- Email: [email protected]
Prenatal alcohol exposure increases TNFα-induced cytotoxicity in primary astrocytes
Alcohol 21 (2000) 63 ± 71
Prenatal alcohol exposure increases TNFa-induced cytotoxicity in primary astrocytes William J. De Vito*, Krisanthi Xhaja, Scott Stone Division of Endocrinology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA, 01655, USA Received 10 December 1999; received in revised form 26 January 2000; accepted 28 January 2000
Abstract We examined the effect of prenatal alcohol exposure (PAE) on tumor necrosis factor-a-(TNFa) induced cell death in primary astrocyte cultures. Flow cytometry revealed that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFa when compared to astrocytes prepared from pair-fed and chow-fed controls. In a number of cell types, TNFa regulates cell growth or death, in part, by the hydrolysis of sphingomyelin to ceramide and sphingosine-1-phosphate (SPP). Using a 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxic assay we found that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFa, sphingomyelinase (SMase), and C2- and C6-ceramide. The increasing cellular concentrations of SPP, a sphingolipid metabolic that induces cell growth, protected the cells from TNFa-induced cell death. N,N-dimethylsphingosine (DMS), which inhibits SPP production, and Noleoylethanolamine, which inhibits acid ceramidases, increased TNFa-induced cytotoxicity in astrocytes prepared from PAE rats. These studies suggest that PAE shifts the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNFa. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Prenatal alcohol exposure; TNFa; Astrocytes; Ceramide
1. Introduction 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. Prenatal alcohol syndrome is associated with growth retardation, central nervous system dysfunction, and is one of the leading causes of mental retardation. Children exposed to ethanol in utero also have defects in host defense and increased incidence and severity of infection with some children with FAS showing long lasting deficiencies in both humoral and cell mediated immunity (Johnson et al., 1981; Cavani et al., 1985). Animal studies have substantiated the immunoteratogenic effect of in utero alcohol exposure. Rodents prenatally exposed to alcohol have been shown to have decreased thymic cellularity, decreased lymphocyte numbers and de-
* Corresponding author. Tel.: +1-508-856-6244; fax: +1-508-8566950. E-mail address: [email protected] (W.J. De Vito).
fects in thymic and splenic proliferation in response to T-cell mitogens (Ewald, 1989; Ewald & Frost, 1987; Ewald et al., 1991). Studies further show that splenic lymphocytes prepared from neonatal and adult animals, which were exposed to alcohol in utero display long-lasting defects in T-cell proliferation (Redei et al., 1989; Normon et al., 1991; Weinberg & Jerrells, 1991; Wong et al., 1992). In utero ethanol exposure also has a marked impact on B-cell development. Among neonatal mice exposed to alcohol in utero there is a marked deficiency in B-cell development and response to lipopolysaccharide (LPS), pokeweed mitogen, phytohemagglutinin, and concanavalin A, which extends passed weaning (Wolcott et al., 1995). Further, in the utero alcohol exposure disrupts the developmental pathway of B-lineage intermediates, thereby leaving the animal immunocompromized at birth (Gottesfeld et al., 1990). Studies clearly show that alcohol is an immunosuppressive drug and that prenatal alcohol exposure (PAE) has marked effects on T- and B-cell development, which compromises the ability of neonatal animals to mount an effective immune response. Little is known, however, about the effects of alcohol and PAE on the neuroimmune response. In the CNS, the response to injury and disease involves
0741-8329/00/$ ± see front matter D 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 0 ) 0 0 0 7 8 - 1
64
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
