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Endocrinology Vol. 138, No. 3 922-928
Copyright © 1997 by The Endocrine Society

Articles

Low Concentrations of Ethanol Inhibits Prolactin-Induced Mitogenesis and Cytokine Expression in Cultured Astrocytes

William J. DeVito, Scott Stone and Kouki Mori

Division of Endocrinology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655

Address all correspondence and requests for reprints to: Dr. William J. DeVito, Division of Endocrinology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Whereas the immunosuppressive effects of chronic alcohol use have been well documented, little is known about the effect of ethanol on the neuroimmune response. We previously demonstrated that PRL is a potent mitogen and induces the expression of several inflammatory cytokines, including tumor necrosis factor-{alpha} (TNF{alpha}) in cultured rat astrocytes. The aim of this study was to examine the effects of ethanol on PRL-induced mitogenesis and TNF{alpha} expression in cultured rat astrocytes. We found that low concentrations of ethanol blocked PRL-induced increases in [3H]thymidine incorporation and TNF{alpha} levels. In contrast, ethanol had no effect on platelet-derived growth factor- or fibroblast growth factor-induced increases in [3H]thymidine incorporation. Radioligand binding analysis revealed that ethanol did not effect PRL receptor binding. We also examined the effect of prenatal alcohol exposure (PAE) on PRL-induced mitogenesis and cytokine expression. PAE during the last 5 days of gestation blunted the PRL-induced increase in [3H]thymidine incorporation and TNF{alpha} levels in cells grown in the absence of ethanol in the culture medium. Addition of ethanol to primary PAE astrocyte cultures resulted in a modest increase in basal [3H]thymidine incorporation, but completely blocked the PRL-induced increase in [3H]thymidine incorporation and TNF{alpha} levels. In contrast, platelet-derived growth factor- and serum (10%)-induced increases in [3H]thymidine incorporation remained intact. Together, these data indicate that ethanol blocks PRL-induced mitogenesis and the expression of TNF{alpha} in cultured rat astrocytes and are consistent with the possible inhibition of the astrocytic response by ethanol in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALCOHOL ABUSE is one of the leading causes of death in the United States. Alcohol, either directly or indirectly, affects every organ and tissue of the body. The debilitating effects of alcohol exposure are dramatically seen in children born to alcoholic women. Fetal alcohol syndrome (FAS) consists of growth retardation and central nervous system (CNS) dysfunction and is one of the leading causes of mental retardation. Despite its frequency, now estimated at 0.97 cases/1000 live births in the general obstetric population and 4.3% among heavy drinkers (1), little is known about the cellular and molecular effects of FAS.

The immunosuppressive effects of chronic alcohol use have been well documented. Ethanol use, either acutely or chronically, has multiple effects on T lymphocyte and natural killer cell functions, Ig production, and neutrophil and monocyte activities, resulting in immunosuppression (2, 3, 4). Similarly, fetal alcohol exposure results in immunodeficiency in humans and animals (5). Whereas compelling evidence indicates that alcohol is a immunosuppressive drug and has marked effects on the CNS, the effect of alcohol on the neuroimmune system remains an unexplored area. In the CNS, the responses to injury and disease involve interactions among various cell types (neurons, astrocytes, oligodendroglia, microglia, and infiltrating inflammatory cells), cytokines, growth factors, and membrane-associated proteins (6, 7). Although astrocytes comprise as much as 25% of the cells in the CNS, astrocytes, until recently, have received little attention. Unlike neurons, astrocytes retain the ability to divide and multiply. In response to injury or infection, astrocytes undergo hypertrophy and proliferation and are transformed into reactive astrocytes, which can function as immunocompetent cells by secreting cytokines, expressing histocompatibility complex class I and II antigens, and can present antigens to T cell clones in a major histocompatibility complex-restricted response (8, 9, 10). This process, termed astrogliosis, is the most frequent cellular reaction to CNS injury or infection, and is found in many neurological disorders, including multiple sclerosis, acquired immune deficiency syndrome dementia complex, Alzheimer’s disease, and the animal model for multiple sclerosis, experimental allergic encephalomyelitis (9, 10, 11).

Originally considered a reproductive hormone, it is now clear that PRL plays a role as an immunoregulatory hormone (12, 13, 14, 15, 16). We have shown that in the CNS, PRL is a novel growth factor and is involved in the regulation of astrocyte mitogenesis and cytokine expression (17, 18, 19, 20). Further, using an in vivo model of immune activation (i.e. wounding), we found that a local increase in PRL synthesis at a hypothalamic wound site is involved in regulation of the brain’s neuroimmune response to injury or trauma (19).

