Restraint Stress Alters Chemokine Cytokine mRNA Expression

Experiments were carried out in order to determine the effects of stress on chemokine/cytokine expression in the CNS and spleen (as an example of an immune organ). Groups of male CBA mice were (1) infected/restrained for 7 nights, (2) infected/nonrestrained, (3) noninfected/restrained, or (4) noninfected/nonrestrained. At sacrifice, their brains and spleens were removed and RNA isolated and incorporated in RNase Protection Assay (RPA) to estimate mRNA cytokine and chemokine expression. Infection with TMEV increased the following chemokine expression: lymphotactin (Ltn), interferon-induced protein (IP-10), macrophage inflammatory protein (MIP)-1ß, monocyte chemoattractant protein-1 (MCP-1), and thymus derived chemotactic agent (TCA)-3 in the spleen but not the brain at day 2p.i. The fact that chemokine expression was increased first in the spleen provides evidence that the immune response to TMEV is initiated in the periphery. Ltn, Regulated upon Activation Normal T cell Expressed and Secreted (RANTES), and IP-10 were elevated in both the spleen and the brain at day 7 p.i. and were significantly decreased by restraint in the brain.These chemokines are responsible for the recruitment of CD4+, CD8+ T cells, macrophages, and NK cells and thus may account for the diminished inflammatory cell infiltrate in the CNS of stressed mice and subsequently the reduced viral clearance and increased mortality in virus-infected restraint stressed mice (Mi et al., 2004).

In experiments examining cytokine expression, mice were subjected to the restraint paradigm and, at sacrifice, half the brain taken for viral infec-tivity assays and the other half for RPA analysis of cytokine RNA levels. TMEV infection elevated IFN-y, LT-ß, IL-12p40, IL-6, and IFN-ß in the brain at days 2 and 7. Importantly, restraint attenuated the increases in IFN-Y but elevated IFN-ß. RNA levels of IFN-y, LT-ß, and TNF-a were negatively correlated with viral titers in the CNS such that mice with higher cytokine levels had lower virus levels. Thus, these cytokines may play a role in the clearance of virus from the CNS. TNF-a protein levels, as measured by Western blots, gave similar results to the RPA data for this cytokine. Interestingly, stress increased the anti-inflammatory cytokine IL-10 in the spleen, which may contribute to the decrease in proinflammatory cytokine production (Mi et al., 2006b).

The cytokines altered by restraint stress in Theiler's virus infection have pleotropic effects and have vital roles in the neuropathogenesis of this disease. Lymphotoxin-ß, a membrane-bound form of lymphotoxin, plays a critical role in the resistance to intracellular pathogens including Theiler's virus (Lin et al., 2003). LT-ß induces IFN-ß and also increases cytotoxic T-cell activity, which are both important mediators of viral clearance from the CNS. IFN-y is an important inflammatory mediator produced by NK cells and T cells, which contributes on the one hand to viral clearance and on the other hand to development of demyelination in TVID. The suppressive effect of stress was first detected at day 2p.i. and attenuated at day 7p.i. Stress increased the anti-inflammatory cytokine IL-10 and decreased proinflammatory cytokines in the spleen. The increase in IL-10 may have contributed to the decrease in proinflammatory cytokines. Interestingly, stress also caused an increase in IFN-P expression in the brain, which may result from the higher levels of virus within the CNS of these mice, and this may compensate for the impaired viral clearance caused by decreased production of proinflammatory cytokine during stress.

ELISA assays examined the effects of restraint stress on IL-1P and TNF-a levels in serum. No detectable levels of IL-1P were observed in any of the groups of mice, but interestingly, restraint stress induced high levels of TNF-a in the serum of both infected and uninfected mice (Welsh et al., 2004).

To summarize our findings with regard to the effects of restraint stress on the expression of chemokines and cytokines: stress reduced the expression of chemokines responsible for the recruitment of CD4+, CD8+ T cells, macrophages, and NK cells;namely, Ltn, RANTES, and IP-10.Virus-induced IFN-y expression was also decreased by stress. IFN-y,TNF-a, and LT-P levels were negatively correlated with viral replication in the brain. These cytokines have important roles in the initiation of immune system activation and also have effective antiviral activities, and therefore lower levels of expression may also result in increased viral replication within the CNS.

Natural killer (NK) cells are known to be important in the early clearance of TMEV as demonstrated by depletion studies (Paya et al., 1989) and are also exquisitely sensitive to stress. Therefore we examined the effect of restraint stress on NK cell activity in CBA mice infected with TMEV. Twenty-four hours postinfection, restraint stress significantly reduced virus-induced NK cell activity in TMEV-infected CBA mice (Welsh et al., 2004) when compared with infected/nonrestrained mice. Decreased NK cell activity may also contribute to the reduced ability to clear virus.

In order to characterize the alterations in spleen cell populations that occur over time after TMEV infection and restraint stress, we conducted flow cytometric analysis experiments on splenocytes using combinations of the following directly labeled antibodies: (1) CD3-FITC, CD19-PE, CD45-PECy5 (leukocyte marker); (2) CD3-FITC, CD8-PE, CD4-PECy7; (3) F4/80-FITC (macrophage marker), DX5-PE (NK cell marker), CD45-PECy5. Preliminary results indicate that at both day 3 and day 7 p.i. in the spleen, restraint stress reduces NK cells and B cells while increasing numbers of T cells overall. No significant differences were seen in macrophages or between CD4+ or CD8+ cells (unpublished observations).

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