RESUMO
Nutrition has been shown to have a significant impact on aging. Caloric Restriction (CR), i.e., undernutrition not malnutrition, significantly increases the survival of laboratory animals by retarding/delaying the aging process. CR has beneficial effects on various physiological systems, including the immune system. Overall, the immunological status of rodents fed a restricted diet is superior to the immunological status of the non-restricted animals. It is believed that CR might retard aging and immunosenescence through a mechanism involving changes in signal transduction and gene expression. Recent studies from our laboratory support the view that the mechanism of CR involves changes in the activation of the upstream signaling molecules and cytokine gene expression that are altered with age.
Assuntos
Envelhecimento/imunologia , Restrição Calórica , Animais , Citocinas/imunologia , Citocinas/metabolismo , Dieta Redutora , Ingestão de Energia , Metabolismo Energético , Perfilação da Expressão Gênica , Humanos , Macaca mulatta , CamundongosRESUMO
The process of lymphocyte proliferation and apoptosis is known to be linked to oxidative stress. In the present study, we have used a new transgenic mouse model to investigate the effect of human Cu/Zn superoxide dismutase (Cu/Zn-SOD) overexpression on activation-induced lymphocytes proliferation and apoptosis. Cu/Zn-SOD activity was 3.5-fold higher in the spleen of the transgenic mice overexpressing Cu/Zn-SOD (Tg-Cu/Zn-SOD) compared to the wild-type littermates. Proliferative response of lymphocytes to lipopolysaccharide (LPS), Concanavalin A (Con A), and anti-CD3 was measured by [3H]-thymidine incorporation. Activation-induced apoptosis was determined by incubating the T cells with anti-CD3 (primary stimulus) for 72 h, followed by restimulation with Con A (secondary stimulus) for various times. Apoptosis was assessed by measuring DNA fragmentation using a spectrofluorimetric assay and monitoring the expression of the specific apoptotic markers (Fas/CD95 receptor and Fas/CD95 ligand (Fas-L) using flow cytometry. There was no significant difference in proliferative response of lymphocytes to LPS, Con A, or anti-CD3 in transgenic mice overexpressing human Cu/Zn superoxide dismutase (Tg-Cu/Zn-SOD) compared to wild-type littermates. In addition, no significant difference was observed in lymphocyte populations and subsets between Tg-Cu/Zn-SOD mice and wild-type littermates. However, splenic T cells from Tg-Cu/Zn-SOD mice exhibited a significantly (p <.05) higher level of activation-induced DNA fragmentation than T cells from wild-type littermates. The increase in DNA fragmentation was paralleled with an increase in the proportion of T cells expressing Fas and Fas-L molecules. The possible consequences of Cu/Zn-SOD overproduction on activation-induced apoptosis are discussed.
Assuntos
Apoptose/fisiologia , Ativação Linfocitária/fisiologia , Superóxido Dismutase/biossíntese , Linfócitos T/fisiologia , Animais , Células Apresentadoras de Antígenos , Divisão Celular , Células Cultivadas , Concanavalina A/farmacologia , Proteína Ligante Fas , Citometria de Fluxo , Humanos , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Baço/enzimologia , Superóxido Dismutase/genética , Receptor fas/metabolismoRESUMO
The aim of this study was to determine if aging or dietary restriction (DR) alters activation-induced cell death, which is known to regulate cell proliferation and eliminate the high number of activated cells during an immune response. Splenic T cells were isolated from young (4-6 months) and old (25-26 months) Fischer 344 rats that had free access to food, ad libitum (AL), and from dietary-restricted (DR) old (25-26 months) rats that beginning at 6 weeks of age were fed 60% (40% food-restricted) of the diet consume by the AL rats. T cells were incubated with anti-CD3 antibody, or staphylococcal enterotoxin B (primary stimulus) for 72-96 h, followed by restimulation with anti-CD3 (secondary stimulus) for 72 h. Activation-induced apoptosis was assessed by DNA fragmentation and the expression of Fas/CD95 receptor and Fas ligand (Fas-L) was measured by flow cytometry. We found that the amount of DNA fragmentation was significantly (P<0.05) higher in the stimulated and restimulated T cells from AL old rats and DR old rats compared to young rats. The increase in DNA fragmentation with age was paralleled by an increase in the proportion of the cells expressing Fas and Fas-L. However, DR had no significant effect on the age-related increase in DNA fragmentation or the expression of Fas or Fas-L. We also measured the levels of Bcl-2 and Bax protein and found that the level of Bcl-2 decreased and Bax increased with age and that DR had no effect on the age-related changes in the level of Bcl-2 or Bax protein. These results demonstrate that aging but not DR alters activation-induced apoptosis in rat T cells.
