Impaired signal transduction in mitogen activated rat splenic lymphocytes during aging

Mechanisms of Ageing and Development

113 (2000) 85–99

Impaired signal transduction in mitogenactivated rat splenic lymphocytes during aging

Min Li, Robin Walter, Claudio Torres, Felipe Sierra *Center for Gerontological Research, MCP Hahnemann Uni6ersity, 2900 Queen Lane, Philadelphia,

PA 19129, USA

Received 30 June 1999; received in revised form 18 October 1999; accepted 20 October 1999

Abstract

Mitogen activated protein kinases (MAPK) are activated by a wide variety of signalsleading to cell proliferation and differentiation in different cell types. With aging, there is amarked decrease in proliferation of T-lymphocytes in response to a variety of mitogens.Several age-related changes in the activation of MAPK pathways in T-lymphocytes activatedvia the T-cell receptor (TCR) have been described in different species. This way, some TCRproximal defects in tyrosine kinase activity have been delineated. In this study, we have usedrat splenic lymphocytes to measure the effect of aging on the activation of two MAP kinasefamilies: ERK and JNK. In order to bypass the receptor-proximal age-dependent defectspreviously described, we used phorbol ester (PMA) and Ca2+ ionophore (A23187) asco-mitogens. Our results demonstrate that splenic lymphocytes from old rats have adisturbance in the activation of the ERK and JNK MAPK signal transduction pathways,that are located downstream of the receptor-proximal events. At least part of the age-relateddefect leading to decreased ERK activity appears to be located upstream of ERK itself, sinceactivation of MEK is also impaired. On the other hand, the observed defects in MAPKactivation do result in decreased activation of downstream events, such as c-Jun phosphory-lation. Thus, we conclude that aging of splenic lymphocytes results in a functional decline insignal transduction, and at least some of these defects are located downstream of the

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Abbre6iations: CaI, calcium ionophore; Con A, concanavalin A; ERK, extracellular signal regulatedprotein kinase; IL-2, interleukin-2; IL-2R, IL-2 receptor; JNK, c-Jun N-terminal kinase; MAPK,mitogen activated protein kinases; PMA, phorbol-12-myristate 13-acetate; TCR, T-cell receptor; WCE,whole cell extract.

* Corresponding author. Present address: Lankenau Medical Research Center, 100 Lancaster Avenue,Wynnewood, PA 19096, USA. Tel.: +1-610-6458583; fax: +1-610-6452290.

E-mail address: [email protected] (F. Sierra)

0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

PII: S0047 -6374 (99 )00096 -2

M. Li et al. / Mechanisms of Ageing and De6elopment 113 (2000) 85–9986

receptor-proximal events previously described by others. The impaired activity of these twoMAP kinase pathways is likely to play a role in the diminished lymphoproliferation observedin old individuals. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Aging; Signal transduction; ERK; JNK; Lymphocytes

1. Introduction

Age-associated decreases in lymphoproliferation in response to mitogens such asconcanavalin A (Con A) or phorbol-12-myristate 13-acetate (PMA) plus calciumionophore (A23187) have long been observed (Kruisbeek, 1976; Kay et al., 1979;Makinodan and Kay, 1980; Holbrook et al., 1989; Goonewardene and Murasko,1993). The decreased proliferation of lymphocytes correlates with decreased IL-2production (Miller, 1995; Pahlavani and Richardson, 1996), which is required forT-cell proliferation. A significant decline in IL-2 production after Con A orphytohemagglutinin (PHA) stimulation was found in different rat strains, includingBrown Norway and F344 (Bash, 1983; Cheung et al., 1983; Goonewardene andMurasko, 1993). Unlike the consistent observation of decreased IL-2 production inmice and humans, there are some discrepancies in the literature concerning IL-2activity, as well as IL-2 mRNA levels and IL-2 receptor cell surface expression inlymphocytes from young and old rats stimulated with either Con A or PMA plusCaI. While some authors have observed a decline with age in these parameters(Thoman and Weigle, 1981; Nagel et al., 1988; Ernst et al., 1989; Pahlavani andRichardson, 1996), other have observed no differences (Wu et al., 1986; Holbrooket al., 1989; Murasko and Goonewardene, 1990). This suggests that some factorsother than decreased expression of IL-2 and IL-2 receptors might also account forthe diminished proliferative capacity of aged rat lymphocytes following mitogeninduction.

