β-endorphin modulation of interferon-γ, perforin and granzyme B levels in splenic NK cells: Effects of ethanol

https://doi.org/10.1016/j.jneuroim.2005.03.015Get rights and content

Abstract

The effects of ethanol and beta-endorphin (β-EP) on productions of cytolytic factors granzyme B, perforin and IFN-γ in splenic rat NK cells were determined. Intracranial administration of β-EP increased protein and mRNA levels of cytolytic factors in NK cells. Chronic ethanol feeding reduced the basal and β-EP-induced levels of cytolytic factors in NK cells. In vitro treatment of β-EP on NK cells increased the levels of perforin, granzyme B and IFN-γ and their mRNA transcripts, whereas ethanol pre-treatment prevented β-EP effects on cytolytic factors in these cells. These results suggest that β-EP and ethanol interact to regulate NK cell functions.

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

References (81)

  • J.W. Heusel et al.

    Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells

    Cell

    (1994)
  • M.R. Irwin et al.

    Central corticotropin-releasing factor suppresses natural killer cytotoxicity

    Brain Behav. Immun.

    (1987)
  • D.A. Jans et al.

    Nuclear transport of granzyme B (fragmentin-2). Dependence of perforin in vivo and cytosolic factors in vitro

    J. Biol. Chem.

    (1996)
  • H. Jiang et al.

    Distinct patterns of cytokine gene suppression by the equivalent effective doses of cyclosporine and tacrolimus in rat heart allografts

    Immunobiology

    (2000)
  • K. Kontani et al.

    Involvement of granzyme B and perforin in suppressing nodal metastasis of cancer cells in breast and lung cancers

    Eur. J. Surg. Oncol.

    (2001)
  • S.M. Levy et al.

    Persistently low natural killer cell activity and circulating levels of plasma beta endorphin: risk factors for infectious disease

    Life Sci.

    (1991)
  • H.G. Ljunggren et al.

    In search of the ‘missing self’: MHC molecules and NK cell recognition

    Immunol. Today

    (1990)
  • N. Mozzanica et al.

    Association between circadian rhythms of endogenous hypothalamic opioid peptides and of natural killer cell activity

    Int. J. Immunopharmacol.

    (1991)
  • L.M. Oswald et al.

    Opioids and alcoholism

    Physiol. Behav.

    (2004)
  • A.E. Panerai et al.

    The beta-endorphin inhibition of mitogen-induced splenocytes proliferation is mediated by central and peripheral paracrine/autocrine effects of the opioid

    J. Neuroimmunol.

    (1995)
  • P.M. Plotsky

    Opioid inhibition of immunoreactive corticotropin-releasing factor secretion into the hypophysial–portal circulation of rats

    Regul. Pept.

    (1986)
  • H. Salih et al.

    Cellular distribution of β-endorphin-like substance in the rat pituitary and brain

    Life Sci.

    (1979)
  • B.R. Seizinger et al.

    Differential effects of acute and chronic ethanol treatment on particular opioid peptide systems in discrete regions of rat brain and pituitary

    Pharmacol. Biochem. Behav.

    (1983)
  • N.A. Shahabi et al.

    Expression of delta opioid receptors by splenocytes from SEB-treated mice and effects on phosphorylation of MAP kinase

    Cell. Immunol.

    (2000)
  • B.M. Sharp et al.

    Delta-opioid suppression of human immunodeficiency virus-1 expression in T cells (Jurkat)

    Biochem. Pharmacol.

    (1998)
  • S.E. Street et al.

    Perforin and interferon-gamma activities independently control tumor initiation, growth, and metastasis

    Blood

    (2001)
  • C. Tsigos et al.

    Hypothalamic–pituitary–adrenal axis, neuroendocrine factors and stress

    J. Psychosom. Res.

    (2002)
  • W.M. Yokoyama et al.

    Natural killer cell activation receptors in innate immunity to infection

    Microbes Infect.

