Introduction

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Chronic or repeated exposure to alcohol can result in a variety of health problems, including damage to the brain. Because the brain is particularly susceptible to ethanol toxicity, understanding the pathogenesis of alcohol-related brain damage becomes very important for developing more effective treatments and preventative measures for alcohol abuse. Glial cells are the most prevalent cell type in the brain. Interactions between the numerous glial cells and neurons are tantamount to a fully functional brain. Astrocytes are a crucial target of ethanol during CNS development and are profoundly affected by prenatal ethanol exposure. Ethanol can affect DNA, RNA, and protein synthesis in primary cultures of rat cortical astrocytes and can suppress astrocyte mitogenesis (Aroor and Baker, 1997). Ethanol also reduces the capacity of astrocytes to secrete growth factors (Valles et al., 1994), induces oxidative stress in astrocytes (Montoliu et al., 1995), and alters the development, content, and distribution of several cytoskeletal proteins, including transcription of the astrocyte marker glial acidic fibrillary protein (Valles et al., 1997). Thus, ethanol-induced alterations in astrocyte gene expression could be important mechanisms underlying the CNS dysfunction observed after prenatal exposure to ethanol (Guerri and Renau-Piqueras, 1997). Chronic alcohol abuse also affects adult glial cells. Human alcoholic brains show clinical and pathological evidence of significant astrocyte activation and glial cell loss in both gray and white matter regions (Hunt and Nixon, 1993; Korbo, 1999).

It is likely that chronic ethanol effects on astrocyte gene expression play a role in the pathogenesis of alcohol-related brain damage. Identity of these genes and their products can provide insight into the etiology of alcohol-related brain damage and possibly alcoholism itself. Genes that play a role in host defense mechanisms may be particularly important targets related to the pathophysiology of alcohol abuse and alcoholism (Cook, 1998); however, there is a paucity of data regarding alcohol and CNS host defense genes and mechanisms.

We have previously demonstrated that the inducible nitric-oxide synthase is one host defense gene in the astrocyte-derived C6 cell line susceptible to ethanol exposure (Syapin, 1995; Militante et al., 1997; Syapin et al., 2001). To identify additional ethanol-responsive genes potentially related to host defense mechanisms and alcohol-related brain damage, we have used differential display of mRNA (Liang and Pardee, 1992) to screen the expressed genes in chronic ethanol-treated rat C6 glial cells after activation with bacterial LPS combined with PMA. With this technique we have detected several differentially regulated genes possibly involved in altered CNS host defense mechanisms or other responses to chronic ethanol (L. Ren and P. J. Syapin, unpublished data), including unsuspected genes such as that coding for fibronectin (Ren et al., 2000).

In this article, we report our findings on an important host defense gene whose expression is differentially regulated by acute and chronic ethanol exposure in activated C6 glial cells, the chemokine MCP-3. Chemokines are low-molecular-weight secreted proteins and represent a subfamily of cytokines with strong chemotactic activity produced in response to proinflammatory signals such as LPS, TNF, and interleukin-1. MCP-3 is a member of the CC or -chemokine subfamily that includes the closely related MCP-1, MIP-1, and regulated upon activation normal T cell expressed and secreted. MCP-3 is a broad-spectrum chemokine, being chemotactic for monocytes, lymphocytes, eosinophils, basophils, natural killer cells, and dendritic cells (Rollins, 1997). Inducible expression of MCP-3 by astrocytes has been documented previously (Hesselgesser and Horuk, 1999; Hua and Lee, 2000), but we are unaware of any studies demonstrating interactions of MCP-3 expression with ethanol exposure in astrocytes or any other cell type. Given the large number of cells that are attracted by MCP-3, its suppression by ethanol would be expected to severely dampen immune and inflammatory responses, an effect that may contribute to the pathogenesis of alcohol-related brain damage.

