Influence of exercise on the immune function of rats of various ages

The purpose of this study was to determine whether exercise could prevent the age-related decline in mitogenesis, which has been well documented in rats, mice, and humans. At 1, 6, 12, and 18 mo of age, male Fischer F344 rats were subjected daily to swimming exercise for 6 mo. At the end of the 6-mo training period, spleen lymphocytes were isolated from the exercised rats and from age-matched sedentary controls. The induction of lymphocyte proliferation was measured with the mitogens concanavalin A (ConA) and lipopolysaccharide (LPS). In addition, the ability of the lymphocytes to produce interleukin 2 (IL 2) in response to ConA induction was measured. ConA- and LPS-induced proliferation decreased 41-63% between 7 and 25 mo of age in both exercised and sedentary control rats. ConA-induced IL 2 production decreased 42 and 62% between 7 and 25 mo of age for exercised and sedentary control rats, respectively. Although the age-related decline in mitogen-induced proliferation and IL 2 production was smaller in exercised rats, this was due to a lower level of mitogenesis and IL 2 production in lymphocytes from young exercised rats. Exercise resulted in a significant decrease (23-32%) in mitogen-induced lymphocyte proliferation and IL-2 production in 7-mo-old exercised rats compared with 7-mo-old sedentary rats. However, in the 18- and 24-mo-old rats, mitogen-induced lymphocyte proliferation and IL 2 production was not significantly different between exercised and sedentary control rats.     

The expression of granulocyte/macrophage colony-stimulating factor in activated mouse lymphocytes declines with age

The expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) was studied in spleen lymphocytes isolated from male C57BL/6J mice of 6, 20, and 29 months of age. GM-CSF expression (biological activity and mRNA level) was maximum after culturing the lymphocytes for 45 hr with concanavalin A and phorbol myristate acetate. The induction of both GM-CSF activity and mRNA levels was observed to decline over 60% between 6 and 29 months of age. The age-related decline in the level of GM-CSF paralleled the age-related decline in the mRNA levels of interleukin-2 and interleukin-3.     

Interleukin 2 and concanavalin A stimulate interferon-gamma production in a murine cytolytic T cell clone by different pathways

We identified a variant murine cytolytic T lymphocyte (CTL) clone which, in contrast to the parent clone and all other murine T cell populations tested, was found to have acquired spontaneously the ability to produce interferon-gamma (IFN-gamma) in response to recombinant interleukin 2 (rIL-2). IFN-gamma production in response to concanavalin A (Con A), which was characteristic of all T cell populations tested, was preserved in this variant. The IFN produced by the variant in response to either stimulus was active in both a macrophage-activating factor assay and an anti-viral assay. Both activities induced by either stimulus could be blocked by monoclonal anti-IFN-gamma antibodies. Upon Northern blot analysis using an IFN-gamma-specific cDNA probe, the IFN-gamma RNA isolated from variant cells stimulated with Con A or IL-2 were found to migrate equivalently. The unusual pattern of responsiveness in this variant CTL was exploited to compare the mechanisms involved in induction of IFN-gamma production by Con A or IL-2. Striking differences were observed. Unlike IFN-gamma production induced by Con A, IFN-gamma production induced by IL-2 was not accompanied by an elevation of intracellular Ca2+ levels, did not require physiologic extracellular Ca2+ levels, and was not inhibited by the immunosuppressive agent cyclosporin A. Thus, in this variant CTL clone, conditions that have ordinarily been associated in an obligate manner with lymphokine gene expression were found instead to be related to the specific mode of stimulation.     

The age-related decline in interleukin-3 expression in mice

Spleen lymphocytes form 5- to 37-month-old C57BL/6J mice were stimulated by concanavalin A (con A) in vitro, and the interleukin-3 (IL-3) expression was assessed by measuring the IL-3 activity in culture supernatants and the cytoplasmic IL-3 mRNA levels. The activity and mRNA level of IL-3 was maximum at 20 hr after culturing in the presence of con A. The IL-3 activity in the culture supernatants and the IL-3 mRNA level in lymphocytes declined 70% to 80% between 5 and 37 months of age. Northern blot analysis revealed no change in the size of IL-3 mRNA between young and old mice. When the expression of IL-3 and interleukin-2 (IL-2) by con A-stimulated lymphocytes was compared, both interleukins showed a similar declined with age.     