Toward understanding the effect of alcohol exposure on the neuroimmune response, we examined the effect of ethanol on PRL-induced mitogenesis and cytokine expression in cultured rat astrocytes. We found that exposure of cultured astrocytes to low concentrations of ethanol markedly inhibit PRL-induced astrocyte proliferation and cytokine expression. Further, PRL-induced mitogenesis and cytokine expression were markedly inhibited in primary astrocyte cultures prepared from rats exposed to alcohol prenatally. These observations suggest that prenatal alcohol exposure (PAE) has deleterious effects on astrocyte progenitor cells, which may impair astrocyte function in the developing and adult brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Astrocytes were prepared from cerebral hemispheres of 1-day-old rat pups as previously described (20). Cells were originally seeded at 2 x 105 cell/cm2 in DMEM containing 10% calf serum and cultured for 7 days at 37 C under an atmosphere of 5% CO2 and 95% air. Cells were subcultured every 7 days and were used between the second and fifth passages. Before use, cells were dispersed by trypsin and counted in a hemocytometer, and cell viability was determined by exclusion of trypan blue.

Membrane preparation and PRL binding
To characterize the PRL receptors in crude membrane fractions (microsomes) were prepared as previously described (21). Briefly, cells were homogenized in 25 mM Tris-HCl, pH 7.4, including 0.25 M sucrose, 1 mM phenylmethylsulfonylfluoride, and 100 kallikrein inhibitory units aprotinin, and centrifuged for 15 min at 1,000 x g at 4 C. The resulting supernatants were centrifuged for 30 min at 100,000 x g at 4 C. PRL binding studies were performed in duplicate using [125I]rat (r) PRL (SA, 47–62 µCi/µg). After brief sonication, microsomal preparations were incubated with approximately 100,000 cpm [125I]rPRL in binding buffer (100 mM Tris-HCl and 500 mM acetic acid, pH 8.3, containing 10 mM MgCl2 and 0.1% BSA in a total volume of 500 µl) as described by Muccioli et al. (22). Incubations were performed at room temperature for 24 h, and receptor-bound radioactivity was separated from unbound [125I]rPRL by centrifugation in 3 ml cold binding buffer. Specific binding was determined by calculating the difference between the radioactivity bound in the absence and presence of unlabeled rPRL (1 µg). The maximum binding capacity and apparent dissociation constants were determined by Scatchard analysis of saturation curves from control and ethanol-treated cells using the program Ligand.

Western blot analysis
Western blot analysis of tumor necrosis factor-{alpha} (TNF{alpha}) was performed as previously described (18, 23). Cells were homogenized in 25 mM Tris-HCl, pH 7.4, including 0.25 M sucrose, 1 mM phenylmethylsulfonylfluoride, and 100 kallikrein inhibitor units aprotinin, and centrifuged for 15 min at 1,000 x g at 4 C. The resulting supernatant was centrifuged for 30 min at 100,000 x g at 4 C. The resulting microsomal fractions were dissolved in sample buffer (0.65 M Tris, pH 6.8; 4% SDS; 20% glycerol; 10% ß-mercaptoethanol; and 0.01% bromophenol blue), and 100 µg protein were separated by SDS-PAGE on 0.75-mm thick slab gels, using a 4% polyacrylamide stacking gel and a 15% resolving gel. Electrophoresis was carried out at 20 mA for approximately 2 h using a Hoefer sturdier electrophoresis apparatus (Hoefer Scientific Instruments, San Francisco, CA). Proteins were electrophoretically transferred onto nitrocellulose at 100 V for 2 h at 4 C. The blots were incubated with TBS containing 2% dry fat-free milk, 5% BSA, and 0.1% Tween-20, pH 7.0, for 6 h and then incubated with anti-TNF{alpha} for 1 h at room temperature. Blots were extensively washed with PBS, developed using ECL detection reagents from Amersham Life Sciences (Arlington Heights, IL) according to the manufacturer’s instructions, and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY). Quantification was performed by densitometry.