	Materials and Methods

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Cell Culture. Stock cultures of C6 glioma cells (Benda et al., 1968) were originally obtained from the American Type Culture Collection (Manassas, VA) and cultured using methods and supplies described previously (Syapin, 1995; Militante et al., 1997). In brief, cultures were grown in high glucose-containing Dulbecco's modified Eagle's medium with 5% (v/v) fetal bovine serum added. Experimental cultures were plated at the appropriate cell density for their intended time of growth as described in Syapin (1995) and grown in medium containing 2.5% (v/v) fetal bovine serum. All cultures were maintained at 37°C inside humidified incubators with 5% CO₂, 95% air. Culture medium was replenished 2 to 3 days after seeding and every other day thereafter, unless otherwise noted.

Ethanol Treatment. Cells were treated with ethanol as for previous studies (Syapin et al., 1999, 2001; Ren et al., 2000). Concentrations used for acute exposure were from 50 to 300 mM. Chronic exposure was to 50 mM ethanol. Chronic treatment began upon initiating the experimental cultures and continued for up to 9 days before cell activation and sample collection. The cultures were grown in 1.8-liter sealed rectangular storage containers (Rubbermaid, Wooster, OH) containing a 300-ml reservoir of aqueous ethanol at the same concentration as in the medium. The atmosphere inside the containers was equilibrated with compressed 5% CO₂/95% air just before sealing and placement inside a water-jacketed 37°C CO₂ incubator. Control cells were handled the same way except the reservoir contained only water.

Cell Activation. LPS (Escherichia coli; Sigma-Aldrich, St. Louis, MO) stock solutions were prepared at 1 mg/ml in cartridge-purified deionized water, filter-sterilized, and stored frozen at 20°C. An LPS working solution (50 µg/ml) was prepared from the stock into fresh assay medium (serum-free Dulbecco's modified Eagle's medium) and used within the hour. Stock PMA (Sigma-Aldrich) was dissolved in acetone (Fisher Scientific, Houston, TX) at 0.5 mg/ml and stored at 20°C. The PMA working solution (40 µg/ml) was prepared fresh on ice into assay medium under subdued lighting and used immediately thereafter. The low level of acetone added to the cells as vehicle was tested and found not to affect the response. C6 glial cells were activated by simultaneous exposure to 500 ng/ml LPS and 400 ng/ml PMA, as described previously (Syapin, 1995; Militante et al., 1997). Activation was initiated by removing the growth medium, rinsing the culture dish once with assay medium, adding fresh assay medium, and then adding aliquots of the LPS and PMA. For cells exposed to ethanol, the assay medium contained the appropriate ethanol concentration during all steps and the subsequent 24-h incubation. Controls consisted of unstimulated cells exposed and not exposed to ethanol. Human recombinant TNF (Collaborative Biomedical Products, Bedford, MA) stock solutions (6 µg/ml) were prepared in sterile phosphate-buffered saline containing 0.2% bovine serum albumin (Sigma-Aldrich) and stored at 4°C. An aliquot of the TNF stock solution was diluted 1:1 (v/v) with assay medium and added to a final concentration of 30 ng/ml. Exposure to the stimulating agents was for 24 h in this study.

mRNA Differential Display. The isolation of total cellular RNA, treatment with DNase I, and performance of the reverse transcription and polymerase chain reaction for differential display were performed exactly as described previously (Ren et al., 2000). Briefly, we used the HIEROGLYPH mRNA profile kit, the FluoroDD TMR-fluorescent-anchored primer adapter kit for the HIEROGLYPH mRNA profile kit system, a Genomyx LR programmable DNA sequencer, and a Genomyx SC fluorescence scanner (Beckman Coulter, Inc., Fullerton, CA). Conditions for electrophoresis and gel band reamplification were exactly as described previously (Ren et al., 2000). The reamplified PCR product was used as the template for direct DNA sequencing on an ABI prism genetic analyzer (PerkinElmer Life Sciences, Boston, MA).