Assuntos
Envelhecimento/fisiologia , Apoptose , Linfócitos T/fisiologia , Animais , Antígenos de Superfície/biossíntese , Antígenos de Superfície/metabolismo , Western Blotting , Complexo CD3/farmacologia , Divisão Celular , Fragmentação do DNA , Dieta , Enterotoxinas/farmacologia , Proteína Ligante Fas , Citometria de Fluxo , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Endogâmicos F344 , Superantígenos/farmacologia , Proteína X Associada a bcl-2 , Receptor fas/biossíntese , Receptor fas/metabolismoRESUMO
Ceramide is a physiological mediator of extracellular signals that control various cellular functions, including proliferation and apoptosis. In the present study, we examined the effects of cell-permeable ceramide analog, N-acetyl-sphingosine (C(2)-ceramide) on the induction of proliferation and interleukin-2 (IL-2) synthesis in T cells from young and old rats. Splenic T cells from 6- and 24-month-old Fischer 344 rats were treated with C(2)-ceramide and then incubated with anti-CD3 antibody for 24 or 48 h. The induction of proliferation and IL-2 production by anti-CD3 was significantly (P<0.001) lower in T cells from old rats compared to T cells from young rats. C(2)-ceramide treatment resulted in suppression of proliferation and IL-2 production in a concentration-dependent manner. The suppressive effect of C(2)-ceramide on proliferation and IL-2 production was greater in T cells from old rats than T cells from young rats. We investigated whether this decreased responsiveness was due to induction of program cell death (apoptosis) and found that there was a significant increase in DNA fragmentation in C(2)-ceramide treated and anti-CD3 stimulated T cells from both young and old rats. The increase in DNA fragmentation was paralleled with an increase in caspase-3 activation. C(2)-ceramide-induced caspase-3 activation and DNA fragmentation was significantly (P<0.5) higher in stimulated T cells from old rats compared to stimulated T cells from young rats. These results suggest that the sphingomyelin-ceramide signaling pathway may play an important regulatory role in the well-documented age-related decline in immune function.
Assuntos
Envelhecimento/imunologia , Interleucina-2/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Envelhecimento/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Inibidores do Crescimento/farmacologia , Imunossupressores/farmacologia , Masculino , Ratos , Ratos Endogâmicos F344 , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
BACKGROUND: Activation-induced apoptosis is believed to limit cell proliferation and eliminate the high number of activated cells during an immune response. METHODS: Activation-induced apoptosis was investigated in splenic T cells isolated from young (6 months) and old (24 months) male Fischer 344 rats. The cells were incubated with anti-CD3, concanavalin A or staphylococcal enterotoxin B (primary stimulus) for 72 h, followed by restimulation with anti-CD3 or concanavalin A (secondary stimulus). Apoptosis was assessed by DNA fragmentation assay and DNA gel electrophoresis. The expression of the apoptotic marker CD95 was analyzed by flow cytometry and the relative levels of CD95 ligand, Bcl-2 and Bax protein were measured by immunoblotting. RESULTS: It was shown that DNA fragmentation was very low in the unstimulated T cells from both young and old rats. However, the level of DNA fragmentation was 45-55% greater in the activated T cells from old rats than in the activated T cells from young rats. The increase in DNA fragmentation was paralleled by an increase in the proportion of cells expressing the CD95 molecule. The proportion of CD95+ cells was approximately 40% higher in T cells from old rats than in T cells from young rats. In addition, it was found that the expression of CD95 ligand and Bax increased and Bcl-2 decreased in the activated T cells from old rats compared to the activated T cells from young rats. CONCLUSION: Our data suggest that the increase in sensitivity of T cells to apoptosis with age may contribute to age-associated immune dysfunction and disorders.