[3H]Thymidine incorporation
For studies of [3H]thymidine incorporation into DNA, astrocytes were grown in serum-free medium in 12-well tissue culture plates. After 2 days, the medium was replaced with DMEM containing 1% serum and supplemented with test reagents as indicated. After 18 h, the medium was removed and replaced with RPMI 1640 medium containing [methyl-3H]thymidine (5 µCi/ml), and cells were incubated for an additional 3 h. Labeling was stopped by aspirating the medium and washing the cells three times with ice-cold PBS and three times with 10% ice-cold trichloroacetic acid. Trichloroacetic acid-precipitable material was solubilized with 1 ml 2% SDS. Cell-associated radioactivity was then counted in a scintillation spectrometer.

PAE
Nulliparous females were individually housed each evening with a male until a vaginal smear indicated day 1 of pregnancy. They were then housed individually and maintained in controlled lighting (lights on from 0700–1900 h). One group of rats received 5% ethanol in their drinking water for the last 5 days of gestation. The addition of 5% ethanol to the drinking water had no effect on water intake, litter size, or the body weights of the females or the pups. Animals were maintained in accordance with the NIH Guidelines for the Care and Use of Animals approved by the institutional animal care and use committee at the University of Massachusetts Medical Center.

Statistical analyses
The effect of ethanol on growth factor-induced increases in [3H]thymidine incorporation were analyzed by two-way randomized factorial ANOVAs. Tests of simple main effects were used to determine differences between two groups. The effects of PAE and in vitro ethanol on PRL- or growth factor-induced increases in [3H]thymidine incorporation were analyzed by three-way randomized factorial ANOVAs. When interactions were significant, tests of simple main effects were used to determine differences between two groups. The size of the region of rejection of the null hypothesis was set by an {alpha} error of 5%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ethanol on PRL-induced thymidine incorporation in cultured astrocytes
Preliminary studies were performed to determine the relationship among [3H]thymidine incorporation, cell number, and DNA content in PRL-stimulated astrocytes. In PRL-stimulated astrocytes, under the experimental condition described above, we found that all three measures increased in parallel (data not presented). Further, thymidine incorporation had the least variability, and thus was selected as the index of cell proliferation in our studies. In the presence of 1% serum, astrocytes plated at low densities grow at a slow rate. Addition of PRL (1 nM), 10% serum, or other growth factors to subconfluent astrocytes results in marked increases in cell number, DNA synthesis, and thymidine incorporation. As illustrated in Fig. 1Go, preincubation of astrocytes with low concentrations of ethanol for 2 days resulted in a dose-dependent increase in the percent inhibition of thymidine incorporation in PRL-stimulated cells (P < 0.05). Incubation of astrocytes with 100 mM ethanol for 2 days in the presence or absence of PRL did not result in cell detachment and had no effect on cell viability, as assessed by trypan blue exclusion. Further, PRL receptor binding was not affected by 100 mM ethanol (Fig. 2Go). Scatchard analysis revealed similar binding affinities (7.9 ± 0.8 vs. 8.2 ± 0.9 fmol/mg protein) and binding capacities (0.24 ± 0.05 vs. 0.27 ± 0.04 nM) in control and ethanol-treated astrocytes. In the above experiment, ethanol was added to the medium 2 days before and during PRL stimulation. To determine whether ethanol preexposure was required, ethanol was added to astrocyte cultures at different times before and during PRL stimulation. As shown in Table 1Go, preexposure of astrocytes for 6 h before PRL stimulation resulted in a significant decrease in thymidine incorporation. The maximal effect of ethanol pretreatment was observed when ethanol was added 18 h before, but not during, PRL stimulation. Addition of ethanol for the 4 h subsequent to the addition of, but not before, PRL stimulation resulted in a small, but significant, decrease in thymidine incorporation.



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  		Figure 1. Effect of ethanol on PRL-induced [3H]thymidine incorporation in cultured astrocytes. Data are the mean ± SEM of three separate experiments.

 


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  		Figure 2. Effect of ethanol on [125I]PRL specific binding to membranes prepared for control and ethanol-treated astrocytes. Data are the mean ± SEM of three separate experiments.

 

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  		Table 1. Effect of ethanol exposure on PRL-induced thymidine incorporation in cultured astrocytes

 
Next we determined the specificity of the inhibitory effect of ethanol on astrocyte proliferation. Subconfluent astrocytes were stimulated with PRL (1 nM), fibroblast growth factor (FGF; 1 ng/ml), or platelet-derived growth factor (PDGF; 50 ng/ml) in the presence or absence of ethanol. As illustrated in Fig. 3Go, addition of PRL, FGF, or PDGF to subconfluent astrocytes resulted in marked increases in thymidine incorporation. As described above, addition of ethanol (50 mM) to PRL-stimulated astrocytes markedly blunted thymidine incorporation. In contrast, in the presence of ethanol (50 mM), there was no inhibition of FGF- or PDGF-induced increases in thymidine incorporation.



