts prepared from unstimulated splenocytes from four individual mice of each age group. The membrane was probed sequentially with anti-ERK1, ERK2, JNK1, and p38 antibodies. The results presented in Fig. 4 indicate that there are no significant age-dependent changes in the steady-state level of any of these proteins. Thus we conclude that the changes in activity cannot be ascribed to variations in the steady-state level of the kinases, but rather, either to their degree of activation by upstream effectors, or to their inactivation by relevant phosphatases. The steady-state levels of these other relevant molecules have yet to be investigated.

View larger version (14K): [in this window] [in a new window]   Figure 4. The steady-state level of mitogen-activated protein kinase proteins is not affected by age in mouse spleen lymphocytes. As a way to determine the steady-state levels of different signal transduction-related proteins, aliquots (30 µg) of whole cell extract obtained from unstimulated splenocytes obtained from individual young and old mice (n = 4 for each age) were subjected to Western blot analysis as described in the Methods section. The membrane was probed sequentially with anti-extracellular signal-related protein kinase (ERK)1, ERK2, c-jun N-terminal protein kinase (JNK)1, and p38 antibodies. The values have been standardized against ß-actin (from antiactin antibody), whose levels do not change significantly as a function of age, and data are presented relative to the mean level observed in splenocytes from young animals, which were arbitrarily given a value of 1. The values given represent the mean ± standard deviation for young (black bars) and old (open bars) animals.

	Discussion

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In agreement with previous reports, we found a decreased proliferative capacity in spleen cells obtained from old animals than in those obtained from young animals. Furthermore, our results indicate that splenocytes from old mice present a defect within the two MAPK pathways studied (ERK and JNK), which is located downstream of receptor activation. In the case of ERK, we observed a marked diminution of nearly 50% in ERK2 activity, whereas ERK1 activity showed no decrease, but only a little significant decline in maximal activation. Although this impairment in ERK activation was observed in unfractionated spleen cells, our results are in agreement with the impairment in ERK activity observed in mouse CD4⁺ T cells (⁽¹⁹⁾; for review see 24). Furthermore, these results are also consistent with other reports showing an age-related decrease in ERK activity in other cell types, such as hepatocytes ^{(41)(42)(43)(44)}.

A decrease in JNK activation as shown here has also been described in human ^(23)(27) and murine T cells ^(29)(30). In this pathway we also found a series of defects, including a reduction in the activity of p54^JNK, with a much smaller (and not statistically significant) reduction in the activity of p46^JNK. For both isoforms, we observed an early deactivation in comparison with young animals. Our results are in agreement with data previously reported by Kirk and Miller ^(29)(30). They detected a reduction in JNK activation that was specifically observed after proliferative stimuli, but not after stress-induced activation of lymphocytes ⁽³⁰⁾. Our results suggest that the different isoforms of ERK and JNK might be activated and inactivated by means of different mechanisms. By using knock-out animals, Kane and colleagues ⁽¹⁾ and Pawelec and Solana ⁽⁴⁵⁾ recently described that each MAPK isoform might have a different function during T-cell development ^(1)(45).

Although there is considerable disagreement with respect to Ras activation in T cells from old mice and rats ^(23)(24)(30), our protocol avoids this step in the pathway by bypassing Ras activation. Under these conditions, our results demonstrate a defect located downstream, possibly at the level of ERK itself. A similar conclusion can be drawn for the JNK pathway. Alternatively, the defect might be controlled by age-related changes in the levels and activity of MAPK phosphatases, and we are currently exploring this possibility. An interesting point is the analogy that exists between previous and our observations and T-cell anergy ^(26)(28)(46). Indeed, it has been reported that incomplete stimulation of T cells leads to a state of unresponsiveness (anergy), characterized by a decrease in both Ras and ERK activities, without affecting the protein levels of these proteins ^(47)(48).