Considerable progress has been achieved in understanding the mechanisms bywhich mitogens stimulate gene transcription, IL-2 production and eventually cellproliferation. Several signaling pathways, including ERK and JNK pathways, PKCand Ca2+/calcineurin, are activated upon T-cell receptor ligation (Crabtree andClipstone, 1994). Specifically, both the ERK and JNK pathways are known to becritical for lymphocyte proliferation (DeSilva et al., 1998; Lafont et al., 1998; Swatet al., 1998).

The limited studies available suggest that age-related disturbances in signaltransduction pathways might explain the diminished IL-2 gene expression. Thus, ithas been shown that tyrosine phosphorylation of several proteins is affected duringaging in purified T-lymphocytes (Ghosh and Miller, 1995; Utsuyama et al., 1997;Whisler et al., 1997). Consistent with these results, a significant age-related reduc-tion of ERK activation was observed in mouse splenic T-cells stimulated via theTCR using anti-CD3 antibodies (Gorgas et al., 1997), in Con A induced splenicT-lymphocytes from rats (Pahlavani et al., 1998) and in peripheral blood T-cellsfrom humans (Whisler et al., 1996). Similarly, an age-related decline in JNK

M. Li et al. / Mechanisms of Ageing and De6elopment 113 (2000) 85–99 87

activation in response to anti-CD3 plus PMA was observed in human peripheralT-lymphocytes (Liu et al., 1997). These studies suggest some age-related impairmentin the ERK/JNK MAPK pathways via the TCR/CD3 complex.

Signaling via the TCR results in a variety of biochemical signaling events thatinclude a rise in intracellular free calcium and activation of protein kinase C. Thesetwo signals can also be generated by CaI and by activators of protein kinase C,such as PMA (Nau et al., 1988). This combination provides a unique opportunityto circumvent the early events in TCR activation, which are known to be defectiveduring aging. Together, these compounds act as effective co-mitogens (Larsen,1990). Most studies have indicated that proliferation is significantly impaired(30–50%) in aged lymphocytes stimulated with either Con A or PMA plus A23187in different species (Thoman and Weigle, 1988; Holbrook et al., 1989; Negoro andHara, 1992; Utsuyama et al., 1997). However, the difference in responsiveness toPMA plus CaI between young and old T-cells is less pronounced than thatobserved in response to receptor-dependent stimuli. This has led some authors tosuggest that the components of the activation pathways that lie downstream fromthe generation of calcium and diacylglycerol (DAG) may be relatively unimpairedby aging (Miller, 1986, 1995). Nevertheless, there appears to be a significant portionof the age-related decrease in responsiveness that still can not be overcome bybypassing the early events through the use of PMA plus Ca2+ as inducers.Therefore, it seems that, besides the major blockade in TCR-dependent signaltransduction located just after the TCR, an additional blockade might also bepresent at sites lying downstream of these events.

To further understand the mechanisms underlying these phenomena, we havestudied the activation of the MAPK signaling cascades in total splenocytes isolatedfrom F344 rats. Lymphocytes from young, middle-aged and old rats were stimu-lated for 15 min with two different concentrations of PMA plus A23187. The lowconcentration of inducers has been reported to induce optimal splenic lymphocyteproliferation with decreased proliferative capacity in lymphocytes from old animals(Holbrook et al., 1989). The high concentration of PMA plus A23187 has also beenused in the literature, for studies designed to investigate the activation of differentsignaling cascades in Jurkat cells (Su et al., 1994). Our data indicate the presence ofan age-related blockade in MAPK signal transduction, located downstream of Rasactivation, which results in decreased phosphorylation of key transcription factorsrequired for further downstream events.

2. Materials and methods

2.1. Animals

Young (6 months), middle-aged (15 months) and old (24 months) Fisher F344male, ad libitum fed rats were purchased from the Natonal Institutes on Aging(NIA). Upon arrival, they were housed in our specific pathogen free facility for 2weeks before use. The mean lifespan of male F344 rats is �24 months. Rats with


tumors or with spleen or other organ enlargements were excluded from the studies.In addition, serum levels of the acute phase protein, a1-acid glycoprotein (AGP)were determined as a measure of inflammatory processes (data not shown). Ratsthat exhibited a high level of expression of AGP were eliminated from the study.