RT-PCR Analysis of MCP-3 mRNA from Total RNA. Total RNA was extracted using the guanidinium isothiocyanate procedure (Chomczynski and Sacchi, 1987). Aliquots (0.2 µg) of RNA from each treatment group were reverse transcribed with random primers (Promega, Madison, WI) and 1 µl of the cDNA mixture was subjected to PCR using specific oligonucleotide primers (5'-GGT ACC ACT CTC TTT CTC CAC CAT G-3' and 5'-AAG CTT ACA GCG GTG AGG AAT TTT GC-3') shown previously to detect rat MCP-3 mRNA (Natori et al., 1997). The same cDNA mixture was also subjected to PCR in a separate tube using a pair of primers (5'-ACG TCA ACA CTG CTC TAC A-3' and 5'-CTT TGC CAT AGT CCT TAA C-3') specific for ribosomal S12 RNA (Ayane et al., 1989). The expected size of the amplification products was 370 base pairs for MCP-3 and 311 base pairs for S12. A number of PCR cycles and denaturation temperatures were examined with scanning densitometry to ascertain a linear working range for both PCR products. After electrophoretic separation on agarose, ethidium bromide-stained gels were photographed with a Polaroid camera system. Quantification was achieved by band densitometry of photographed gels using an Alpha imager (Alpha Innotech Corporation, San Leandro, CA) followed by normalization to the corresponding S12 ribosomal protein cDNA band density as reported previously (Ren et al., 2000). S12 mRNA served as an internal standard to verify whether equal quantities of cDNAs were amplified and allowed for an estimation of the integrity of the extracted RNA. This abundant RNA does not seem to change significantly upon addition of ethanol or LPS plus PMA to cultured C6 cells and is adequately stable (Fig. 7B). Significant within-group biological variation in the MCP-3 response to stimuli was seen in some experiments (Fig. 3B), leading to larger than usual standard errors.

RT-PCR Analysis of MCP-3 mRNA from Nuclear RNA. Nuclei isolation, nuclear run-on transcription using nonradioactive dNTPs, and extraction and precipitation of nuclear RNA were carried out according to standard procedures (Greenberg and Bender, 1997). After the reverse transcription of nuclear RNA, PCR analysis was used to detect MCP-3 mRNA with normalization to S12 mRNA as described above.

Determination of MCP-3 mRNA Stability. To measure RNA stability, control cultures were first stimulated with LPS plus PMA for 24 h to induce MCP-3 mRNA expression. After induction, the media were removed from the cultures, and they were rinsed once with fresh 37°C assay medium. This was followed by application of new assay medium containing 25 µg/ml 5,6-dichlorobenzimidazole riboside (DBR), an inhibitor of mRNA synthesis (Tamm et al., 1976). The cultures were then placed in individual storage containers, equilibrated with 5% CO₂, 95% air, and returned to the incubator as described under "Ethanol Treatment". To examine the effect of ethanol on RNA stability, the stimulated cultures were separated into control and ethanol-treated groups. The ethanol-treated group was handled in the same way as the controls except the assay medium and reservoir inside the storage containers contained 200 mM ethanol. At designated times after DBR addition control and ethanol-treated cultures were removed from the incubator and processed as described under "RT-PCR Analysis of Total RNA". The DBR (Sigma-Aldrich or ICN Pharmaceuticals Biochemicals Division, Aurora, OH) was prepared as a 12.5-mg/ml stock solution in dimethyl sulfoxide (Sigma-Aldrich) and stored at 20°C between uses.

