β-endorphin modulation of interferon-γ, perforin and granzyme B levels in splenic NK cells: Effects of ethanol☆
Introduction
Among the several components of the immune system, natural killer (NK) cells provide first line defense by their unique cytolytic action. NK cells can act against target cells without the requirement of prior sensitization or major histocompatibility restriction (Ljunggren and Karre, 1990). NK cells are crucial until specific immunity is activated against pathogens (Yokoyama and Scalzo, 2002, French and Yokoyama, 2003). NK cell activity is primarily involved in defense against viral infections, control of tumor growth and metastasis (Barlozzari et al., 1983, Gorelik et al., 1982, Schantz et al., 1987, Lotzova, 1993, Brittenden et al., 1996, Smyth et al., 2001). The cytolytic activity of NK cells involves the synergistic action of the pore-forming protein perforin and the serine protease granzyme B to cause apoptosis of target cells (Heusel et al., 1994, Shresta et al., 1995, Jans et al., 1996). In addition, the cytolytic function of NK cells is also increased by interferon gamma (IFN-γ; Barry and Bleackley, 2002), one of the major cytokines released by NK cells. Through these cytokines, NK cells not only control viral infections in a non-cytolytic way but also activate dendritic cells and helper T-cells to shape the specific immune response. This well-orchestrated NK cell surveillance can be disturbed by several endogenous and exogenous agents.
Alcohol is one of the exogenous agents that can modulate the body's immune defense mechanism and can exacerbate malignancy and morbidity of several infections like human immunodeficiency virus (HIV-1), hepatitis-C (HCV) and mycobacterium infections (Roselle et al., 1995, Smith-Warner et al., 1998, Bagasra et al., 1993). Alcohol consumption increases the progression of HIV-1 infection and potentiates HCV infection toward hepatic cancer. There are several studies that indicate a strong association between alcohol consumption and cancer of the upper digestive and respiratory tracts (Pitot and Dragan, 1995, Seitz et al., 1998a, Seitz et al., 2004), hepatocarcinogenesis (Stickel et al., 2002), and colorectal and breast cancer (Seitz et al., 1998a, Seitz et al., 1998b). Most of the studies done in humans and animals provide evidence that alcohol impairs the immune system and causes defects in the cellular components of the innate and adaptive immune system (Cook, 1998, Szabo, 1999). Alcohol-induced immunosuppression can be partly due to abnormal NK cell functions in humans and animals (Boyadjieva et al., 2001, Wu et al., 1994, Wu and Pruett, 1997, Gallucci et al., 1994, Laso et al., 1997).
Alcohol effects on immune cells are dose- and time-dependent. A single binge dose of ethanol that increases blood concentrations of ethanol to .25–.50% (wt / vol) suppresses NK cell cytolytic activity in mice (Wu et al., 1994). However, administration of a moderate dose of ethanol that increases blood levels of ethanol to 0.1% (wt / vol) for a period of 7 days has no significant effect on NK cell cytolytic activity in rats. Chronic administration of the same dose of ethanol for 2 or 3 weeks has been shown to produce marked decrease in NK cell cytolytic activity (Boyadjieva et al., 2001). Alcohol effects are known to be manifested through the alteration of various neurotransmitters and neuropeptides of the central nervous system (CNS) and the peripheral systems (Herz, 1997). Among these neuropeptides, the opioidergic system has been shown to be a major target of ethanol (Oswald and Wand, 2004). Beta-endorphin (β-EP), an endogenous opioid peptide, is found in the hypothalamus, the pituitary (Ogawa et al., 1979, Salih et al., 1979) and peripheral cells such as lymphocytes (Blalock, 1999). β-EP is shown to increase NK cell cytolytic function in splenocytes (Mathews et al., 1983, Gatti et al., 1993, Boyadjieva et al., 2001). We and many other investigators showed the dual effect of the moderate dose of ethanol that increases blood ethanol concentration to 0.1% (wt/vol) on β-EP release; acute exposure of this dose of ethanol stimulates β-EP release whereas chronic ethanol treatment decreases CNS and peripheral levels of β-EP (Gianoulakis et al., 1989, Schulz et al., 1980, Genazzani et al., 1982, Seizinger et al., 1983, Gianoulakis and Barcomb, 1987, Boyadjieva and Sarkar, 1999, Boyadjieva et al., 2001). Although the acute exposure to moderate dose of ethanol does not affect NK cell cytolytic activity, the chronic exposure of this dose of ethanol decreases the NK cell cytolytic response and cytotoxic factor production by β-EP in the spleen (Boyadjieva et al., 2001, Dokur et al., 2004). The effects of ethanol or β-EP on the cytotoxic factors in the NK cells that regulate the cytolytic function of these cells have not been determined. Hence, in this study, we investigated the effects of β-EP and ethanol on perforin, granzyme B and the IFN-γ mRNA and protein levels in enriched NK cells, both in vivo and in vitro.
Section snippets
Animal feeding design
Male Fischer-344 rats of 150–175 g body weights were maintained on a 12-h light/dark cycle (lights on 0700 hours and lights off 1900 hours) and fed ad libitum with rodent chow, fed a liquid diet containing ethanol (8.7% v / v) or pair-fed an isocaloric liquid diet (Bio-Serv, Frenchtown, NJ). Graduated ball barrel cylinders containing the freshly prepared diet were placed in the animal cages 1 h before lights off at 1900 hours for 2 weeks. Animals were given free access to water. Animal surgery
The effect of β-EP perfusion on the mRNA levels of perforin, granzyme B and IFN-γ in enriched splenic NK cells
We have previously shown that Fischer-344 male rats fed an ethanol-containing liquid diet for a period of 2 weeks showed increased levels of blood alcohol (131.4 ± 21 mg/dl; Dokur et al., 2003). We have also shown that β-EP perfusion into the PVN increases splenic NK cell cytolytic activity as well as the splenic production of cytolytic factors in ad lib-fed and pair-fed rats, but not in ethanol-fed rats (Boyadjieva et al., 2001, Dokur et al., 2004). In this study using a similar ethanol feeding
Discussion
The present study was conducted with enriched NK cells to determine the effects of β-EP on NK cell functions. We found that β-EP increased the protein and mRNA levels of granzyme B, perforin and IFN-γ in enriched NK cells when perfused into the PVN of the hypothalamus in animals. β-EP also increased protein levels of these cytolytic activity-regulatory factors in NK cells in primary cultures, suggesting that the opioid peptide increases the production of granzyme B, perforin and IFN-γ by acting
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This investigation was supported by National Institutes of Health Grant AA12642.