Assuntos
Apoptose , Ativação Linfocitária , Linfócitos T/fisiologia , Animais , Complexo CD3/imunologia , Concanavalina A/farmacologia , Fragmentação do DNA , Enterotoxinas/farmacologia , Masculino , Ratos , Ratos Endogâmicos F344 , Linfócitos T/efeitos dos fármacos , Receptor fas/análiseRESUMO
The age-related decrease in immunologic function is believed to be the major predisposing factor contributing to increased morbidity and mortality with age. Hence, the restoration of immunologic function is expected to have a beneficial effect in reducing pathology and maintaining a healthy condition in advanced age. Among various intervention strategies, caloric restriction (CR) has been shown to be the most powerful modulator of aging process. It is the most efficacious means of increasing longevity and reducing pathology. Several mechanisms have been proposed to explain its beneficial and robust action on various physiological systems, including the immune system. Experimental evidence suggests that CR increases longevity and reduces pathology through its action on the immune system. The observation that CR attenuates immunosenescence has provided a rationale for studying whether CR exerts its action through modulation of gene expression. The available data indicate that the effect of CR on signal transduction and gene expression can vary considerably from gene to gene and from one signaling molecule to another. This review summarizes the studies on the influence of CR on aging immune system and discusses the current state of knowledge on the molecular mechanisms responsible for the immunomodulatory action of caloric restriction.
Assuntos
Envelhecimento/imunologia , Ingestão de Energia , Animais , Humanos , Linfócitos T/imunologiaRESUMO
The effect of the DNA polymerase-beta (beta-pol) deficiency on mitogenic response and cytokine production was studied in spleen lymphocytes from 4-5- and 20-22-month-old beta-pol(-/+) mice and their age-matched wild-type littermates. The proliferative response of lymphocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [3H]thymidine incorporation, and the induction of cytokine production (interleukin (IL)-2, IL-4, and interferon necrosis factor (IFN)-gamma) was assessed by enzyme-linked immunosorbent assay. There was no significant difference in Con A- or LPS-induced proliferation or cytokine production in young beta-pol(-/+) mice compared with young wild-type littermates or in old beta-pol(-/+) mice compared with old wild-type littermates. However, mitogen-induced proliferation and cytokine production changed significantly with age. The proliferative response to Con A and to LPS, and the IL-2 production was significantly lower, and IL-4 and IFN-gamma levels were significantly higher in lymphocytes from old beta-pol(-/+) mice and old wild-type mice than in lymphocytes from young beta-pol(-/+) mice and young wild-type littermates. In addition, flow cytometric analysis showed no significant differences between young beta-pol(-/+) mice and young wild-type littermates or between old beta-pol(-/+) mice and old wild-type littermates in the proportion of B- and T-cell populations, and T-cell subsets. However, the number of lymphocytes expressing CD4+ phenotype slightly decreased and the proportion of lymphocytes expressing CD44/Pgp-1 (memory) phenotype increased with age. Thus, we found no evidence for alteration in immune function in DNA polymerase-beta deficient mice, although they exhibit a decline in immunologic function with age.