Restricted expression of mitogen-induced high affinity IL-2 receptors in aging mice

Several lines of indirect evidence suggest that the number and/or affinity of IL-2R expressed by activated T lymphocytes declines with age and that this decline is implicated in the age-related proliferative impairment of Ag or mitogen-stimulated T cells. In an attempt to provide a direct demonstration of such a defect, various experimental approaches were used to analyze the expression of high and low affinity IL-2R as well as their functional properties in relation to age in purified populations of murine T lymphocytes. IL-2R were induced by Con A-activation which involves a transmembrane signaling mechanism or by exposure to phorbol dibutyrate (PDBu) which bypasses such a pathway. Consistent with the previously reported age-related defect in signal transduction, a major deficiency in the expression of high affinity IL-2R was observed in mitogen-activated cells derived from aged animals. As expected, PDBu-induction circumvented the transmembrane signaling defect and resulted in the restoration of a measurable amount of high affinity IL-2R expressed by cells from aged mice early after activation. The functional properties of the IL-2R expressed as a consequence of Con A or PDBu induction were investigated by assessing the proliferative response induced through the high affinity IL-2R as compared to that mediated by the beta-chain alone. Although Con A-induction resulted in a decreased expression of high affinity IL-2R by T lymphocytes derived from aged mice, the ability of these receptors as well as that of their beta-chain component to transmit a proliferative signal was identical in both age groups. In contrast, PDBu induced in both cell populations the expression of functionally aberrant IL-2R, unable to signal for proliferation unless excessively high concentrations of rIL-2 were available. The quantitative minimal estimate of the frequency of Con A-activated, IL-2-responsive cells showed a fourfold age-associated decrease, confirming the inability of a subpopulation of T lymphocytes from aged mice to express a sufficient density of high affinity IL-2R as a consequence of mitogenic activation.     

Interactions of aging and annual rhythms on the recovery of cryopreserved murine splenic lymphocytes

The physiological status of donor organisms is an often overlooked factor in cryopreservation experiments. Murine splenic lymphocytes exhibit systematic changes in function which are endogenous and influence recoveries of viable and functional frozen-thawed cells over the life span of mice. One of these changes is the decline in the performance of unfrozen cells as organisms age. Superimposed on the age-related decline in lymphocyte functions are circannual rhythms in T- and B-cell mitogenesis, and the properties of these rhythms also change with age. Splenocytes from young, 15-month-old and 23- to 27-month-old C57BL/6 mice were cryopreserved and tested for recovery of mitogenic responses to activation by the T-cell mitogens, phytohemagglutinin and concanavalin A, and the B-cell mitogen, lipopolysaccharide. Tritiated thymidine incorporation by activated, dividing cells was determined after 72 hr of in vitro culture. Seasonal patterns in recovery of viable and functional cryopreserved cells from young mice resembled those of their unfrozen controls (6). By 15 months of age, the responses after freeze-thaw stress decreased to the levels observed for cells obtained from senescent mice, and seasonal patterns were no longer observed. In these middle-aged mice, intracellular changes in lymphocytes that are equivalent to those in senescent animals resulted in irreparable structural-functional injury during cryopreservation.     

Impact of ageing on proteasome structure and function in human lymphocytes

Key actors of the immune response, lymphocytes exhibit functional deficits with advancing age. For instance, the age-related decline in lymphocyte proliferation may be related to alteration in the degradation of crucial proteins such as cell-cycle regulators. Degradation of these proteins is mediated by the ubiquitin-26S proteasome system. The proteasome is also the major "housekeeping" proteolytic complex responsible for eliminating intracellular damaged proteins. To investigate the occurrence of proteasome structural and functional age-related alterations, 26S proteasome was purified from peripheral blood lymphocytes of 20-63-year-old donors. Changes in peptidase activity were measured and modifications in the proteasome particle structure were analysed using bi-dimensional electrophoresis. We found the age-related decline of 26S proteasome-specific activity to be associated with an increased yield of post-translational modifications of proteasome subunits, while proteasome content and subunit composition were unchanged. In particular, some catalytic and assembly subunits of the 20S proteasome were preferentially modified with age. Western blotting of proteasome subunits resolved by bi-dimensional electrophoresis showed some of these modified subunits to be glycated, conjugated with a lipid peroxidation product and/or ubiquitinated. In conclusion, it is suggested that structural alterations of proteasome subunits may contribute to the observed decline of proteasome activity with age and could play a major role in immune senescence.     