	Results

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Differential Display of MCP-3 mRNA in LPS/PMA-Stimulated C6 Cells. The differential display of mRNA technique (Liang and Pardee, 1992) was used to isolate genes that are up- or down-regulated in chronic 50 mM ethanol-treated rat C6 glial cells with and without activation by LPS/PMA. One of the mRNAs found to be regulated under these conditions was a cDNA referred to as band 20C42 (Fig. 1). This band was detected using the TMR-anchored primer 5'-ACG ACT CAC TAT AGG GCT TTT TTT TTT TTG G-3' and the arbitrary primer 5'-ACA ATT TCA CAC AGG AGA CCA TTG CA-3' in the RT-PCR. After reamplification of this band, sequence analysis of the PCR product revealed 96% nucleotide sequence identity in a 430-base pair fragment that overlapped the 3' end of the rat MCP-3 mRNA sequence (Wang et al., 1999). This result clearly indicated that this differentially expressed cDNA corresponded to that of the rat MCP-3 transcript. Figure 1 is from the differential display gel. It shows that expression of this mRNA was markedly increased by 24 h LPS/PMA stimulation of control C6 cells. In addition, it shows that 9 days of chronic exposure to 50 mM ethanol before LPS/PMA stimulation seemed to significantly reduce its expression.

View larger version (71K): [in this window] [in a new window]   Fig. 1.   Identification of gene expression changes by differential display in 24-h LPS/PMA-stimulated rat C6 glial cells treated chronically with 50 mM ethanol. mRNA differential display was carried out using an anchored primer (5'-ACG ACT CAC TAT AGG GCT TTT TTT TTT TTG G-3') and an arbitrary primer (5'-ACA ATT TCA CAC AGG AGA CCA TTG CA-3'). PCR products were resolved in a 5.6% denaturing polyacrylamide gel in the following order: lanes A, control of untreated and unstimulated C6 glial cells; lanes B, LPS/PMA-stimulated C6 glial cells; lanes C, ethanol-treated C6 glial cells; and lanes D, LPS/PMA-stimulated C6 glial cells treated with ethanol. The band indicated by an arrowhead (designated as band 20C42) showed a marked change in expression in response to LPS/PMA stimulation and ethanol treatment. M denotes nucleotide size marker lane, with values shown above bands.

Time Course of LPS/PMA-Stimulated MCP-3 mRNA Expression in Chronic Ethanol-Treated C6 Glial Cells. To confirm the differential display data and to obtain further information on 50 mM ethanol effects on MCP-3 mRNA expression in C6 glial cells, we compared expression levels in control cells and cells exposed to 50 mM ethanol for 1, 5, and 9 days with and without stimulation with LPS/PMA. For these and all subsequent studies specific MCP-3 primers were used during PCR (see Materials and Methods). As shown in Fig. 2, chronic 50 mM ethanol treatment for 9 days significantly decrease normalized MCP-3 mRNA expression in LPS/PMA-stimulated C6 glial cells (P < 0.05), whereas basal expression in unstimulated cells was unchanged by chronic ethanol exposure.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Differential expression of MCP-3 mRNA in 24-h LPS/PMA-stimulated rat C6 glial cells treated with ethanol for different times using semiquantitative PCR. PCR was carried out using the primer pairs for MCP-3 and S12 listed under Materials and Methods. A, representative ethidium bromide-stained PCR gel. Total RNA was isolated from cells treated with and without 50 mM ethanol for the number of days designated after 24-h stimulation with and without LPS/PMA, as shown above lanes. M denotes DNA molecular size marker lane. B, normalization of MCP-3 PCR product to corresponding S12 PCR product as IDV ratios (means with S.E.M.; n = 3/group). One-way ANOVA indicated significant differences between stimulated groups (p = 0.0028). , p < 0.001 versus control (0-day)-stimulated group by Dunnett's multiple comparison test. Results are representative of three independent experiments.