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  		Figure 3. Effect of ethanol (50 mM) on PRL-, PDGF-, and FGF-induced [3H]thymidine incorporation in cultured astrocytes. Subconfluent astrocytes were stimulated with vehicle (PBS), PRL (1 nM), FGF (1 ng/ml), or PDGF (50 ng/ml) in the presence or absence of ethanol. Data are the mean ± SEM of three separate experiments. *, P < 0.05 vs. PRL-stimulated astrocytes in the absence of ethanol.

 
Effect of PAE on thymidine incorporation in primary astrocyte cultures
Figure 4Go illustrates the effect of PAE on PRL-induced mitogenesis in primary cultures of astrocytes. In primary astrocyte cultures obtained from control rats, PRL induced a dose-dependent increase in thymidine incorporation. Addition of ethanol (50 mM) before and during PRL stimulation resulted in approximately 50% inhibition of thymidine incorporation at all the three PRL concentrations. In primary astrocyte cultures obtained from rats exposed to ethanol prenatally and cultured in the absence of ethanol, there was a significant decrease in thymidine incorporation compared to that of control cultures. Addition of ethanol to cultures obtained from rats exposed prenatally to ethanol resulted in a marked decrease in thymidine incorporation compared to that in cultures prepared form rats receiving prenatal ethanol treatment, but not exposed to ethanol during the culture period. Further, PRL-induced thymidine incorporation in astrocyte cultures prenatally exposed to ethanol and exposed to ethanol during culture was completely blocked at all the three PRL concentrations.



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  		Figure 4. Effect of PAE on PRL-induced [3H]thymidine incorporation in primary astrocytes. Primary astrocyte cultures were obtained from control rats and rats exposed to 5% ethanol in their drinking water during the last 5 days of gestation. All cultures were grown in the absence of ethanol for 4 days and then cultured in the presence or absence of ethanol (50 mM) for 3 days. Data are the mean ± SEM of three separate experiments. *, P < 0.05 vs. PRL-stimulated astrocytes in the absence of ethanol.

 
We also determined the specificity of the inhibitory effect of PAE on astrocyte proliferation. As illustrated in Fig. 5Go, addition of PRL (1 nM), 10% serum, or PDGF (10 ng/ml) to control astrocytes resulted in similar increases in [3H]thymidine incorporation. The inhibitory effect of ethanol on [3H]thymidine incorporation in PRL-, 10% serum-, and PDGF-stimulated cultures, however, was stimulus dependent. That is, addition of ethanol (50 mM) to control astrocytes inhibited PRL-induced [3H]thymidine incorporation. In contrast, [3H]thymidine incorporation induced by serum or PDGF was not affected. Similarly, [3H]thymidine incorporation in PRL-stimulated PAE astrocytes, grown in the absence of ethanol, was significantly less than that in control cultures, whereas [3H]thymidine incorporation in 10% serum and PDGF-stimulated astrocytes was similar to that in control astrocytes. Addition of ethanol to PAE astrocytes resulted in a slight increase in basal [3H]thymidine incorporation. Addition of ethanol to PAE astrocytes, however, completely blocked PRL-induced thymidine incorporation and significantly inhibited serum-induced [3H]thymidine incorporation. In contrast, ethanol (50 mM) did not inhibit PDGF-induced [3H]thymidine incorporation.



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  		Figure 5. Effect of PAE on PRL (1 nM)-, 10% serum-, or PDGF (10 ng/ml)-induced [3H]thymidine incorporation in primary astrocytes. Primary astrocyte cultures were obtained from control rats and rats exposed to 5% ethanol in their drinking water during the last 5 days of gestation. All cultures were grown in the absence of ethanol for 4 days and then cultured in the presence or absence of ethanol (50 mM) for 3 days. Data are the mean ± SEM of three separate experiments. *, P < 0.05 vs. PRL-stimulated astrocytes in the absence of ethanol.