Assuntos
Envelhecimento/imunologia , Citocinas/biossíntese , DNA Polimerase beta/metabolismo , Ativação Linfocitária , Linfócitos/imunologia , Animais , DNA Polimerase beta/deficiência , DNA Polimerase beta/genética , Citometria de Fluxo , Interferon gama/biossíntese , Interleucinas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologia , Baço/imunologiaRESUMO
The signaling cascade mediated by Ras (p21ras) and MAPK (mitogen-activated protein kinase) and calcium/calmodulin regulating enzymes, calcineurin (CaN) and CaMK-IV, are considered to be essential for T-cell growth and function. In the present study, the effect of aging and caloric restriction (CR) on the induction of Ras and MAPK activation by concanavalin A (ConA) was studied. Splenic T cells were isolated from young (4-6 months) and old (22-24 months) rats that had free access to food (control group), and from caloric restricted old (22-24 months) rats that beginning at 6 weeks of age were fed 60%(40% caloric restriction) of the diet consumed by the control rats. We found that the induction of Ras activity in T cells isolated from control old rats was lower (P<0.001) than that in control young rats. However, the levels of Ras activity in T cells isolated from CR old rats were similar to the levels in the age-matched control rats. The induction of MAPK activity in T cells isolated from control old rats and CR old rats was significantly less than in T cells isolated from control young rats, and caloric restriction significantly (P<0.05) reduced the age-related decline in MAPK activation. We also measured the induction of CaN and CaMK-IV activities by ConA in T cells from control young and old and CR old rats. The induction of both CaN and CaMK-IV activity decreased with age. Caloric restriction significantly (P<0.05) reduced the age-related decline in CaN activity, but had no significant effect on CaMK-IV activity. The changes in Ras/MAPK activation and in CaN and CaMK-IV activity with age or with CR were not associated with alterations in their corresponding protein levels. Thus, caloric restriction has a differential effect on the activation of the upstream signaling molecules that are altered with age.
Assuntos
Envelhecimento/metabolismo , Calcineurina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linfócitos T/enzimologia , Animais , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Ingestão de Energia , Ativação Enzimática , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
A decline in the induction of heat shock protein 70 (hsp70) expression with age has been shown to occur in a variety of tissues from male rodents. Because the age-related change in the expression of many genes often differ in male and female rodents, we have measured the induction of hsp70 expression in hepatocytes and splenocytes from young/adult (4-8 months) and old (20-22 months) female Fischer 344 rats. Hepatocytes and splenocytes isolated from old female rats showed a marked decrease in the induction of hsp70 mRNA and protein levels by heat shock when compared to hepatocytes and splenocytes isolated from young/adult female rats. Because the heat shock transcription factor HSF1 mediates the heat-induced transcription of hsp70, the effect of age on HSF1 was also studied. The ability of extracts from heat-shocked splenocytes to bind to the heat shock element (HSE) decreased with age. Interestingly, the levels of HSF1 protein were similar in splenocytes and hepatocytes from old female rats compared to young/adult female rats, even though the levels of HSE-binding were lower for splenocytes isolated from old rats. In this study, we show an age-related decline in the expression of hsp70, and this decline was similar to what we had previously observed in male Fischer 344 rats.
Assuntos
Envelhecimento/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Fígado/metabolismo , Baço/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Fígado/citologia , Ratos , Ratos Endogâmicos F344 , Baço/citologiaRESUMO
We have previously shown that the DNA binding activity of the transcription factor NFAT which plays a predominant role in IL-2 transcription decreases with age. Because the transactivation (dephosphorylation and nuclear translocation) of the NFAT-c (cytoplasmic component of the NFAT complex) is mediated by the calcium/calmodulin-dependent phosphatase, calcineurin (CaN), and because Ca2+/calmodulin-dependent kinases (CaMK-II and IV/Gr) have been shown to play a critical role in calcium signaling in T cells, it was of interest to determine what effect aging has on the activation and the levels of these calcium regulating enzymes. The induction of calcineurin phosphatase activity, and CaMK-II and IV/Gr activities, were studied in splenic T cells isolated from Fischer 344 rats at 6, 15, and 24 months of age. In addition, the changes in the protein levels of these enzymes were measured by Western blot. The calcineurin phosphatase activity and CaMK-II and IV kinase activities were at a maximum after the cells were incubated with anti-CD3 antibody for 5-10 minutes. The induction of calcineurin activity by anti-CD3 and by calcium ionophore (A23187) declined 65 and 55%, respectively, between 6 and 24 months of age. The induction of CaMK-IV activity, but not CaMK-II activity by anti-CD3, was significantly less (by 54%) in T cells from old rats compared to T cells from young rats. The decline in the activation of these enzymes with age was not associated with changes in their corresponding protein levels. These results demonstrate that alterations in calcineurin phosphatase activity and CaMK-IV activity may contribute to the well-documented age-related decline in T cell function.