2.2. Spleen lymphocyte isolation

Animals were decapitated, and spleens were removed and homogenized in aTenbroeck tissue grinder (Fisher) in RPMI 1640 (Whittaker Bioproducts). The cellsuspension was centrifuged at 400×g for 10 min at 4°C, the pellets were resus-pended in 5 ml RPMI and were overlaid on Ficoll (1-Step™ 1.077/265 Animal;Accurate Chemical & Scientific) and centrifuged at 800×g for 20 min at roomtemperature. Splenocytes were carefully collected from the interphase, washed andresuspended in RPMI.

2.3. Cell stimulation

After two washes in RPMI, splenocytes from individual rats were resuspended at5×106 cells/ml in RPMI medium containing 10% fetal calf serum, 2 mM glu-tamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Aliquots of 107 cells fromeach age group were placed in 35-mm tissue culture plates and kept at 37°C, 5%CO2. Cells were stimulated with low concentration of PMA plus A23187 (10 ng/mland 0.05 mg/ml, respectively) or high concentration of PMA plus A23187 (50 ng/mland 1 mg/ml, respectively) for 15 min. Uninduced splenocytes were used as control.

2.4. Whole cell extract preparation

To prepare whole cell extracts (WCE), the cells were pelleted at 16 000×g for 10s, and resuspended in lysis buffer (25 mM Hepes, pH 7.7, 0.3 M NaCl, 1.5 mMMgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 2 mM sodium pyrophosphate, 0.5 mMDTT, 20 mM b-glycerophosphate, 5 mg/ml leupeptin, 10 mg/ml aprotinin, 100 mg/mlPMSF, and 0.1 mM sodium orthovanadate (modified from (Su et al., 1994). Aftershaking the cell suspension at 4°C for 30 min, the extract was clarified bycentrifugation at 12 000×g for 10 min. The supernatants (WCE) were collected andprotein concentration was measured by the Bradford method as commercialized byBioRad.

2.5. Western blot

Aliquots (35 mg) of WCE were used for Western blot analysis. Samples weredenatured by boiling in Laemmli SDS loading buffer, reduced with 5% b-mercap-toethanol, and fractionated by SDS–PAGE using 10% mini gels. Proteins werethen electrophoretically transferred to nitrocellulose filters (BA 85, Schleicher andSchuell) for 60 min at 100 V in a BioRad mini electrotransfer apparatus. The blotswere reversibly stained with Ponceau S (0.05% Ponceau S in 3% TCA) for 1 min,


followed by two washes with ddH2O. After photography, the membranes wereblocked with BSA buffer (1% BSA, 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1%Tween 20) for 1 h at room temperature, and probed with horseradish peroxidaseconjugated anti-phosphotyrosine monoclonal antibody (PY-20, Transduction Lab-oratories) for 1 h at room temperature. After washing three times for 15 min inTNT buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20), detection wasperformed using the enhanced chemiluminescence (ECL) system from Amersham,according to the instructions supplied by the manufacturer.

As a control for loading, the membranes were probed with anti-actin antibody(ICN). Western blot analysis with the anti-active ERK antibody was performedexactly as suggested by the manufacturer (Promega), while the analysis withanti-phospho JNK, anti-phospho MEK1/2, and anti-phospho c-Jun were doneaccording to the protocol provided by New England Biolabs.

2.6. Data analysis

Films were scanned and quantitated using the ImageQuaNT image quantitationsoftware. The activities of ERK, JNK and MEK1/2 are expressed as fold inductionrelative to their uninduced cells of each age. The values represent the mean9S.D.from three independent experiments. Statistical comparison of the values relative toyoung animals was performed by a two-tailed, paired Student’s t-test.