interactions among various cell types (neurons, astrocytes, oligodendroglia, microglia, and infiltrating inflammatory cells), cytokines, growth factors, and membrane-associated proteins (Hatten et al., 1991; Eng et al., 1992). Astrocytes comprise as much as 25% of the cells in the CNS; however, until recently, astrocytes have received little attention. Unlike neurons, astrocytes retain the ability to divide and multiply. In the CNS, astrocytes can function as immunocompetent cells by secreting cytokines, and by expressing MHC class I and II antigens and adhesion molecules (Chung et al., 1991). In response to injury or infection, astrocytes undergo hypertrophy and proliferation and are transformed into reactive astrocytes. 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 (MS), acquired immune deficiency syndrome dementia complex, Alzheimer's disease, and the animal model for MS, experimental allergic encephalomyelitis (Hickey et al., 1985; Benveniste, 1992; Martin et al., 1992). The in vitro exposure of primary astrocyte cultures to ethanol have many of the same morphological and biochemical changes found in primary cultures prepared from animals that were prenatally exposed to ethanol (Davies & Vernadakis, 1984; Renau-piqueras et al., 1988, 1989; Guerri et al., 1990; Saez et al., 1991). This has allowed for the examination of the direct effects of ethanol on glial cells. We have shown that ethanol decreases cytokine- and hormoneinduced astrocyte proliferation, as well as the expression and secretion of tumor necrosis factor-alpha (TNFa) (DeVito et al., 1997), the cell surface expression of ICAM-1 in human astrocytoma cells (DeVito et al., 2000), and blocks the prolactin-induced activation of the JAK/STAT pathway in cultured astrocytes (DeVito & Stone, 1999). Further, we have recently shown that the pre-exposure of astrocytes to low concentrations of ethanol increases their susceptibility to the cytotoxic effect of TNFa (DeVito et al., 2000). In this study, we examined the effect of PAE on the cytotoxic effect of TNFa in primary astrocyte cultures. Here, we show that primary astrocytes, which were prepared from rats prenatally exposed to alcohol and grown in the absence of ethanol, are sensitized to the cytotoxic effect of TNFa. Further, our data suggest that TNFa-induced metabolism of sphingolipids is shifted to favor the production of ceramide resulting in cell death. 2. Materials and methods 2.1. Materials Recombinant rat TNFa was obtained from the Genzyme (Cambridge, MA). Other materials were purchased from the following sources: bacterial sphingomyelinase (SMase; Staphylococcus aureus), C2-ceramide, C6-ceramide, dihydroC2-ceramide N,N-Dimethylsphingosine (DMS), D,L-dithio-
threitol (DTT), N-oleoylethanolamine, from Sigma (St. Louis, MO); calf serum from Gibco BRL (Grand Island, NY). All other chemicals and reagents were obtained from commercial sources and were of reagent or molecular biology grade. 2.2. Cell culture The astrocytes were prepared from cerebral hemispheres of 1-day-old rat pups as previously described (DeVito et al., 1992). Cells were seeded at 2 105 cell/cm2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum and cultured at 37°C, under an atmosphere of 5% CO2, 95% air. Cells were grown to confluence and used after 14 days in culture. 2.3. 3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay Cell viability was determined by the MTT assay. Astrocytes were seeded in 96-well plates and treated as described in the figure legends. MTT was dissolved in PBS at a concentration of 5 mg/ml. From this stock solution, 20 ul per 100 ul of medium was added to each well, and the plates were incubated at 37°C for 4 h. Acid ±isopropanol (100 ul of 0.04 M HCl in isopropanol) was added to lyse the cells. After 15 min at room temperature, the plates were read on a Labsystems Multiskan MS plate reader at a test wavelength of 595 nm and a reference wavelength of 650 nm. 2.4. Prenatal alcohol exposure Nulliparous females (150 ± 210 g) were individually housed each evening with a male until a vaginal smear, which was indicated as day 1 of pregnancy. They were then housed individually under controlled lighting, lights on between 0600 and 1900 h. Prospective mothers were fed a chow diet (Purina 5008) until gestational (G) day 7. At this time, the rats were weight-matched and separated into the three dietary groups: a chow (Ch) diet, a control (Ct) diet or a ethanol diet (Et). The control and ethanol diets were highprotein, liquid diets (Lieber ± DeCarli), which are nutritionally balanced to meet the specific needs of pregnant rats (Bioserve, Frenchtown, NJ). Both diets were fortified with Vitamin Diet Fortification Mixture and Salt Mixture. Commencing on day 7 of gestation, the dams were fed an alcohol-containing liquid diet (No 1265, Bio-Serv, Frenchtown, NY) containing 2.2% (v/v) ethanol at day 7 and 4.5% v/v ethanol at day 14. In the control diet, an isocaloric amount of dextrin-maltose was substituted for the ethanol. Tap water was available ad libitum. The chow diet consisted of standard rat chow and water ad libitum. Liquid diets were provided in 120-ml graduated feeding tubes. All ethanol-fed rats were paired by weight with a rat fed with control diet, and the two were pair-fed. That is, each control-fed rat received the same volume of liquid diet as that consumed
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
the previous day by the weight-matched ethanol-fed rat. Thus, during their pregnancies, both the control- and ethanol-fed rats consumed diets of identical caloric content. The amount of liquid ingested by the control- and ethanol-fed females was determined at 0900 h each morning and at 1700 h each evening. If an ethanol-fed rat required more than the estimated 100 ml of diet, additional diet was added at 1700 h to insure that each ethanol-fed rat received an adequate supply of diet to meet an individual animals needs. After recording the past day's food consumption, the ethanol-fed and control-fed rats received their daily food allotments. Throughout the gestation period, chow-fed rats were fed chow and water ad libitum. All pregnant rats were weighed on alternative days, and only those weight-matched controlfed and ethanol-fed rats that maintained a weight within 10% of the chow-fed rats were kept in the study. At birth, the pups were counted and weighed. Maternal blood samples were collected by orbital venipuncture under general anesthesia (pentobarbital 50 mg/kg, i.p.) on the morning of birth from control- and ethanol-fed rats. Pooled blood samples was collected from the pups of each litter during the preparation of the primary cultures. The concentration of ethanol in the blood samples was measured using a Sigma diagnostic kit (#332 UV), according to the manufacturer's instructions. All experimental procedures were approved by the University of Massachusetts Medical School institutional animal care and use committees. 2.5. DNA content The cells were collected by centrifugation and transferred into 1 ml of Hanks' buffered salt solution and then into tubes containing 10 ml of 70% ethanol on ice. The cell were stored in the fixative at ÿ20°C for 24 h. The cells were then centrifuged at 800 g for 5 min and the ethanol was thoroughly removed. The cells were stained with propidium iodide and the florescence of individual cells was measured by flow cytometry using a FACScan instrument (Becton Dickinson, Sunnyvale, CA). 2.5.1. Statistical analysis The effect of PAE and ethanol on cell viability were analyzed with a randomized factorial analysis of variance. The test of simple main effects was used to test for differences between two groups. The size of the region of rejection of the null hypothesis was set by an alpha error of 5%. 3. Results 3.1. Effect of PAE on DNA content in TNFa stimulated primary astrocytes The exposure of rats to ethanol during gestation resulted in blood alcohol concentrations in dams and pups ranging
65
between 52 and 78 mg/dl, and 31 and 56 mg/dl, respectively on the day of birth. To examine the effect of PAE on TNFainduced DNA fragmentation, flow cytometric analysis of cellular DNA content was preformed on primary astrocytes prepared from control, pair-fed and ethanol-fed rats. Flow cytometric analysis of cellular DNA content from astrocytes prepared from rats fed a chow diet shows a typical cell cycle DNA profile with a primary G1 DNA peak and a smaller G2M DNA peak (Fig. 1A). The flow cytometric analysis of cellular DNA content from astrocytes prepared from chowand pair-fed rats showed that incubation of astrocytes with TNFa for 18 h did not increase the number of cells having a DNA content less (M1) than those cells in G1 (Fig. 1B and C). In astrocytes