Dose-Dependent Effects of Ethanol on MCP-3 mRNA Expression during 24-h LPS/PMA Stimulation. As can be seen from the previous results (Fig. 2), 1 and 5 days of exposure to 50 mM ethanol did not reduce MCP-3 mRNA expression in LPS/PMA-stimulated C6 glial cells. Because the 1-day ethanol exposure coincided with the 24-h LPS/PMA stimulation, this treatment was equivalent to an acute ethanol exposure. Further investigations were aimed at identifying acute effects of ethanol on MCP-3 mRNA expression in C6 glial cells. Therefore, C6 cells were stimulated with LPS/PMA in the presence of 0, 50, 100, 150, 200, and 300 mM concentrations of ethanol, followed 24 h later by RT-PCR analysis of total RNA to measure MCP-3 mRNA in the cells. As shown in Fig. 3, acute treatment with 50, 100, and 150 mM ethanol could not significantly alter LPS/PMA-stimulated MCP-3 mRNA expression, whereas statistically significant inhibition was observed at 200 and 300 mM concentrations.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Effect of acute ethanol exposure on expression of MCP-3 mRNA in LPS/PMA-stimulated C6 cells using semiquantitative PCR. PCR was carried out using the primer pairs for MCP-3 and S12 listed under Materials and Methods. A, representative ethidium bromide-stained PCR gel. Samples were isolated from cells stimulated with and without LPS/PMA for 24 h in the presence or absence of ethanol as shown above lanes. B, normalization of MCP-3 PCR product to corresponding S12 PCR product as IDV ratios (means with S.E.M.; n = 3/group). One-way ANOVA indicated significant differences between stimulated groups (p = 0.0082). , p < 0.05 and , p < 0.01 versus control (0 mM)-stimulated group by Dunnett's multiple comparison test. Results are representative of two independent experiments.

Effect of Acute and Chronic Ethanol Exposure on TNF-Stimulated MCP-3 mRNA Expression. TNF is a cytokine that can also stimulate MCP-3 mRNA expression in C6 glial cells. To determine whether the effects of ethanol are stimulus-specific, we also performed acute and chronic ethanol exposure studies on cells activated by TNF. Data for acute ethanol experiments are shown in Fig. 4. The results are virtually identical to those obtained when LPS/PMA was used to induce MCP-3 mRNA (compare Figs. 3 and 4). Ethanol concentrations of 200 mM and greater elicited a statistically significant reduction in normalized MCP-3 mRNA. As seen previously, ethanol had no effect on basal MCP-3 mRNA levels. Additional studies indicated that exposure to IL-1 (2 ng/ml) also induced MCP-3 mRNA expression and that acute treatment with 200 mM ethanol was able to inhibit this induction (data not shown).

View larger version (60K): [in this window] [in a new window]   Fig. 4.   Expression of MCP-3 mRNA in 24-h TNF-stimulated rat C6 glial cells treated with different concentrations of ethanol using semiquantitative PCR. PCR was carried out using the primer pairs for MCP-3 and S12 listed under Materials and Methods. A, representative ethidium bromide-stained PCR gel. Samples were isolated from cells stimulated with and without TNF for 24 h in the presence or absence of ethanol as shown above lanes. B, normalization of MCP-3 PCR product to corresponding S12 PCR product as IDV ratios (means with S.E.M.; n = 3/group). One-way ANOVA indicated significant differences between stimulated groups (p = 0.0123). , p < 0.05 and , p < 0.01 versus respective control (0-day)-stimulated group by Dunnett's multiple comparison test. Results are representative of two independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Differential expression of MCP-3 mRNA in TNF-stimulated C6 glial cells chronically exposed to 50 mM ethanol. PCR was carried out using the primer pairs for MCP-3 and S12 listed under Materials and Methods. A, representative ethidium bromide-stained PCR gel. Total RNA was isolated from cells treated with and without 50 mM ethanol for the number of days designated after 24-h stimulation with and without TNF, as shown above lanes. M denotes DNA molecular size marker lane. B, normalization of MCP-3 PCR product to corresponding S12 PCR product as IDV ratios (means with S.E.M.; n = 3/group). One-way ANOVA indicated significant differences between stimulated groups (p = 0.0002) and between unstimulated groups (p = 0.0009). , p < 0.05 and , p < 0.001 versus respective control (0-day)-stimulated group by Dunnett's multiple comparison test. Results are representative of two independent experiments.