By having the full complement of splenic cells, our model recapitulates rather faithfully the conditions found in vivo during lymphocyte activation. However, a clear problem arises from the use of relatively nonspecific inducers such as PMA, plus A23187 treatment leads to a similar level of activation in both cases. The effect of aging on both of these main populations of lymphocytes has been well studied. There is currently little evidence for a dramatic change in the number or type of B lymphocytes with aging in either human blood ⁽⁴⁹⁾ or mouse spleen ⁽⁵⁰⁾, but changes in the proportion of B-cell subsets have been reported (reviewed in ⁽¹⁹⁾). In the case of T cells, the main age-related change described is a dramatic shift in the proportion of naive and memory T cells within the CD4⁺ and CD8⁺ pools ⁽⁵¹⁾. This has been described in both mice and humans. Recently, it has been reported that the age-associated accumulation of memory T cells could be the result of chronic activation of PKC, which should promote the generation of memory T cells and inhibit the elimination of previously generated memory cells ⁽⁵²⁾. Interestingly, it has been observed that naive and memory CD4⁺ T cells from young mice are equally able to convert ERK2 to its slower migrating form in response to anti-CD3 stimulation ⁽²⁰⁾.

In summary, although the defects we have observed are likely to represent changes in all or most of the cell types present in our mixed cell population, a dramatic functional and phenotypic change during aging has been observed primarily in the T-cell compartment ^(13)(49). For this reason, we believe that the defects in signal transduction we have observed are more physiologically relevant in these cells. In contrast, it is clear that proliferation and differentiation of both cell types is critical to effective humoral and cell-mediated immune responsiveness to pathogens and perhaps tumors. Therefore, we expect our studies to be of high significance to our further understanding of immunosenescence and the medically relevant immunodeficiency in the old.

	Acknowledgments

 
This work was supported by Grants AG14232 and AG 13902 from the National Institute on Aging. We are gratefully indebted to our colleagues Drs. Bruce Troen, Mary Kay Francis, and Craig Volker for critical reading of the manuscript.

Received February 16, 2001

Accepted September 2, 2001

	References

Top Abstract Methods Results Discussion References

 