IL-7 regulation of cytotoxic lymphocytes: pore-forming protein gene expression, interferon-gamma production, and cytotoxicity of human peripheral blood lymphocytes subsets.

The effects of IL-7 on the generation of cytolytic human peripheral blood lymphocytes (PBL) were investigated. Induction of T-cell pore-forming protein (PFP) mRNA and cytotoxic potential by IL-7 was both slow and minor compared with that observed in IL-2-cultured T cells. IL-7 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in T cells. Clearly, however, both IL-7 and IL-2/IL-7 induced the PFP gene expression and cytotoxic potential of CD8+ T cells and not CD4+ T cells. In addition, neither monoclonal antibodies (mAb) to the p55 or p75 IL-2-receptor subunits had any effect upon IL-7 induction of CD8+ T-cell cytotoxicity, indicating that IL-7 induction of cytotoxic CD8+ T cells was IL-2 independent. IL-7 induction of CD3- large granular lymphocyte (LGL) and PB gamma delta T-cell cytotoxicity was also delayed and reduced compared with that effected by IL-2. IL-7 (10 or 1000 U/ml, 72 hr) enhanced the NK and LAK cytotoxic of LGL and PB gamma delta T cells. By contrast IL-7 or IL-2 augmented the redirected cytotoxic potential of PB gamma delta T cells, but not that of LGL, and neither lymphokine had any effect on constitutive PFP mRNA expression in either lymphocyte subset. In addition, IL-7 induction of LGL IFN-gamma production was weak and delayed compared with that effected by IL-2 and neither IL-2 nor IL-7 stimulated IFN-gamma production in PB gamma delta T cells. Therefore, overall the effects of IL-2 and IL-7 on various cytotoxic human PBL were qualitatively similar, but quantitatively and kinetically different.     

Molecular characterization of the interleukin-1 receptor (IL-1R) on monocytes and polymorphonuclear cells.

Primary human monocytes and monocytic cells express an interleukin 1 receptor (IL-1R) which is similar in molecular weight and IL-1 binding characteristics to the IL-1R expressed on B lymphocytes (type II). Northern blot analysis of monocytic cells using a cDNA probe from the recently isolated type II IL-1R indicates that this mRNA is detectable by 4 h and accumulates for at least 24 h following treatment with IL-1R inducing drugs. The time course of induction of this mRNA is slower than that of the type I IL-1R mRNA which is also transcribed in monocytic cells but does not appear to be translated. Sequence analysis of a monocyte-derived cDNA corresponding to the type II IL-1R mRNA shows that the monocyte and B-cell mRNAs are identical. Comparison of monocyte IL-1R peptide maps with those of the type II IL-1R suggests that the two surface IL-1R are identical. This was confirmed serologically using a polyclonal antiserum raised against the type II IL-1R. Data are presented which indicate that primary human neutrophils can also be induced to express abundant type II IL-1R.     

Age-related defect in signal transduction during lectin activation of murine T lymphocytes