Assuntos
Envelhecimento/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/farmacologia , Ativação Linfocitária/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Linfócitos T/fisiologia , Envelhecimento/metabolismo , Animais , Western Blotting , Calcimicina/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Células Cultivadas , Ativação Linfocitária/efeitos dos fármacos , Masculino , Monoéster Fosfórico Hidrolases/efeitos dos fármacos , Testes de Precipitina , Ratos , Ratos Endogâmicos F344 , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
T cells from young (6 months) and old (24 months) male Fischer 344 rats were isolated and exposed to three different oxidative stress conditions: (a) reactive oxygen species generated by xanthine-xanthine oxidase (X/XO), (b) hydrogen peroxide (H2O2), and (c) hyperthermia (43 degrees C for 1 h). After oxidative stress treatment, the induction of proliferation and IL-2 production by concanavalin A (Con A) was measured. Exposure of T cells to X/XO or H2O2 resulted in suppression of proliferation and IL-2 expression, and the suppressive effect was more pronounced in T cells from young rats than in T cells from old rats. Similarly, hyperthermia caused inhibition of proliferation and IL-2 expression in T cells from young and old rats. Addition of antioxidant to cultured cells only slightly attenuated the effects of X/XO and H2O2 on T cell function; however, antioxidant had no effect on heat shock-mediated inhibition of proliferation in young or old rats. Because IL-2 plays a crucial role in T cell proliferation and because the transcription factor NFAT (nuclear factor of activated T cell) plays a major role in the regulation of IL-2 transcription, the induction of NFAT as well as NF-KB and AP-1 DNA binding activities in nuclear extracts of the X/XO-treated and untreated control cells was measured using a gel shift assay. The ability of nuclear extracts to bind NFAT or NF-KB oligonucleotide decreased in the X/XO-treated cells from young and old rats compared to the untreated controls. Therefore, these data imply that reactive oxygen species generated by the X/XO system alter the distal step of mitogen-mediated signal transduction, i.e., transcription factors that regulate IL-2 transcription.
Assuntos
Envelhecimento , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/fisiologia , Animais , Antioxidantes/farmacologia , Células Cultivadas , Concanavalina A/farmacologia , Radicais Livres , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Interleucina-2/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344 , Fatores de Transcrição/metabolismo , Xantina/metabolismo , Xantina Oxidase/metabolismoRESUMO
Although it is well established that the functional properties of T cells decrease with age, its biochemical and molecular nature is poorly understood. The available data suggest that changes in the signal transduction machinery are responsible for the impairment of T cell function during aging. T cell activation is initiated when an antigenic peptide is recognized by the antigen receptor of T cells. This recognition event promotes sequential activation of a network of signaling molecules such as kinases, phosphatates, and adaptor proteins that couple the stimulatory signal received from T cell receptor (TCR) to intracellular signaling pathways. The coordinate activation of these signaling molecules is sufficient to stimulate the activation of transcription factors and the expression of the immediate-early genes that are crucial in regulation of T cell function.