Effect of Ethanol on MCP-3 Nuclear Gene Transcription. To investigate whether the suppressive effect of acute and chronic ethanol on total cellular MCP-3 mRNA induction was due to changes in transcriptional activity, we examined MCP-3 RNA levels in isolated nuclei. We followed procedures for the standard nuclear run-on transcription assay, but used RT-PCR to detect the transcripts rather than membrane hybridization. As shown in Fig. 6A, MCP-3 RNA transcripts were greatly reduced in nuclei isolated from cells acutely exposed to 200 mM ethanol during stimulation. When the experiment was repeated using cells chronically exposed to 50 mM ethanol for 9 days, identical results were obtained (Fig. 6B). The effect of acute and chronic ethanol exposure on nuclear MCP-3 RNA was very pronounced. In fact, there were no statistical differences between stimulated and unstimulated values from ethanol-exposed cells. This is in contrast to the control group where the nuclear content of MCP-3 RNA was significantly increased by LPS/PMA stimulation.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effect of ethanol on differential expression of nuclear MCP-3 RNA in LPS/PMA-stimulated and unstimulated cells. Total nuclear RNA isolation and semiquantitative PCR were carried out as described under Materials and Methods. A, nuclear MCP-3 RNA content normalized to nuclear S12 RNA content from cells stimulated in the presence and absence of 200 mM ethanol. One-way ANOVA indicated significant differences between groups (p = 0.0177). , p < 0.05 versus either unstimulated group or the ethanol-stimulated group by Newman-Keuls multiple comparison test. No other comparisons were statistically significant (p > 0.05). B, nuclear MCP-3 RNA content normalized to nuclear S12 RNA content from cells treated with and without 50 mM ethanol for 9 days before stimulation with and without LPS/PMA. One-way ANOVA indicated significant differences between groups (p = 0.0061). , p < 0.01 versus either unstimulated group or the ethanol-stimulated group by Newman-Keuls multiple comparison test. No other comparisons were statistically significant (p > 0.05). Values are mean integrated density value ratios with S.E.M. (n = 3/group). Results are representative of three independent experiments.

Effect of Acute Ethanol on MCP-3 mRNA Stability. Experiments were also performed to determine whether ethanol had any direct effects on the stability of MCP-3 mRNA. Control cultures were stimulated with LPS/PMA for 24 h to enhance MCP-3 mRNA levels, followed by treatment with the adenosine analog DBR to inhibit further mRNA synthesis. Ethanol (200 mM) was added along with the DBR to examine its effects. MCP-3 mRNA levels were determined every 3 h over a 12-h period. As seen in Fig. 7A, normalized MCP-3 mRNA levels were relatively unchanged in control cells over the 12-h post-DBR, whereas acute ethanol exposure led to significant degradation by 9- and 12-h post-DBR. These results suggest that posttranslational stabilization of MCP-3 message occurs after LPS/PMA exposure and that acute ethanol can disrupt the stabilization process.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effect of acute ethanol on MCP-3 mRNA stability. After being activated with LPS/PMA for 24 h, C6 glial cells were treated with DRB, an inhibitor of RNA synthesis, in the absence (control) and presence (ethanol) of 200 mM ethanol. Cellular total RNA was isolated at different times after treatment and semiquantitative PCR was carried out using the primer pairs for MCP-3 and S12 listed under Materials and Methods. A, MCP-3 mRNA content normalized to nuclear S12 mRNA content from unstimulated cells and cells stimulated in the absence and presence of 200 mM ethanol. One-way ANOVA indicated significant differences between ethanol-exposed groups (p = 0.0019). , p < 0.05 and , p < 0.001 versus 0-h-stimulated group by Dunnett's multiple comparison test. Values are mean integrated density value ratios with S.E.M. (n = 3/group). Results are representative of four independent experiments. B, integrated band density values (arbitrary units) obtained by densitometry (see Materials and Methods) for S12 mRNA used for normalization purposes in A. One-way ANOVA indicated no significant differences for stimulated or stimulated + ethanol groups (p > 0.05), nor between unstimulated, stimulated, and stimulated + ethanol groups at time 0 h (p > 0.05). Values (n = 6/group) are mean absolute integrated band densities (in arbitrary units) with S.E.M. from two experiments in which all conditions were matched, including PCR cycles.