Interleukin 2 (IL-2) production and recognition are clearly involved in the age-associated proliferative defect of mitogen-stimulated T lymphocytes. The external signal delivered by mitogens is transmitted across the membrane via the release of two messenger molecules, diacylglycerol and inositol 1,4,5-trisphosphate (IP3), involved in the activation of protein kinase C (PK-C) and the elevation of cytosolic free Ca2+. In that Ca2+ mobilization and PK-C activation appear to be crucial events in the production of IL-2 and the expression of IL-2 receptors, a defect in transmembrane signaling would result in decreased synthesis and response to IL-2. We therefore examined PK-C activity and translocation, generation of inositol 1,4,5-trisphosphate, and cytosolic Ca2+ levels as a function of age in murine G0 T lymphocytes before and after exposure to mitogenic doses of concanavalin A (Con A). The basal levels and distribution of PK-C before and after direct activation of the enzyme by 2 or 20 nM phorbol myristate acetate were comparable in both age groups indicating no inherent age-associated functional defect in the enzyme. However, the Con A-induced PK-C translocation was reduced by 50% in cells from 24-mo-old animals. The Con A stimulation of G0 T lymphocytes increased free cytoplasmic Ca2+ concentration ([Ca2+]i) and the production of inositol phosphates to the same level, irrespective of the age of the donor. However, basal levels of both of these second messengers were consistently higher in lymphocytes derived from old mice. As a result, the net increase in inositol phosphates and [Ca2+]i was reduced by approximately the same extent as that observed for the translocation of PK-C. These results clearly point to an age-associated defect in the generation of phosphoinositide-derived second messengers and indicate that an alteration in signal transduction plays a primary role in the age-related impairment of the mitogen-induced, IL-2-mediated proliferative response of T lymphocytes.     

Immunomodulatory effects of seabuckthorn (Hippophae rhamnoides L.) against chromium (VI) induced immunosuppression

The present study reports the immunomodulatory effects of seabuckthorn (Hippophae rhamnoides L.) leaf extract on cellular and humoral immune response by studying delayed-type hypersensitivity response, IL-2, IL-4 and γ-IFN levels and antibody titres in chromium-induced immunosuppressed animals. Oral feeding of chromium (30 mg/kg bw) significantly inhibited antibody production and S-RBC induced delayed-type hypersensitivity response. Administration of leaf extract (100 mg/kg bw) along with chromium significantly inhibited chromium-induced immunosuppression. To understand the immunomodulatory mechanism of leaf extract, in vitro studies were carried out using rat lymphocytes. Addition of chromium resulted in a significant decrease in lymphocyte size and increased ROS generation. The leaf extract of seabuckthorn significantly inhibited chromium-induced reactive oxygen species (ROS) generation and maintained the cell size identical to that of control cells. Chromium treatment markedly inhibited the mitochondrial transmembrane potential by larger lymphocytes in particular, while the leaf extract restored the same significantly. Chromium also inhibited significantly concanavalin A (ConA) induced IL-2, IL-4 and γ-IFN production in rat lymphocytes. The leaf extract (100 μg/ml) alone stimulated IL-2 and γ-IFN production even in the absence of ConA and also inhibited chromium-induced decline in IL-2 and γ-IFN production but it did not change IL-4 production. These observations suggest that the leaf extract of seabuckthorn has significant immunomodulatory activity and specifically activates the cell-mediated immune response. (Mol Cell Biochem 278: 101–109, 2005)     

Activation of the TLR/MyD88/NF-κB Signal Pathway Contributes to Changes in IL-4 and IL-12 Production in Piglet Lymphocytes Infected with Porcine Circovirus Type 2 In Vitro

Porcine circovirus type 2 (PCV2) causes immunosuppression in pigs. One causative factor is an imbalance in cytokine levels in the blood and lymphoid tissues. Many studies have reported changes in cytokine production, but the regulatory mechanisms involved have not yet been elucidated. In this study, we investigated alteration and regulation of IL-4 and IL-12 production in lymphocytes following incubation with PCV2 in vitro. The levels of IL-4 decreased and levels of IL-12 increased in lymphocyte supernatants, and the DNA-binding activity of NF-κB and the expression of p65 in the nucleus and p-IκB in the cytoplasm of lymphocytes increased after incubation with PCV2. However, these effects were reversed when lymphocytes were coincubated with PCV2 and the NF-κB inhibitor BAY11-7082. In addition, the expression of MyD88 protein increased and the expression of mRNA for the toll-like receptors (TLRs) TLR2, TLR3, TLR4 and TLR9 was upregulated when lymphocytes were incubated with PCV2. However, no change was seen in TLR7 and TLR8 mRNA expression. In conclusion, this study showed that PCV2 induced a decrease in IL-4 and an increase in IL-12 production in lymphocytes, and these changes were regulated by the TLR-MyD88-NF-κB signal pathway.     