 
Effect of PAE on PRL-induced expression of TNF{alpha} in primary astrocyte cultures
We have shown that in passaged astrocytes grown in 1% serum, TNF{alpha} was not detected by Western blot analysis (18). Similarly, we found in primary astrocytes that TNF{alpha} levels were below the limit of detection (Fig. 6Go). In control astrocytes, 4 h after the addition of PRL (1 nM), TNF{alpha} levels were easily detected. Preincubation of astrocytes with ethanol resulted in a marked decrease in the PRL-induced expression of TNF{alpha}. In PAE astrocytes grown in the absence of ethanol, PRL-induced TNF{alpha} levels were less than those in control astrocytes. Addition of ethanol to PAE astrocytes completely blocked PRL-induced expression of TNF{alpha}.



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  		Figure 6. Effect of PAE on PRL-induced increase in TNF{alpha} levels in cultured astrocytes. Primary astrocyte cultures were obtained from control rats and rats exposed to 5% ethanol in their drinking water during the last 5 days of gestation. All cultures were grown in the absence of ethanol for 4 days and then cultured in the presence or absence of ethanol (50 mM) for 24 h. Cell were stimulated with vehicle or PRL (1 nM) for 4 h and harvested. The data are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Compelling evidence indicates that alcohol is an immunosuppressive drug; however, the effect of alcohol on the neuroimmune system remains an unexplored area. Originally considered a reproductive hormone, it is now clear that PRL also plays a role in the regulation of humoral, cell-mediated, and autoimmune responses (12, 13, 14, 15). Using a homogeneous population of cultured astrocytes, we have shown that at physiological concentrations, PRL (10 pM to 1 nM) is a potent mitogen, increasing cell number, [3H]thymidine incorporation, and inducing the rapid expression of several inflammatory cytokines, including interleukin-1{alpha}, TNF{alpha}, and transforming growth factor (17, 18, 19, 20). Using this model, the primary goal of the present study was to determine the effects of alcohol on the regulation of PRL-induced mitogenesis and cytokine expression in cultured astrocytes. We found that incubation of astrocytes in the presence of ethanol (10–60 mM) resulted in a dose-dependent decrease in PRL-induced mitogenesis and TNF{alpha} levels. In contrast, ethanol did not affect the mitogenic action of PDGF or serum (10%). Competitive inhibition binding studies revealed binding affinities and binding capacities similar to those previously described in brain (21, 22). Further, neither binding affinity nor binding capacity was affected by ethanol exposure in cultured astrocytes, indicating that the inhibitory effect of ethanol on PRL-induced mitogenesis and cytokine expression was not due to a decrease in PRL receptor binding.

Our results are consistent with other reports demonstrating direct effects of ethanol on glial cells. For example, a brief exposure of astrocytes obtained from embryonic chick cerebra to ethanol impairs cell proliferation, protein synthesis, and glutamine synthesis (24). In C6 cells, ethanol delays cAMP- and dexamethasone-induced morphological and biochemical responses and cell proliferation (25, 26). Similarly, ethanol inhibits insulin-like growth factor I (IGF-I)-induced proliferation in C6 glial cells (27). This is in contrast, however, to the effect of ethanol in 3T3 fibroblasts, in which ethanol enhances the mitogenic effect of IGF-I (28). In primary astrocyte cultures, ethanol treatment results in marked morphological and biochemical alterations (29). In addition, ethanol decreases DNA and protein synthesis (24, 30, 31), serotonin uptake (32), and secretion of nerve growth factor (33); results in the alteration of cytoskeleton organization (29, 34); delays morphological maturation (29, 31); retards differentiation (35); and affects superoxide dismutase activity, thereby increasing vulnerability to free radical damage (36).

Animal models clearly show debilitating effects of PAE on CNS development, including gross malformation, microencephaly, heterotropias, errors of migration, neuronal depletion, abnormal cell death, and cerebrovascular damage (35). Primary astrocyte cultures obtained from animals prenatally exposed to ethanol and cultured in the absence of ethanol show morphological and biochemical changes similar to those of astrocyte cultures prepared from nonethanol-treated controls but grown in the presence of ethanol (24, 29, 30, 31, 34). Here we examined the effect of PAE on PRL-induced mitogenesis and cytokine expression. We found that in PAE astrocytes cultured in the absence of ethanol, PRL-induced mitogenesis and TNF{alpha} expression were markedly inhibited. Further, when PAE astrocytes were grown in the presence of ethanol, PRL-induced mitogenesis and TNF{alpha} expression were completely blocked. To determine the specificity of the inhibitory effect of PAE on PRL-induced mitogenesis, control and PAE astrocytes were stimulated with equivalent doses of PRL, serum, or PDGF in the presence or absence of ethanol. We found that the inhibitory effect of ethanol on astrocyte proliferation is stimulus dependent. That is, in PAE astrocytes grown in the absence of ethanol, PRL-induced, but not serum- or PDGF-induced, proliferation was inhibited. Furthermore, we found that exposure of PAE astrocytes to ethanol in vitro resulted in a marked inhibition of PRL-induced thymidine incorporation and cytokine expression, suggesting that PAE astrocytes may be sensitized to the inhibitory effects of ethanol.