1. Kane LP, Lin J, Weiss A, 2000. Signal transduction by the TCR for antigen. Curr Opin Immunol 12:242-249. [Medline]
2. Myung PS, Boerthe NJ, Koretzky GA, 2000. Adapter proteins in lymphocyte antigen-receptor signalling. Curr Opin Immunol 12:256-266. [Medline]
3. Genot E, Cantrell DA, 2000. Ras regulation and function in lymphocytes. Curr Opin Immunol 12:289-294. [Medline]
4. Crabtree GR, 1989. Contingent genetic regulatory events in T lymphocyte activation. Science 243:355-361. [Abstract/Free Full Text]
5. Owaki H, Varma R, Gillis B, et al. 1993. Raf-1 is required for T cell IL2 production. EMBO J 12:4367-4373. [Medline]
6. DeSilva DR, Jones EA, Favata MF, et al. 1998. Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy. J Immunol. 160:4175-4181. [Abstract/Free Full Text]
7. Su B, Jacinto E, Hibi M, Kallunki T, Karin M, Ben-Neriah Y, 1994. JNK is involved in signal integration during costimulation of T lymphocytes. Cell 77:727-736. [Medline]
8. Kyriakis JM, Avruch J, 2001. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807-869. [Abstract/Free Full Text]
9. Minden A, Karin M, 1997. Regulation and function of the JNK subgroup of MAP kinases. Biochim Biophys Act 1333:F85-F104. [Medline]
10. Hibi M, Lin A, Smeal T, Minden A, Karin M, 1993. Identification of an oncoprotein and UV responsive kinase that binds and potentiates the activity of the c-jun activation domain. Genes Dev 7:2135-2148. [Abstract/Free Full Text]
11. Deacon K, Blank JL, 1997. Characterization of the mitogen-activated protein kinase kinase 4(MKK4)/c-junNH₂-terminal kinase 1 and MKK3/p38 pathways regulated by MEK kinase 2 and 3. MEK kinase 3 activates MKK3 but does not cause activation of p38 kinase in vivo. J Biol Chem 272:14,489-14,496. [Abstract/Free Full Text]
12. Kay MMB, Mendoza J, Diven J, Denton T, Union N, Lajiness N, 1979. Age-related changes in the immune system of mice of eight medium and long-lived strains and hybrids. I. Organ, cellular, and activity changes. Mech Ageing Dev 11:295-346. [Medline]
13. Makinodan T, Kay MM, 1980. Age influence on the immune system. Adv Immunol 29:287-330. [Medline]
14. Murasko DM, Goonewardene IM, 1990. T-cell function in aging: mechanism of decline. Ann Rev Gerontol Geriatr 10:71-96. [Medline]
15. Pawelec G, Warner W, Adibzadeh M, Engel A, 1999. T cell immunosenescence in vitro and in vivo. Exp Gerontol 34:419-429. [Medline]
16. Pahlavani MA, Richardson A, 1996. The effect of age on the expression of interleukin-2. Mech. Ageing Dev 89:125-154. [Medline]
17. Pahlavani MA, 1998. T cell signaling: effect of age. Front Biosc 3:d1120-d1133.
18. Pawelec G, Effros RB, Caruso C, Remarque E, Barnett Y, Solana R, 1999. Tcell and aging. Front Biosc 4:d216-d269.
19. Miller RA, 2000. Aging and immune function. Paul WE, , ed.Fundamental Immunology 947-966. Lippincott-Raven, Philadelphia.
20. Ghosh J, Miller RA, 1995. Rapid tyrosine phosphorylation of Grb2 and Shc in T cells exposed to anti-CD3, anti-CD4, and anti-CD45 stimuli: differential effects of aging. Mech Ageing Dev 80:171-187. [Medline]
21. Whisler RL, Chen M, Liu B, Newhouse YG, 1999. Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorilations of -chains and p59^fyn/p65^lck in human T cells. Mech Ageing Dev 111:49-66. [Medline]
22. Pahlavani MA, Harris MD, Richardson A, 1998. Activation of p21^ras/MAPK signal transduction molecules decreases with age in mitogen-stimulated T-cells from rats. Cell Immunol 185:39-48. [Medline]
23. Liu B, Carle KW, Whisler RL, 1997. Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals. Cell Immunol 82:79-88.
24. Kirk CJ, Miller RA, 1998. Analysis of Raf-1 activation in response to TCR activation and costimulation in murine T-lymphocytes: effect of age. Cell Immunol 190:33-42. [Medline]
25. Whisler RL, Newhouse YG, Bagenstone SE, 1995. Age-related reductions in the activation of mitogen-activated protein kinase P44^mapk/ERK1 and P42^mapk/ERK2 in human T cells stimulated via ligation of the T cell receptor complex. Cell Immunol 168:201-210.
26. Gorgas G, Butch ER, Guan KL, Miller RA, 1997. Diminished activation of the MAP kinase pathway in CD3-stimulated T lymphocytes from old mice. Mech Ageing Dev 94:71-83. [Medline]