Orally administered Kampo (Japanese herbal) medicine, "Juzen-Taiho-To" modulates cytokine secretion in gut associated lymphoreticular tissues in mice.

The influence of the oral administration of Juzen-Taiho-To (JTT; Si-Quan-Da-Bu-Tang in Chinese), a Kampo (Japanese herbal) medicine, on the cytokine production in mice were investigated. Lymphocytes from spleen (SPN), mesenteric lymph node (MLN) and Peyer's patches (PP) from mice, which received orally administered JTT for 2 weeks, were stimulated with concanavalin A (Con A), and the resulting conditioned medium was tested for cytokine production by ELISA. Administration of JTT caused enhancement of interferon g (IFN-gamma) and interleukin-4 (IL-4) production to some extent but decreased IL-5 production from the SPN. On the other hand, notable changes in cytokine production were observed in lymphocytes from MLN and PP. Administration of JTT increased IFN-gamma production remarkably, as well as IL-5 secretion from both MLN and PP, whereas IL-2 secretion was plainly reduced. The ratio of IFN-gamma and IL-4 was shifted to Th1 dominant in MLN and PP, however changed little in SPN. The flow cytometric analysis revealed that the population of CD3+, CD4+, CD45R/B220+, and CD45RBlowCD4+ cells in each lymphocyte was not changed significantly after oral administration of JTT. These findings demonstrate that the lymphocytes from gut associated lymphoreticular tissues (GALT) are more sensitive to produce cytokines on cytokine production than those from SPN by oral administration of JTT, and that the modulation of cytokine production may be due to functional change of lymphocytes but not change in lymphocytes population.     

Production of and responsiveness to interleukin 2 in autoimmune BXSB mice

BXSB male mice serve as one of several murine models of human systemic lupus erythematosus. T-cell abnormalities in these mice involve decreased production of and responsiveness interleukin 2 (IL-2) and are age-related. The studies presented here investigated the mechanism of these T-cell defects. The results suggest that excessive suppressor-T-cell activity as well as soluble inhibitors of IL-2 production and activity, including PGE, are not responsible for the low levels of IL-2 observed in culture supernatants of Con A-stimulated lymphocytes from "old" (3-6 months) BXSB male mice. Supplementation of Con A-stimulated lymphocyte cultures from BXSB male mice with human IL-1 or normal murine accessory cells did not augment IL-2 production. Reduced proliferative responses were observed in bulk cultures of Con A- or alloantigen-stimulated "old" BXSB male lymphocytes, which were not enhanced by exogenous IL-2. Limiting dilution analysis revealed reduced frequencies of Con A- and alloantigen-inducible IL-2-reactive T cells in these mice. These results suggest intrinsic defects in the ability of T cells from "old" BXSB male mice to be activated to produce and respond to IL-2.     

VLDL modulates the cytokine secretion profile to a proinflammatory pattern.

Human very-low-density lipoprotein (VLDL) inhibits DNA synthesis in lymphocytes activated by the nonspecific mitogen concanavalin A (Con A). We studied the effects of VLDL on lymphocyte activation (IL-2 receptor expression), cell cycle progression, and production of IL-2 and of IL-4 (a proinflammatory and an anti-inflammatory interleukin, respectively) to understand why an atherogenic lipoprotein inhibits cell proliferation. After 48 h of stimulation with the mitogen, VLDL decreased the population of cells bearing IL-2 receptor and the population of T-cells that progress through the cell cycle, increasing the population of T-cells in G(0)/G(1). Cells cultured in the presence of Con A and VLDL produced higher levels of IL-2 and lower levels of IL-4 than cells cultured without VLDL. These results suggest that VLDL inhibits lymphocyte proliferation by reducing IL-2 receptor and enhancing the levels of IL-2. Probably, one atherogenic effect of VLDL is to modulate the cytokine secretion profile of lymphocytes to a predominantly proinflammatory response.     