	Discussion

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To gain a more comprehensive understanding of ethanol-mediated gene regulation in activated astroglial cells, we used the technique of mRNA differential display to isolate LPS/PMA-induced transcripts whose expression seemed modulated in rat C6 glial cells by chronic exposure to 50 mM ethanol. The C6 glial cell line was used because it possesses extensive chemical and functional analogy to normal rat brain astrocytes and has served as a useful astroglial cell model for decades, including studies on acute and chronic ethanol effects. Also, its clonal nature affords very reproducible results, an important advantage for mRNA profiling studies. Herein, we report having identified the chemokine MCP-3 as one such gene whose mRNA expression is increased by LPS/PMA stimulation in C6 cells and decreased by ethanol exposure. To our knowledge, this is the first description of an effect of ethanol on MCP-3 expression.

MCP-3 is a member of the chemokine superfamily of mediators involved mainly in the immune and inflammatory process (Oppenheim et al., 1991; Baggiolini et al., 1994) but serves other functions as well (Luster, 1998). Astrocytes can secrete numerous cytokines, including chemokines such as MCP-3, and it is widely accepted that these cells actively participate in an integrative communicative pathway between resident immune cells of the CNS and those of the periphery (Aschner, 1998). Thus, chemokine expression by astrocytes may contribute to leukocyte infiltration within the CNS during injury and inflammation.

The present results are the first to demonstrate an effect of ethanol on expression of the -chemokine MCP-3. However, previous studies have observed ethanol effects on related members of the -chemokine family. For example, acute ethanol consumption attenuated ex vivo stimulation of interleukin-8 and MCP-1 induction by human leukocytes (Szabo et al., 1999), whereas Kupffer cells isolated from rats injected with ethanol and LPS had reduced MIP-1 and MCP-1 mRNA expression (Bautista, 2001). Interestingly, LPS-induced Kupffer cell expression of mRNA for the -chemokine regulated upon activation normal T cell expressed and secreted was enhanced by ethanol under the same conditions (Bautista, 2001). Consistent with the present results, LPS-induced expression of MIP-1 was attenuated in murine alveolar macrophages after chronic ethanol exposure (Standiford and Danforth, 1997), whereas expression profiling using DNA microarrays identified MCP-1 as an ethanol-responsive gene that is down-regulated after chronic exposure of human SH-SY5Y neuroblastoma cells to 100 mM ethanol (Thibault et al., 2000). Interestingly, chronic ethanol consumption has been found to increase selected hepatic cytokines (Tilg and Diehl, 2000), including the -chemokines MCP-1, MIP-1, and MIP-1 (Afford et al., 1998). Therefore, -chemokines seem to be important cellular targets of both in vivo and in vitro ethanol exposure.

Ethanol treatment seemed to transcriptionally inhibit stimulated expression of the MCP-3 gene, based on reductions in nuclear MCP-3 mRNA levels. However, the sensitivity of inducible MCP-3 expression to chronic ethanol exposure seems to be less than that of two other inducible genes previously studied under identical conditions, those for fibronectin (Ren et al., 2000) and iNOS (Syapin, 1995). For both fibronectin and iNOS, we observed significant reductions in steady-state mRNA levels after just 5 days of chronic exposure to 50 mM ethanol (Ren et al., 2000; P. J. Syapin, unpublished data), whereas 5 days of exposure did not significantly reduce MCP-3 mRNA. However, levels for all three transcripts are significantly reduced with 9 days of chronic exposure. Interestingly, inducible expression of MCP-3 and fibronectin mRNA is similarly sensitive to acute ethanol exposure, with both requiring at least 200 mM ethanol for inhibition. This contrasts with the acute sensitivity of iNOS mRNA to as little as 100 mM ethanol (Militante et al., 1997). The explanation for, and consequences of, this divergent sensitivity of host defense-related genes to ethanol exposure remain to be determined.