3. Results

3.1. Protein tyrosine phosphorylation in response to mitogen stimulation is impairedduring aging in rat splenocytes

It has been established that the rate and extent of total protein tyrosinephosphorylation (P-Tyr) is impaired as a function of age in T-lymphocytes acti-vated via the TCR in mouse, human and rat (Shi and Miller, 1992; Ghosh andMiller, 1995; Whisler et al., 1997; Pahlavani et al., 1998). We wanted to investigateif this age-related defect was apparent for specific proteins in rat splenocytesactivated by PMA plus A23187. Because of our interest in signaling molecules, wefocused on the 40–50-kDa molecular weight range. The results presented in Fig. 1Aindicate that, in this range, protein tyrosine (Tyr) phosphorylation also declineswith age after PMA plus A23187 induction of rat splenocytes. A decline in Tyrphosphorylation of proteins in this molecular weight range is already observed inmiddle-aged animals, and this decrease becomes more dramatic in splenocytesisolated from old rats. Therefore, we conclude that tyrosine phosphorylation in the40–50-kDa range in response to PMA plus A23187 is impaired during aging inrats. The same membranes were stripped and reprobed with anti-actin antibody asan internal control (Fig. 1B). The figure shown is representative of three indepen-dent experiments that yielded consistent results.


3.2. Signal transduction is impaired in splenocytes from old rats induced withPMA/A23187

3.2.1. ERKSince ERK activation is dependent upon tyrosine and threonine phosphorylation

(Charest et al., 1993; Sugiura et al., 1997), we measured the phosphorylation ofERK proteins after PMA plus A23187 induction in rat splenocytes. For this, thesame membranes used in Fig. 1 were stripped and reprobed with an anti-activeERK antibody that recognizes dually phosphorylated (tyrosine and threonine)ERK1 (44 kDa) and ERK2 (42 kDa), but does not significantly recognize thecorresponding inactive form of the enzymes. The results shown in Fig. 2A indicatethat both ERK activities (ERK1 and ERK2) are induced in response to PMA plusA23187 in splenocytes isolated from rats of all age groups. However, phosphoryla-tion of both ERK1 and ERK2 is slightly but reproducibly diminished in bothmiddle-aged and old animals. The same membranes were stripped and reprobedwith an anti-ERK2 antibody (Transduction Labs). The basal and induced ERK2protein levels did not change (Fig. 2A).

As before, the data shown in Fig. 2A are a representative of three independentexperiments. Quantitation of this data (Fig. 2B) indicates that the fold inductionrelative to the basal levels of both ERK1 and ERK2 activities is significantlydiminished in both the middle and old age groups, as compared to their youngercounterparts (PB0.05). Thus, the results indicate that, in response to PMA andCaI, the capacity of ERK activation is diminished with age.

Fig. 1. Protein tyrosine phosphorylation in response to mitogen induction is impaired in rat splenocytesduring aging. Splenocytes from three individual animals of each age group, young (6 months),middle-aged (15 months) and old (24 months) rats were stimulated with different concentration ofPMA/A23187 for 15 min. WCE were prepared, aliquots (35 mg) were resolved on 10% SDS–PAGE gelsand Western analysis was performed. Lane 1: untreated; lane 2: PMA (10 ng/ml) plus A23187 (0.05mg/ml); lane 3: PMA (50 ng/ml) plus A23187 (1 mg/ml). Panel A: Tyrosine phosphorylated proteins weredetected using anti-phospho-tyrosine antibody (P-Tyr, PY 20, Transduction Laboratories). Panel B:Anti-actin antibody (ICN) was used to reprobe the same membrane as an internal control. The resultsshown are representative of three independent experiments.


Fig. 2. ERK phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. Panel A:The same membrane as in Fig. 1 was stripped and re-probed with either anti-active ERK antibody (top)or anti-ERK2 antibody (bottom). Panel B: Quantitation of three independent experiments similar to theone shown in panel A. Phosphorylated ERK1 or ERK2 (P-ERK1 or P-ERK2) is represented as foldinduction relative to uninduced cells in each age group. The values represent the mean9S.D. Starsrepresent values that display a statistically significant difference relative to young rats (PB0.05).