27. Beiqing L, Carle KW, Whisler RL, 1997. Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals. Cell Immunol 182:79-88. [Medline]
28. Li M, Walter R, Torres C, Sierra F, 2000. Impaired signal transduction in mitogen activated rat splenic lymphocytes during aging. Mech Ageing Dev 113:85-99. [Medline]
29. Kirk CJ, Freilich AM, Miller RA, 1999. Age-related decline in activation of JNK by TCR- and CD28-mediated signals in murine T-lymphocytes. Cell Immunol 197:75-82. [Medline]
30. Kirk CJ, Miller RA, 1999. Age-sensitive and -insensitive pathways leading to JNK activation in mouse CD4(+) T-cells. Cell Immunol 197: (2) 83-90. [Medline]
31. Larsen CS, 1990. Activation of human T lymphocytes by phorbol-12, 13-dibutyrate and ionomycin. Scand J Immunol 3:353-360.
32. Utsuyama M, Wakikawa A, Tamura T, Nariuchi H, Hirokawa K, 1997. Impairment of signal transduction in T cells from old mice. Mech Ageing Dev 93:131-144. [Medline]
33. Franklin RA, Tordai A, Mazer B, Terada N, Lucas JJ, Gelfand EW, 1994. Activation of MAP2-kinase in B lymphocytes by calcium ionophores. J Immunol 153:4890-4898. [Abstract]
34. Miller RA, 1996. The aging immune system: primer and prospectus. Science. 273:70-74. [Abstract]
35. Li Y, Baccam M, Waters SB, Pessin JE, Bishop GA, Koretzky GA, 1996. CD40 ligation results in protein kinase C-independent activation of ERK and JNK in resting murine splenic B cells. J Immunol 157:1440-1447. [Abstract]
36. Furukawa T, Meydani SN, Blumberg JB, 1987. Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech Ageing Dev 38: (2) 107-117. [Medline]
37. Kirschmann DA, Murasko DM, 1992. Splenic and inguinal lymph node T cells of aged mice respond differently to polyclonal and antigen-specific stimuli. Cell Immunol 139:426-437. [Medline]
38. Shi J, Miller RA, 1992. Tyrosine-specific protein phosphorylation in response to anti-CD-3 antibody is diminished in old mice. J Gerontol Biol Sci 47:B147-B153.
39. Marshall CJ, 1994. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Op Genet Dev 4:82-89. [Medline]
40. Torres C, Li M, Walter R, Sierra F, 2000. Modulation of the ERK pathway of signal transduction by cysteine proteinase inhibitors. J Cell Biochem 80: (1) 11-23. [Medline]
41. Xu X, Bennett SA, Ingram RL, Sonntag WE, 1995. Decreases in growth hormone receptor signal transduction contribute to the decline in insulin-like growth factor I gene expression with age. Endocrinol 136:4551-4557. [Abstract]
42. Liu Y, Guyton KZ, Gorospe M, et al. 1996. Age-related decline in mitogen-activated protein kinase activity in epidermal growth factor-stimulated rat hepatocytes. J Biol Chem 271:3604-3607. [Abstract/Free Full Text]
43. Palmer HJ, Tuzon CT, Paulson KE, 1999. Age-dependent decline in mitogenic stimulation of hepatocytes. J Biol Chem 274: (16) 11,424-11,430. [Abstract/Free Full Text]
44. Zhen X, Uryu K, Cai G, Gerard PJ, Friedman E, 1999. Age-associated impairment in brain MAPK signal pathways and the effect of caloric restriction in Fischer 344 rats. J Gerontol Biol Sci 54A:B539-B548. [Abstract]
45. Pawelec G, Solana R, 1997. Immunosenescence. Immunol Today 18: (11) 514-516. [Medline]
46. Li W, Whaley CD, Mondino A, Mueller DL, 1996. Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells. Science 271:1272-1276. [Abstract]
47. Fields PE, Gajewski TF, Fitch FW, 1996. Blocked ras activation in anergic CD4+ T cells. Science. 271:1276-1278. [Abstract]
48. Telander DG, Malvey EN, Mueller DL, 1999. Evidence for repression of IL-2 gene activation in anergic T cells. J Immunol 162:1460-1465. [Abstract/Free Full Text]
49. Utsuyama M, Hirokawa K, Kurashima C, et al. 1992. Differential age-changes in the numbers of CD4⁺ CD45 RA⁺ and CD4⁺ CD 29⁺ T cell subsets in human peripheral blood. Mech Ageing Dev 63:57-68. [Medline]
50. Subbarao B, Morris J, Kryscio RJ, 1990. Phenotypic and functional properties of B lymphocytes from aged mice. Mech Ageing Dev 51:223-241. [Medline]
51. Miller RA, 1994. Nathan Shock Memorial Lecture 1992 aging and immune function: cellular and biochemical analyses. Exp Gerontol 29:21-25. [Medline]
52. Ohkusu K, Du J, Isobe KI, et al. 1997. Protein kinase C-mediated chronic signal transduction for immunosenescence. J Immunol 159:2082-2084. [Abstract/Free Full Text]

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