Assuntos
Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Linfócitos T/fisiologia , Fatores Etários , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Genes ras/fisiologia , Humanos , Fosforilação , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Sistemas do Segundo MensageiroRESUMO
Dietary restriction (DR) is the only effective experimental manipulation known to retard aging in rodents, and this manipulation has been shown to alter a variety of processes that change with age. However, there is no information on the effect of DR on macrophage Function. In the present study, the effect of aging and DR on the ability of alveolar macrophages (AMs) to express the heat shock gene, hsp70 was studied. AMs were isolated by lavage from the lungs of young (4-6 months) and old (24-26 months) rats fed either ad libitum (AL) or a restricted diet (60% of AL). There was no age-related change in the number of cells recovered from young and old rats fed AL. However, the number of cells recovered from the lungs of the DR rats was reduced, and this decrease was statistically significant in young rats. The expression of heat shock protein 70 (hsp70) was measured by the level of the hsp70 mRNA transcript in total RNA isolated from AMs cultured under two conditions: in suspension and after adherence to plastic. When AMs were incubated at 37 degrees C in suspension, no detectable hsp70 expression was observed; however, hsp70 expression was induced at 37 degrees C when the AMs adhered to the plastic culture dishes. Hsp70 mRNA levels were rapidly induced by heat shock (43 degrees C, 1 h) in AMs cultured both in suspension and on plastic. The induction of hsp70 expression did not change significantly with either age or DR in AMs cultured in suspension. In contrast, the induction of hsp70 mRNA levels by AMs adherent to plastic culture plates decreased approximately 70% with age, and hsp70 induction was greater in AMs isolated from DR rats; this difference was statistically significant in young rats. The induction of hsp70 by heat shock (43 degrees C, 1 h) also decreased with age in the adherent AMs, and DR increased the induction of hsp70 expression three- to fourfold in adherent AMs from both young and old rats.
Assuntos
Envelhecimento/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Macrófagos Alveolares/metabolismo , Animais , Células Cultivadas , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Signal transduction is ubiquitously involved in the initiation of physiological signals that lead to growth and proliferation of cells. The signaling cascade mediated by the mitogen-activated protein kinase (MAPK) is considered essential for T cell growth and function. Therefore, it was of interest to determine the influence of age on the induction of MAPK in mitogen-activated T cells. T cells from young (4-6 months) and old (24-26 months) rats responded to concanavalin A (Con A) stimulation by increasing MAPK, c-jun amino terminal kinase (JNK), and p21ras activities. The time course of induction of MAPK/JNK and p21ras activities was similar in T cells isolated from young and old rats. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and p21ras activities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. Although the relative protein levels of p42 and p44 MAPK did not change with age, the proportion of the phosphorylated p44 MAPK decreased with age. In addition, it was found that the in vitro kinase activities of the T cell receptor-associated protein tyrosine kinase Lck (p56Lck) and ZAP-70 but not Fyn (p59Fyn) were lower in T cells from old rats than in T cells from young rats. The decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels. Thus, our results demonstrate that aging alters the activation of the signal transduction cascade that leads to T cell activation.
Assuntos
Envelhecimento/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Linfocitária/fisiologia , Proteínas Quinases Ativadas por Mitógeno , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Animais , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/imunologia , Concanavalina A/farmacologia , Ativação Enzimática/imunologia , Indução Enzimática/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Masculino , Fosforilação , Testes de Precipitina , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Ratos , Ratos Endogâmicos F344 , Linfócitos T/enzimologia , Linfócitos T/imunologia , Fatores de Tempo , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
Caloric restriction has been the subject of intensive research and is known to be the most efficacious means of increasing longevity and reducing pathology. Caloric restriction has been found to influence a wide variety of age-sensitive immunological parameters such as interleukin-2 (IL-2) gene expression, and overall, the immunological status of rodents fed a caloric restriction diet is superior to the immunological status of the non-restricted animals. IL-2 is a growth promoting cytokine that plays a critical role in immune function. The expression of IL-2 has been shown to decrease with age, and the decrease in IL-2 expression parallels the age-related decrease in immune function. The focus of this review article is to discuss the studies on the influence of caloric restriction on IL-2 expression and the recent findings on the mechanisms by which caloric restriction enhances IL-2 gene expression. A number of studies have demonstrated that caloric restriction alters the expression of the IL-2 gene at the level of transcription. The increase in IL-2 expression correlates with an increase in binding activity of the transcription factor NFAT which plays a predominant role in IL-2 transcription. In addition, preliminary results suggest that activation of the upstream signaling molecules, the mitogen-activated protein kinase (MAPK) signaling cascade, may play a role in the enhancement of IL-2 transcription.