3.2.2. JNKSince JNK is also an important constituent of the MAPK signaling cascade, we

have studied the effect of age on JNK activation. Fig. 3A shows a representativeWestern blot using an anti-phospho JNK antibody. As in the case of ERK, thisantibody detects dually phosphorylated JNK1 (46 kDa) and JNK2 (54 kDa), butdoes not cross-react with the corresponding phosphorylated form of either ERK orp38 MAPK. The results indicate that JNK is activated only by high, but not by lowconcentrations of PMA plus A23187. Interestingly, the basal level of JNK activityis relatively high in splenocytes from old rats, as compared to cells from young ormiddle-aged animals. In addition, splenocytes from old animals display only a weakinduction of JNK activity in response to the same concentration of inducers that is


effective in cells from younger animals. The induced level of JNK activity in oldanimals is still considerably lower than that observed in younger rats. Furtherreprobing of the same membranes with an anti-JNK1 antibody that does notdistinguish between phosphorylated and dephosphorylated forms of the enzyme

Fig. 3. JNK phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. Panel A:The same membrane in Fig. 1 was stripped and reprobed with anti-phospho JNK antibody (top) oranti-JNK1 antibody (bottom). Panel B: Quantitation of three independent experiments similar to the oneshown in panel A. Phosphorylated JNK1 or JNK2 (P-JNK1 or P-JNK-2) is represented either asarbitrary units (left panels) or as fold induction relative to uninduced cells in each age group (rightpanels). The values represent the mean9S.D. Stars represent values that display a statisticallysignificant difference relative to young rats (PB0.05).


Fig. 4. MEK1/2 phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. PanelA: The same membranes as in Fig. 1 above were stripped and reprobed with anti-phospho-MEK1/2antibody (ser217/221). Panel B: Quantitation of three independent experiments similar to the one shownin panel A. Phosphorylated MEK1/2 (P-MEK1/2) is represented as fold induction relative to uninducedcells in each age group. The values represent the mean9S.D. Stars represent values that display astatistically significant difference in fold induction relative to young animals (PB0.05).

indicates that the steady state level of JNK1 does not change as a function of age(Fig. 3A). Quantitation of the data obtained from three independent experiments isshown in Fig. 3B. It indicates that the absolute activities of JNK1 and JNK2 arestatistically diminished in cells from old animals, but not in those from middle-agedones (PB0.05). Furthermore, the fold induction of JNK2, but not JNK1, relativeto their basal activities is also diminished in splenocytes from old rats as comparedto younger counterparts (PB0.05).

3.3. The age-related defect in ERK acti6ation is paralleled, at least in part, bydecreased acti6ation of MEK

Signaling upstream of ERK involves Ras, Raf-1 and MEK. To date, ERK1/ERK2 are the only known substrates for MEK1/2 (Crews et al., 1992; Franklin etal., 1994b; Zheng and Guan, 1994), while JNK1/JNK2 are the only knownsubstrates for SEK1 (also known as MKK4 or JNKK) (Davis, 1994; Sanchez et al.,1994; Yan et al., 1994). MEK activity is increased upon stimulation with eitheranti-CD3, PMA or calcium influx (Franklin et al., 1994b; Rosen et al., 1994), andwe measured MEK activity to determine whether there are any defects upstream ofERK upon induction with PMA plus CaI. Fig. 4A shows a representative Westernblot using anti-phospho MEK1/2 antibody (New England Biolabs). This antibodyselectively recognizes active MEK only when it is phosphorylated at Ser217/221,and does not cross-react with other related family members such as MKK4, MKK3or MKK6. Hence, it is an excellent marker of MEK1/2 activity. The results indicate


that MEK is activated to a similar extent with both concentrations of PMA plusA23187, and this activation declines considerably in the old. Quantitation of threeindependent experiments (Fig. 4B) indicates that the decrease in MEK activationwith age is statistically significant (PB0.05). Thus, we conclude that MEK1/2activation is also affected by the age of the splenocyte donor, even if the TCR isbypassed. This in turn might affect ERK activation during aging.

3.4. Acti6ation of transcription factors in response to PMA/A23187 is impaired insplenocytes from old rats

Signaling downstream of ERK and JNK results in the activation of severaltranscription factors, including Elk-1, c-myc, c-Fos, c-Jun and ATF-2 (Davis,1995). Some of these have been shown to play a crucial role in the regulation ofdownstream genes such as IL-2. Therefore, it was of primary interest to determinewhether activation of these factors is diminished during aging as a result of thedecreased activation of ERK and JNK. Since we have observed that the age-relateddefect in JNK activation is more pronounced than that of ERK, we concentratedon a transcription factor activated by this pathway, namely c-Jun.