Whereas our studies clearly show that ethanol inhibits PRL-induced mitogenesis and cytokine expression, the cellular mechanisms involved are unknown. In PRL target tissues, the regulation of PRL-induced mitogenesis involves at least two signal transduction pathways, the activation of protein kinase C (PKC), and the tyrosine phosphorylation of several proteins, including the PRL receptor and a member of the Janus (JAK) family of receptor-associated tyrosine kinases (JAK-2). Several lines of evidence support a role for PKC in the regulation of PRL-induced mitogenesis. In Nb2 lymphoma cells, a cell line that is dependent on lactogenic hormones for proliferation (37, 38), PKC is involved in PRL-induced mitogenesis (39, 40, 41). In the rat liver, the mitogenic effect of PRL is linked to an increase in 1,2-diacylglycerol and the subsequent activation of PKC (42). In cultured astrocytes, we have shown that PRL-induced activation of PKC plays an important role in the regulation of PRL-induced mitogenesis and cytokine production (17, 20). Interestingly, in a number of cell types, including astrocytes, low concentrations of ethanol alter ligand-induced activation and the cellular localization of PKC-specific isozymes (43, 44, 45), suggesting that the effects of ethanol on PRL-induced mitogenesis and cytokine expression in cultured astrocytes may involve an ethanol-induced alteration in PRL-induced activation or subcellular localization of specific PKC isozymes.

A notable characteristic of PRL receptors as well as other members of the hematopoietic receptor superfamily is the absence of a consensus sequence indicative of catalytic function, such as protein kinase activity (46). Studies using Nb2 rat lymphoma cells, however, show that PRL induces the rapid tyrosine phosphorylation of several proteins, including JAK-2 (47, 48, 49, 50, 51, 52). Whereas the effect of ethanol on the activation of JAK kinases is unknown, several lines of evidence suggest that ethanol can inhibit receptor protein kinase activity. That is, in A431 human epidermal carcinoma cells, ethanol inhibits the EGF-induced increase in tyrosine kinase activity (53). Similarly, ethanol inhibits IGF-I-induced proliferation in C6 glial cells, an effect that may be mediated by a decrease in IGF-I receptor autophosphorylation (27). Together, these studies suggest that ethanol may inhibit PRL-induced mitogenesis and cytokine expression through alterations in multiple cellular mechanisms, including the activation and subcellular localization of specific PKC isoforms and/or changes in PRL-induced tyrosine phosphorylation. This hypothesis currently is under investigation.

In summary, whereas the immunosuppressive effects of ethanol have attracted considerable attention, the effects of alcohol in the regulation of the neuroimmune system have remained unexamined. Clearly, astrocyte proliferation and the expression of inflammatory cytokines play important roles in the regulation of the brain’s response to injury or infection. A few studies show that ethanol decreases glial cell growth in vitro. In these studies, we show that 1) in cultured rat astrocytes, ethanol markedly inhibits PRL-induced mitogenesis and the expression of TNF{alpha}; 2) ethanol-induced inhibition of PRL-induced mitogenesis is not due to a decrease in PRL binding; 3) in PAE astrocytes grown in the absence of ethanol, PRL-induced mitogenesis and cytokine expression are markedly decreased; 4) in PAE astrocytes grown in the presence of low concentrations of ethanol, the inhibitory effect of ethanol is enhanced, resulting in the complete suppression of PRL-induced mitogenesis and cytokine expression; and 5) the inhibitory effect of ethanol on astrocyte mitogenesis is not a general suppression of astrocyte proliferation. Together, our studies suggest that ethanol functions as an immunosuppressive drug in the CNS though the inhibition of primary components of the neuroimmune response. Further, the inhibitory effect of PAE on PRL-induced mitogenesis and TNF{alpha} levels in the absence of ethanol in vitro suggests that PAE may have disruptive effects on astrocyte progenitor cells.

Received August 7, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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