Assuntos
Restrição Calórica , Interleucina-2/genética , Transcrição Gênica/genética , Animais , Regulação da Expressão Gênica , Modelos Biológicos , Ligação Proteica , Ratos , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The decline in immunologic function with age is associated with an increase in susceptibility to infections and the occurrence of autoimmune diseases and cancers. Hence, the restoration of immunologic function is expected to have a beneficial effect in reducing pathology and maintaining a healthy condition in advanced age. A number of therapeutic strategies have been employed to intervene in the aging immune system. This article reviews the effect of dietary restriction (DR), dehydroepiandrosterone (DHEA) treatment, melatonin (MLT) therapy, and exercise on modulating the immune responses and retarding/reducing immunosenescence. DR has been subject to intensive research and is known to be the most efficacious means of increasing longevity, reducing pathology and enhancing immune function. The circulatory levels of the androgenic hormone DHEA and the pineal hormone MLT decrease with increasing age, and this decrease has been correlated with the age-related decline in the immune system. Therefore, the observation that immunosenescence is associated with low levels of DHEA and MLT has provided a rationale for therapeutic intervention. DHEA treatment and MLT therapy both exhibit immunostimulatory actions and preliminary reports indicate that hormonal (DHEA or MLT) substitution therapy reverses immunosenescence in mice. Similarly, exercise in some studies has been shown to enhance the immune response. However, these findings have not been confirmed by other laboratories. Thus, at the present time, it is difficult to draw any definitive conclusions on the efficacy of DHEA, MLT, and exercise on reversing or restoring the aging immune system.
RESUMO
The objective of this study was to determine if the increase in the induction of interleukin-2 (IL-2) expression with caloric restriction correlates with changes in binding activity of the IL-2-specific transcription factor NFAT (nuclear factor of activated T cells) and/or the ubiquitous transcription factor AP-1 in T cells from male Fischer 344 rats. Splenic T cells were isolated from young (6-month) and old (24-month) rats fed ad libitum and from old (24-month) rats fed a restricted diet (40% caloric restriction) that began at 6 weeks of age. T cells were stimulated with concanavalin A (Con A) and the expression of IL-2 and the DNA binding activity of the transcription factors NFAT and AP-1 were measured in these cells. We found that the induction of IL-2 activity and mRNA levels decreased with age and that caloric restriction significantly (P < 0.05) reduced the age-related decline in IL-2 expression. The ability of nuclear extracts from T cells isolated from old rats fed ad libitum and restricted old rats to bind to the NFAT oligonucleotide or AP-1 oligonucleotide decreased with age. Caloric restriction significantly (P < 0.05) reduced the age-related decline in NFAT but had no significant effect on AP-1 binding activity. We also measured the induction of c-fos and c-jun expression by Con A in T cells from young and old rats fed ad libitum or caloric-restricted diet. The induction of c-fos protein and mRNA levels but not c-jun protein or mRNA levels decreased significantly with age. Caloric restriction significantly (P < 0.05) reduced the age-related decline in c-fos expression but had no significant effect on c-jun expression. Therefore, the increase in IL-2 expression with caloric restriction correlates with an increase in binding activity of transcription factor NFAT and an increase in the expression of c-fos, which is a component of the NFAT-protein complex.
Assuntos
Envelhecimento/imunologia , Proteínas de Ligação a DNA/metabolismo , Ingestão de Energia/imunologia , Interleucina-2/biossíntese , Proteínas Nucleares , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Dietoterapia , Regulação da Expressão Gênica , Interleucina-2/genética , Masculino , Fatores de Transcrição NFATC , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-jun/biossíntese , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Fator de Transcrição AP-1/metabolismoRESUMO
Melatonin (MLT) treatment in vivo has been shown to have immunomodulatory and anti-immunosenescent effects in the mouse model. In the present report, the in vitro effect of MLT on mitogen-induced lymphocyte proliferation and cytokine expression was evaluated in a rat model. Splenic lymphocytes were isolated from young (6 months) and old (24 months) F344 rats and were incubated with MLT in the presence or absence of mitogens. The proliferative response to concanavalin A (ConA) or PMA plus ionomycin was measured in splenocytes or T cells isolated from young and old rats. In addition, the induction of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production was measured in MLT-treated and untreated lymphocytes isolated from young and old rats. The ConA-induced lymphocyte proliferation and IL-2 expression were significantly lower and induction of IFN-gamma production was significantly higher in splenocytes and purified T cells isolated from old rats compared to splenocytes and T cells isolated from young rats. Treatment of lymphocytes with MLT did not significantly alter ConA-induced lymphocyte proliferation or IL-2 or IFN-gamma expression in lymphocytes isolated from either young or old rats. On the basis of these data, we conclude that in vitro MLT treatment had no immunomodulatory effect on lymphocytes from rats.