The results shown in Fig. 5A indicate that c-Jun can be phosphorylated in Ser 63and 73 only by high concentrations of PMA plus A23187, but not by lowconcentrations of these inducers. Its activation, however, was only barely detectablein splenocytes isolated from old animals (PB0.05, Fig. 5B). The pattern lookssimilar to the one observed for JNK activation, which suggests that phosphoryla-

Fig. 5. c-Jun phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. Panel A:The same membranes were stripped and reprobed with anti-phospho-c-Jun antibody. Panel B: Quantita-tion of three independent experiments similar to the one shown in panel A. Phosphorylated c-Jun isrepresented as fold induction relative to uninduced cells in each age group. The values represent themean9S.D. Stars represent values that display a statistically significant difference relative to younganimals (PB0.05).


tion of c-Jun is affected in the old animals as a result of the upstream defect of JNKactivation.

4. Discussion

In this study, we have analyzed the effect of age on both ERK and JNKactivation in unfractionated total rat spleen lymphocytes. Bypassing the earlyevents in either TCR- or B-cell receptor (BCR)-directed signal transduction, PMAplus A23187 can substitute for receptor ligation as a primary signal in cellactivation (Nau et al., 1988; Kanno and Siebenlist, 1996; Rudd, 1996).

ERK activity can be induced by either high or low concentrations of PMA plusA23187, and the induced activities for each age group are comparable with eitherconcentration, indicating that the low concentration of PMA/CaI can induce fullactivation of ERK. In fact, at least in Jurkat cells, PMA alone can cause fullactivation of ERK, with no synergistic effect by A23187. However, CaI does havea synergistic effect with PMA on JNK activation, as only the combination of botheffectors leads to full activation of JNK (Su et al., 1994 and our own data). IL-2synthesis requires co-stimulation with PMA plus CaI (Mohagheghpour et al., 1995),while PMA alone can only induce cell surface expression of IL-2R molecules byT-cells, but not IL-2 synthesis. For these reasons, we used both as comitogens, sothat the data for ERK and JNK activation could be directly comparable. Ourresults suggest that splenic lymphocytes from old rats are afflicted by multipledefects within the MAP kinase pathways, some of which are located downstream ofthose previously described in the literature. In the case of ERK, we have furtherinvestigated the activation of the upstream kinases, MEK1 and MEK2, and foundthem to be impaired as well. While the decrease in MEK activation was moredramatic, and observed later in life, we conclude that, at least in the old animals,part of the diminished activation of ERK can be ascribed to decreased signalingfrom its upstream kinase. Medrano et al. have reported that senescent andterminally differentiated human melanocytes are unable to phosphorylate tyrosineresidues in ERK2 in response to PMA induction, in spite of presenting normalamounts of ERK2 protein (Medrano et al., 1994). In addition, in their experiments,ERK2 did not show the nuclear accumulation observed in proliferatingmelanocytes after PMA activation and remained localized in the perinuclear area Itis likely that a similar situation might apply to aged splenocytes in our study.

Although PMA plus A23187 induce ERK1 and ERK2 activities in rat spleno-cytes even when used at a low concentration, this treatment fails to induce eitherJNK1 or JNK2 activation. Thus, JNK isozymes were activated in a dose-dependentmanner by these inducers. The ability of splenic lymphocytes to phosphorylate JNKis strongly diminished with age. The results suggest that an age-related defect existsin this pathway, even if the TCR is not utilized. Our results also indicate thatsplenocytes from old rats have a higher level of basal JNK phosphorylation, ascompared to either young or middle-aged animals. It is possible that a disturbancein JNK subcellular location with age might prevent dephosphorylation by specific


phosphatases, which would lead to a gradual accumulation of phosphorylated JNKin the cells. However, we observed no evidence of activation of downstreamtranscription factors in resting splenocytes from old animals (Fig. 5B, and data notshown). This could be due to lack of activity of the phosphorylated kinases in vivo,possibly due to anomalous subcellular distribution.