    (2002)
  • L. Azzoni et al.

    Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity

    J. Immunol.

    (2002)
  • O. Bagasra et al.

    Alcohol intake increases human immunodeficiency virus type 1 replication in human peripheral blood mononuclear cells

    J. Infect. Dis.

    (1993)
  • T. Barlozzari et al.

    In vivo role of natural killer cells: involvement of large granular lymphocytes in the clearance of tumor cells in anti-asialo GM1-treated rats

    J. Immunol.

    (1983)
  • M. Barry et al.

    Cytotoxic T lymphocytes: all roads lead to death

    Nat. Rev., Immunol.

    (2002)
  • J.E. Blalock

    Proopiomelanocortin and the immune-neuroendocrine connection

    Ann. N.Y. Acad. Sci.

    (1999)
  • N. Boyadjieva et al.

    Chronic ethanol inhibits NK cell cytolytic activity: role of opioid peptide β-endorphin

    J. Immunol.

    (2001)
  • N.I. Boyadjieva et al.

    β-endorphin modulation of lymphocyte proliferation, effects of ethanol

    Alcohol., Clin. Exp. Res.

    (2002)
  • N.I. Boyadjieva et al.

    Opioid antagonist naltrexone disrupts feedback interaction between mu and delta opioid receptors in splenocytes to prevent alcohol inhibition of NK cell function

    J. Immunol.

    (2004)
  • J. Brittenden et al.

    Natural killer cells and cancer

    Cancer

    (1996)
  • M.J. Brownstein

    A brief history of opiates, opioid peptides, and opioid receptors

    Proc. Natl. Acad. Sci. U. S. A.

    (1993)
  • R. Buzzetti et al.

    A critical assessment of the interactions between the immune system and the hypothalamo–pituitary–adrenal axis

    J. Endocrinol.

    (1989)
  • R. Buzzetti et al.

    A critical assessment of the interactions between the immune system and the hypothalamo–pituitary–adrenal axis

    J. Endocrinol.

    (1989)
  • Cited by (17)

    • In vivo immunoprotective role of Indigofera tinctoria and Scoparia dulcis aqueous extracts against chronic noise stress induced immune abnormalities in Wistar albino rats

      2017, Toxicology Reports
      Citation Excerpt :

      Thereafter, 5 μm thick sections were prepared and stained with haematoxylin and eosin (H&E) and histopathological examination were observed [42] under a light microscope (Olymbus-BX43, Japan). Expression of Granzyme B, Perforin and TNF- α were analyzed by Western blot [43]. Total protein was extracted from the spleen of different group animal and equal amount of protein (40 μg) was separated by 12% SDS-PAGE.

    • Opiate antagonist prevents μ- and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth

      2012, Journal of Biological Chemistry
      Citation Excerpt :

      These opioid peptides regulate NK cell functions by acting directly on the spleen and by altering the neuroendocrine-immune system function (1–3). These opioid peptides are also involved in controlling ethanol-modulating effects on innate immune function (4–6). In the central nervous system, β-endorphin and enkephalins are known to bind to classical DOR and MOR (7).

    • Endogenous opiates and behavior: 2005

      2006, Peptides
      Citation Excerpt :

      Whereas peripherally acting naloxone methiodide decreased interleukin-1-beta-induced Fos activity in the medial PVN CRF neurons and increased Fos in ventrolateral medullary epinephrine cells and NTS NE cells, centrally acting naloxone produced decreases in the PVN, ventrolateral medulla and NTS, but increased Fos in the central amygdala and BNST [139]. Chronic ethanol reduced basal and BEND-induced levels of cytolytic factors in splenic rat NK cells [296]. Splenocyte proliferations were inhibited by methadone in concentration-dependent fashion, by fentanyl in a bell-shaped curve, and were induced in concentration-dependent fashion by NBNI [523].

    View all citing articles on Scopus

    This investigation was supported by National Institutes of Health Grant AA12642.

    View full text