Elsevier

Behavioural Brain Research

Volume 165, Issue 1, 30 November 2005, Pages 110-125
Behavioural Brain Research

Research report
Perturbation of chemokine networks by gene deletion alters the reinforcing actions of ethanol

https://doi.org/10.1016/j.bbr.2005.06.026Get rights and content

Abstract

Microarray analysis of human alcoholic brain and cultured cells exposed to ethanol showed significant changes in expression of genes related to immune or inflammatory responses, including chemokines and chemokine receptors. To test the hypothesis that chemokines exhibit previously undiscovered pleiotropic effects important for the behavioral actions of ethanol, we studied mutant mice with deletion of the Ccr2, Ccr5, Ccl2 or Ccl3 genes. Deletion of Ccr2, Ccl2 (females) or Ccl3 in mice resulted in lower preference for alcohol and consumption of lower amounts of alcohol in a two-bottle choice test as compared with wild-type mice. Ethanol treatment (2.5 g/kg, i.p.) induced stronger conditioned taste aversion in Ccr2, Ccl2 or Ccl3 null mutant mice than in controls. Ccr2 and Ccr5 null mutant mice did not differ from wild-type mice in ethanol-induced loss of righting reflex (LORR), but mice lacking Ccl2 or Ccl3 showed longer LORR than wild-type mice. There were no differences between mutant strains and wild-type mice in severity of ethanol-induced withdrawal. Genetic mapping of chromosome 11 for the Ccl2 and Ccl3 genes (46.5 and 47.6 cM, respectively) revealed that an alcohol-induced LORR QTL region was contained within the introgressed region derived from 129/SvJ, which may cause some behavioral phenotypes observed in the null mice. On the contrary, known QTLs on Chr 9 are outside of 129/SvJ region in Ccr2 and Ccr5 (71.9 and 72.0 cM, respectively) null mutant mice. These data show that disruption of the chemokine network interferes with motivational effects of alcohol.

Introduction

Chemokines constitute a superfamily of small proteins (8–14 kDa) that induce chemotaxis, tissue extravasation and modulate the function of leucocytes during inflammation [51]. Approximately, 50 different human chemokines have been described and these chemokines may interact with18 different chemokine receptors [63], [87]. Upon ligand binding, all chemokine receptors activate G proteins, leading to the dissociation of the G protein heterotrimer into it's α and βγ subunits. G protein triggering initiates several parallel signaling cascades, which finally trigger Ca2+ release from intracellular stores. Transient elevation of intracellular Ca2+ is a well-characterized effect of chemokine stimulation and is widely used to study functional interaction of chemokine receptors and chemokines (see [19], for review). Chemokine-induced activation of different types of kinases, such as phosphatidyl inositol-3-OH kinase [80], the mitogen-activated protein kinase cascade [83] and activation of the janus kinase-signal transducers and activators of transcription pathway [59] are well-documented.

Chemokines are structurally related, with most containing four invariant cysteine residues. Depending on the arrangement of the first two of these cysteines, chemokines are divided into four subfamilies: CXC (α), CC (β), C (γ) and CX3C (σ) [63]. Recent evidence shows the chemokine family is not unique to mammals, with several members also identified in birds, amphibians and fish, including a primitive vertebrate, the lamprey [46].

As chemoattractant molecules, chemokines regulate cell trafficking through interactions with a set of their receptors [87]. For example, some evidence suggest that the migration of autoreactive immune cells via the blood–brain barrier is an early and critical process during the development of inflammatory CNS lesions of experimental allergic encephalomyelitis and multiple sclerosis, and that this transmigration is regulated by chemokines [38], [41]. In addition to chemotaxis, chemokines are also involved in the regulation of T cell differentiation, apoptosis, cell cycle and metastatic processes. Further, chemokines can control the generation of soluble inflammatory products, such as free radicals, nitric oxide, cytokines and matrix metalloproteases [87], [53].