Aging impairs induction of cyclin-dependent kinases and down-regulation of p27 in mouse CD4(+) cells

To define the link between the early activation defects and the impaired proliferation response of cells from old mice, we characterized the influence of age on expression and activity of proteins that participate in cell-cycle regulation. We found that aging led to significant declines in the ability of mouse CD4(+) T cells to respond to CD3 and CD28 stimuli by induction of the cyclin-dependent kinases CDK2, CDK4, and CDK6, whether the defect was assessed by protein level or functional activity. Induction of CDK2 activity was also impaired in cells from old mice that were activated with PMA plus ionomycin, stimuli that bypass the TCR/CD3 complex, or by CD3/CD28 in the presence of IL-2, indicating that the age-related changes lie, at least in part, downstream of the enzymes activated by these stimuli. We also noted an impairment in the ability of CD4(+) cells from old mice to down-regulate the CDK inhibitor p27 after activation, but we found no change in induction of p21, an inhibitor of CDK that may also play other roles in cell-cycle control. Altered CDK activation is likely to mediate the age-related decline in T cell proliferation to polyclonal stimulation.     

Interleukin-2 mRNA expression, lymphokine production and DNA synthesis in glutathione-depleted T cells

The stimulation of DNA synthesis in lymphocyte populations was previously shown to depend strongly on the intracellular glutathione (GSH) level. Since T cell growth is known to depend on interleukin 2 (IL-2), the experiments in this report were designed to determine whether intracellular GSH depletion may inhibit IL-2 production or the IL-2 dependent DNA synthesis. Our experiments revealed that IL-2 production and DNA synthesis of mitogenically stimulated splenic T cells have indeed different requirements for GSH. The addition of relatively high concentrations of GSH (5 mM) to cultures of concanavalin A (Con A)-stimulated splenic T cells was found to augment strongly the DNA synthesis but inhibited the production of IL-2. Moderate intracellular GSH levels, however, are apparently not inhibitory for IL-2 production, since intracellular GSH depletion by cysteine starvation or by graded concentrations of DL-buthionine sulfoximine (BSO) had virtually no effect on IL-2-specific mRNA expression and the production of T cell growth factor (TCGF). The DNA synthesis activity, in contrast, was strongly suppressed after GSH depletion with either method. As in cultures of splenic T cells, GSH depletion had no substantial effect on the induction of IL-2 mRNA and TCGF production in several mitogenically stimulated T cell clones. Taken together, our experiments suggest that complex immune response may operate best at intermediate GSH levels that are not too high to inhibit IL-2 production but sufficient to support DNA synthesis.     

Activation of human lymphocytes by concanavalin A or purified protein derivative results in no alteration of fluorescence polarization of lipid probes although the electrophoretic mobility of the cells is changed

Upon stimulation with either concanavalin A or the tuberculin antigen, purified protein derivative, human peripheral blood lymphocytes, purified on Ficoll-Hypaque, did not exhibit a concomitant lipid fluidity alteration as measured by fluorescence polarization (P) of the lipid probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). This result was independent of the incubation period, ranging from 10 min to 72 h. However, a general reduction in polarization value, from P = 0.287 (maintained for up to 2 h of incubation) to P = 0.225 after 20 h was observed for both experimental and control samples. Moreover, fluorescence polarization studies of the nonpenetrating modified DPH cationic lipid probe, 1-[4′-trimethylaminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), also failed to show any change in lipid fluidity subsequent to a 1–3 h incubation of lymphocytes with concanavalin A. Cell electrophoretic mobility, however, was altered (mean cell mobility increased by 10–15%) in a fast response to stimulation and was observed within several hours of in vitro application of concanavalin A and purified protein derivative. This initial response disappeared with further incubation at 37°C (>3 h) and was followed by a decline of cellular mobility of the concanavalin A-exposed cells after 48 and 72 h of incubation. The unstimulated control cells did not change in mobility as a function of incubation time. The slow decline in mean cell mobility of the experimental cells is believed to be associated with blastogenesis. It is concluded that neither blastogenic transformation nor short term membrane alterations associated with human lymphocyte activation lead to lipid fluidity changes as measured in steady state by the fluorescence polarization of both DPH and TMA-DPH.