In principle, splenocytes from old rats could have a disturbance at the level ofexpression of a JNK-specific MKP. A recent study in hepatocytes has providedevidence that the levels of MKP-1 mRNA are increased in EGF-stimulatedhepatocytes from old rats compared to those from young animals (Liu et al., 1996).Thus, it is reasonable to assume that an alteration in the balance between MAPKand MKP activities might contribute to the age-related defects in MAPK activitieswe have observed in lymphocytes.

Consistent with the observed decrease in JNK activation, our results confirm thatphosphorylation of c-Jun is also impaired upon PMA plus A23187 induction in ratsplenocytes. JNK binds tightly and specifically to the N-terminal region of c-Junand phosphorylates it at Ser63 and 73 (Derijard et al., 1994; Kyriakis et al., 1994).The phospho c-Jun (Ser63) antibody we have used reacts with phosphorylatedc-Jun, but does not cross-react with the corresponding phosphorylated forms ofJunD or JunB.

In this study, we have used a mixed lymphocyte population, consisting primarilyof B-cells, T-cells, and a small amount of macrophages, NK cells, etc. Theinteractions between B-cells and T-cells are complex and bidirectional, as B-cellspresent antigen to T-cells, and also receive signals from T-cells for division anddifferentiation. Such unfractionated splenic lymphocyte populations have thereforebeen used by many investigators, since they represent a situation that is closer tothe in vivo condition, in which different cell types coexist and interact with eachother. However, an obvious problem arises when these cells are used in conjunctionwith relatively non-specific inducers such as PMA plus A23187, which not onlyactivate T-cells but also other cell populations, including B-cells. Both MAPKfamily members under study, ERK and JNK, are known to be activated in B-cells(Franklin et al., 1994a; Weiss and Littman, 1994and our own data). Thus, we cannot infer from our studies which cell population plays a critical role in the decreasein MAPK activation with age. Indeed, it is very likely that a defect in the rateand/or extent of activation of these pathways represents a combined defect ofactivation of both T- and B-cells in our model system.

In summary, our studies have revealed the presence of novel defects in both theERK and JNK MAPK pathways of signal transduction, even when the receptorproximal defects are bypassed by PMA plus CaI. While we did not measureproliferative responses in parallel, it is well established that proliferation is signifi-cantly impaired (30–50%) in aged lymphocytes stimulated with either Con A orPMA plus A23187 in several species (Thoman and Weigle, 1988; Holbrook et al.,1989; Negoro and Hara, 1992; Utsuyama et al., 1997). The defects we haveobserved can be attributed, at least partially, to the decreased activation ofupstream signaling molecules, such as MEK or JNKK. Indeed, we have identifiedage-associated defects in MEK activation. Since both MEK and JNKK are located


at the same levels in their corresponding signaling cascades, we predict that theremight be some age-related defects in JNKK activation as well. Possible mechanismsto explain our results include age-related changes in MAPK phosphatase activities,disturbances in signal molecule subcellular location, and changes in the subpopula-tion of lymphocytes during aging. Our data complement other investigators’findings, and further indicate that the age-related defects in lymphocyte activationare not limited to TCR proximal events, but extend to multiple steps in thesignaling cascades. The outcome of signal transduction and all the related biochem-ical changes in response to lymphocyte activation is the induction of cell prolifera-tion and acquisition of immune functions. It is clear that proliferation anddifferentiation of both T- and B-cells is critical to effective humoral and cell-medi-ated immune responsiveness to pathogens, and that the proliferative response isdiminished during aging. The observed decline in signaling might contributesignificantly to the impairment of immune functions during aging.

Acknowledgements

This work was supported by grants AG14232 and AG00378 from the NIH, andby a pilot project grant from the Nathan Shock Center of Excellence in AgingResearch, AG 13282-05. We are gratefully indebted to Drs Vincent J. Cristofaloand Donna M. Murasko for their unwavering support and to Dr. Mary K Francisfor critical reading of the manuscript. We also thank the Murasko lab for theirinvaluable help in setting the splenocyte model system.

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Impaired signal transduction in mitogen activated rat splenic lymphocytes during aging

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