Assuntos
Envelhecimento/imunologia , Citocinas/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Melatonina/farmacologia , Mitógenos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Células Cultivadas , Citocinas/efeitos dos fármacos , Interferon gama/biossíntese , Interferon gama/efeitos dos fármacos , Interleucina-2/biossíntese , Masculino , Ratos , Ratos Endogâmicos F344 , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Oxidative damage caused by free radicals in vivo is believed to play an important role in the etiology of aging and age-associated degenerative diseases. The most direct evidence supporting this theory is the recent finding that the transgenic Drosophila that overexpress the antioxidant enzymes catalase and superoxide dismutase exhibit an increase in life span. Although the increase in life span in Drosophila by these enzymes is certainly important, the next logical direction is to demonstrate whether increased antioxidant protection occurs similarly in mammals. Several transgenic mouse models that overexpress antioxidant enzymes are currently available. However, one major shortcoming in using these transgenic mice is the difficulty of producing antioxidant overexpression in more than a few tissues. Despite the potential shortcomings of using transgenic mice, these animals provide a unique system in which individual components of a complex system, such as the antioxidant defense system, can be modulated and examined independently. Transgenic mice are therefore potentially powerful tools to study the role of various components of the antioxidant system in the aging process. A parallel direction in the study of free radical roles in aging is to investigate the modulation of transcription factors by oxidative stress. Among these, the transcription factors, NF-κB and AP-1 are implicated in oxidative stress. The activities of these oxidative stress-response transcription factors are regulated by upstream signaling molecules, which involve a cascade of phosphorylation and dephosphorylation events leading to their activation. In this article, we review recent studies that use molecular approaches to investigate the biological role of oxidant stress. Each of these studies potentially provide new insights into the roles of free radicals and free radical damage in the aging process.
RESUMO
The purpose of this study was to determine if food restriction alters the age-related decline in heat shock protein 70 (hsp70) expression in lymphocytes from male Fischer 344 rats. Spleen lymphocytes were isolated from young (6 mo) and old (24 mo) rats that had free access to food (control group) and from food-restricted old (24 mo) rats that, beginning at 6 wk of age, were fed 60% (40% food restriction) of the diet consumed by the control rats. Lymphocytes were either heat shocked (42.5 degrees C for 1 h) or maintained at 37 degrees C (control). The levels of hsp70 protein and mRNA and the induction of interleukin-2 (IL-2) mRNA by concanavalin A (conA) were measured in these rats. In addition, the percentage of viability (as an index of thermosensitivity) of the lymphocytes from these rats was also measured after exposure of cells to different temperatures (first at 42.5 degrees C and then 45 degrees C). We found that the induction of hsp70 protein and mRNA in lymphocytes isolated from control old rats and food-restricted old rats was lower (P < 0.001) than that in control young rats. However, the levels of hsp70 and mRNA in lymphocytes isolated from food-restricted old rats were similar to the levels in age-matched control rats. Lymphocytes from both control old rats and food-restricted old rats were more thermosensitive than lymphocytes from control young rats; however, thermosensitivity of lymphocytes from food-restricted old rats was similar to that of age-matched control rats. The induction of IL-2 mRNA levels by conA was significantly (P < 0.001) lower in old than in young control rats. In contrast to hsp70, which was not affected by food restriction, the induction of IL-2 mRNA was higher (P < 0.05) in food-restricted old rats than in age-matched control rats. Therefore, food restriction has a differential effect on gene expression.