Although originally identified on the basis of their ability to regulate the trafficking of immune cells, the biological role of chemokines goes well beyond this simple description of their function as chemoattractants, and they have been shown to be involved in a number of biological processes, including growth regulation, hematopoiesis, embryonic development and angiogenesis [36]. In vitro studies have demonstrated that, for example, SDF-1α/CXCL12 is a potent chemoattractant for several types of neural cells, including neuronal precursor cells from the external germinal layer [86], cortical neuronal progenitors [47], cerebellar granule neurons [52] and dentate gyrus granular neurons [4]. In vivo studies using genetically manipulated mice deficient in chemokine receptors or their ligand indicate a crucial role for chemokines in the brain development [88]. It is becoming evident that chemokines and their receptors play an important role in glial proliferation in the developing brain [5], [60], [62]. Chemokines may be also involved in the modulation of synaptic transmission in the brain. For example, the stimulation of mouse and rat CXCR2 receptors by the chemokines Gro-α/CXCL1, Gro-β/CXCL2 and IL-8/CXCL8 modulates fast synaptic transmission and long-term synaptic plasticity of the cerebellum through the activation of the extracellular signal-regulated kinase pathway [31], [67]. Such modulation of synaptic activity in the CNS has been broadly described for neutrophic factors, which are characterized by their important roles at different stages of neuronal development and their capacity to alter synaptic transmission in the brain [13], [37]. The similarities between the activities of chemokines on nerve cells to those of classical neurotrophins suggest that neuronal activity may be altered, at least in the cerebellum, as a consequence of neurodegenerative diseases, where chemokine expression is upregulated. Dysregulation of chemokine expression is associated with chronic inflammatory conditions, such as arthritis, atherogenesis, inflammatory bowel syndrome, glomerulonephritis, diabetes, endometriosis, transplant rejection and multiple myeloma [3], [29], [32], [69]. Chemokines and their receptors can be important to the molecular mechanisms of hyperalgesia [17] and diseases, such as Alzheimer's disease [73] and multiple sclerosis [79]. Some studies showed that chemokine receptors play crucial role in human immunodeficiency virus (HIV) infection [71]. Overall, the chemokines are important components of the endogenous host defense system [54], [57].

Alcohol is a frequently abused drug that inhibits numerous immune functions of the host (see [64], [74], for reviews). Enhanced production of CC chemokines is thought to contribute to the development of alcoholic liver disease. For example, elevated monocyte chemotactic protein-1 (Ccl2) levels were observed in sera and tissues of patients with alcoholic liver disease [28]. Prolonged consumption of alcohol is associated with elevated serum levels of proinflammatory cytokines and chemokines in humans and in experimental models of alcohol intoxication [8], [9], [33], [56].

It is interesting to note that microarray studies of brain tissue from human alcoholics [48], [58] and of cultured human cells exposed to ethanol [76] showed changes in expression of genes related to immune or inflammatory responses, including the chemokines (Ccl2, Ccl3) and the chemokine receptors (Ccr5, Ccr1 motif). However, research to date has focused on changes in chemokines (or cytokines) in response to ethanol administration. We asked a different question: do altered levels of chemokines or chemokine receptors affect the behavioral actions of ethanol? The purpose of the present study was to test the hypothesis that mice deficient in either chemokines Ccl2 and Ccl3 (macrophage inflammatory protein-1-alpha) or chemokine receptors Ccr5 and Ccr2 will show changes in preference and consumption of ethanol using a two-bottle choice paradigm. Our findings indicate that endogenous chemokine receptors may contribute to oral self-administration of ethanol, perhaps because they reduce aversive reactions to alcohol.

Section snippets

Animals

Null Ccl3 (−/−), Ccl2 (−/−), Ccr2 (−/−) and Ccr5 (−/−) allele mice were created using homologous recombination as previously described [20], [44], [45], [50]. Ccl3 (−/−) mice purchased from Jackson Laboratories, Bar Harbor, ME. Founders of Ccl2 (−/−) mice were a generous gift from Dr. Barrett J. Rollins (Dana Farber Cancer Institute). Ccr2 (−/−) and Ccr5 (−/−) null mutant mice were from colonies maintained at the University of Texas by Dr. W. Kuziel. The colony of double knockout Ccl2 (−/−) × 

Ethanol preference

In a two-bottle paradigm in which mice could drink either water or an ascending series of ethanol concentrations (3, 6, 9, 12 and 15%), males lacking Ccr2 displayed significantly reduced preference for ethanol (F(1,140) = 36.2, P < 0.0001) and a reduction in the amount of ethanol consumed (F(1,140) = 12.4, P < 0.0001) (Fig. 1a and c). Ccr5 (−/−) mutant males showed no change in ethanol consumption or preference (Fig. 1a and c). As for males, null Ccr2 females showed significant reduction in the amount

Discussion

A summary of the behavioral results obtained from all four mutant strains of mice are presented in Table 3. It is clear that deletion of either chemokines (Ccl2 or Ccl3) or chemokine receptors (Ccr2) leads to substantial reduction of alcohol consumption and preference. A possible explanation for this reduction could be increased aversion to alcohol. Indeed, all three mutants (Ccl2, Ccl3 and Ccr2) demonstrated stronger conditioned taste aversion to ethanol than control mice. Reduced preference

Summary

Deletion of chemokine receptors (Ccr2) or chemokines (Ccl2 and Ccl3) in mice resulted in lower preference for alcohol and consumption of lower amounts of alcohol in a two-bottle choice test as compared with wild-type mice. Ethanol treatment (2.5 g/kg, i.p.) induced stronger conditioned taste aversion in Ccr2, Ccl2 or Ccl3 null mutant mice than in controls. Ccr2 and Ccr5 (chemokine receptor) null mutant mice did not differ from wild-type mice in ethanol-induced loss of righting reflex. In

Acknowledgment

This work was supported by the National Institute of Alcohol Abuse and Alcoholism, NIH (AA U01 13520-INIA Project: AA06399).

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