A possible explanation for the differential sensitivity of MCP-3 expression to ethanol exposure, relative to that of iNOS and fibronectin, may relate to differences in regulatory mechanisms that govern their expression. Because the identical stimulus, LPS/PMA exposure, can be used to induce expression of MCP-3, fibronectin, and iNOS genes in C6 cells, the observed differences in ethanol sensitivity cannot be readily explained by differences in ethanol effects on signal transduction events. Rather, differences in the trans- and cis-acting elements necessary for transcriptional regulation of the individual genes may dictate their sensitivity to ethanol.

To explore this possibility further we have used the program Tfsitescan (http://www.ifti.org) to analyze the nucleotide sequences of the rat MCP-3, fibronectin, and iNOS gene promoter regions acquired from GenBank (accession number AF154245, X05831, and AF042085, respectively) to identify known cis regulatory elements. For this analysis the iNOS region was restricted to the proximal 526 bases upstream of the transcriptional start site because we have recently shown this region to be sufficient to mediate the transcriptional inhibition of iNOS expression by acute ethanol (Syapin et al., 2001). Well over 40 putative cis-regulatory sites were identified in each promoter region, but only 13 different elements, including three for the basal transcription machinery, were common among the three promoter regions. Six additional cis-elements of diverse sequence reported to bind similar trans-acting factors were also identified. Thus, from this in silico analysis there are a large number of putative regulatory sites that may confer sensitivity or resistance to ethanol effects on expression of these three inducible genes. It is interesting, however, that only the iNOS and fibronectin promoters share binding sites for the transcription factor cAMP response element-binding protein, which has been implicated to play a role in chronic effects of ethanol (Yang et al., 1998). Furthermore, both the rat iNOS and fibronectin promoters possess the C-to-T genetic variant of the Sp1 binding cis-element that was previously implicated in alcoholism (Harada et al., 1998). Thus, it is evident that further studies are necessary to identify those transcription factors that mediate ethanol effects on inducible gene expression.

In addition to an apparent effect on MCP-3 transcription, acute ethanol treatment also decreased MCP-3 transcript stability measured 24 to 36 h after induction. Whether MCP-3 transcripts are similarly labile after chronic ethanol exposure has been difficult to determine because expression levels at 24 h of stimulation are already low after chronic exposure. However, preliminary data from one experiment suggests that it is also more labile (P. J. Syapin, unpublished data). The mechanism responsible for ethanol-induced MCP-3 destabilization is not known. The 3'-untranslated region of the rat MCP-3 transcript is known to contain two A+U-rich elements (ARE) associated with mRNA stabilization (Xu et al., 1997), but their functionality has not been documented. Even though our results provide the first evidence for stabilization of the MCP-3 message after stimulated induction in control cells, the data do not directly link the ARE to this process. Further study is required to understand the role of ARE in MCP-3 message stability and whether ethanol interacts with the function of these sites.

In conclusion, using the technique of differential display of mRNA we have discovered that MCP-3 mRNA expression is up-regulated by LPS plus PMA exposure in C6 glial cells and that chronic ethanol exposure can reduce this expression. We have characterized this response further and provide evidence for a dual mechanism whereby acute ethanol exposure reduces steady-state MCP-3 mRNA levels. The specific consequences of this effect are unclear at this time, but may result in altered immune cell CNS trafficking after injury or infection of ethanol-intoxicated individuals.