prepared from rats that were prenatally exposed to alcohol, however, TNFa stimulation significantly (P < 0.05) increased the number of cells with DNA content less than those cells in G1 when compared to chowand pair-fed controls. This indicates that PAE increases the sensitivity of astrocytes to the cytotoxic effect of TNFa, resulting in increased DNA fragmentation and decreased DNA content. Next, we examined the effect of PAE on cell viability using the MTT cytotoxic assay, which is a sensitive index of decreased cell viability. In a series of preliminary studies, we found that the response of astrocytes to TNF and SMase, C6- and C2-ceramide where similar in astrocytes prepared from chow- and pair-fed rats. Therefore, to reduce the number of animals required to complete these studies and to simplify the presentation of the data, we used astrocytes from pair-fed rats as controls. We found that incubation of astrocytes prepared from pair fed-rats with low concentrations of TNFa did not decrease cell viability (Fig. 2). The incubation of astrocytes prepared from rats prenatally exposed to alcohol with low concentrations of TNFa, however, resulted in a dose-dependent decrease in cell viability. We then examined the effect of in vitro ethanol exposure on TNFa-induced cell death in astrocytes prepared from pair-fed and PAE rats (Fig. 2). Astrocytes were cultured in the presence of ethanol (50 mM) for 18 h, and then stimulated with increasing concentrations of TNFa for 18 h. The incubation of astrocytes with ethanol (50 mM) for 36 h had no effect on cell viability (data not shown). In astrocytes prepared from pair-fed controls, pre-incubation with 50 mM ethanol for 18 h increased the TNFa-induced cytotoxicity with TNFa concentrations of 5 ng/ml, or greater, decreasing cell viability. Similarly, in astrocytes prepared from rats prenatally exposed to alcohol, pre-incubation with ethanol resulted in a further increase in TNFa-induced cell death. 3.2. Effect of PAE on SMase- and ceramide-induced cell death in cultured astrocytes Recent studies have revealed that the hydrolysis of sphingomyelin to ceramide is involved in the regulation of the cytotoxic effect of TNFa in a variety of cell types (Natoli et al., 1998). Accordingly, studies were performed
66
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
Fig. 1. Effect of prenatal alcohol exposure on DNA content in TNFa-stimulated cultured rat astrocytes. Astrocytes were prepared from rats fed with a chow diet (Ch), control diet (Ct) or an ethanol diet (Et). Astrocytes were grown to confluence and stimulated with TNFa (100 ng/ml) for 18 h. Data are presented as DNA fluorescence histograms of propidium iodide-stained astrocytes. The data presented are representative of three separate experiments.
Fig. 2. MTT dye-reduction analysis of the effect of prenatal alcohol exposure on cell viability in TNFa-stimulated cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines), or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with TNFa in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control.
to determine if this pathway is involved in TNFa-induced cell death in cultured astrocytes prepared from rats prenatally exposed to alcohol. Astrocytes were incubated with SMase, which results in the hydrolysis of sphingomyelin to ceramide, or C2- or C6-ceramide, membrane-permeable ceramide analogs. In astrocytes prepared from pair-fed control rats, the incubation of cultured astrocytes with increasing concentrations of SMase did not decrease cell viability (Fig. 3A). Similarly, the incubation of astrocytes prepared from pair-fed controls with increasing concentrations of C2- or C6-ceramide did not decrease astrocyte viability (Fig. 3B and C). In contrast, the incubation of astrocytes prepared from rats prenatally exposed to alcohol with SMase, C2- or C6-ceramide resulted in a dose dependent decrease in cell viability (Fig. 3A,B,C, respectively). In astrocytes prepared from pair-fed controls, pre-incubation with ethanol for 18 h sensitized the cells to the cytotoxic effects of SMase, C2-, and C6-ceramide. In astrocytes prepared from rats prenatally exposed to alcohol, the in vitro ethanol exposure increased the cytotoxic effects of C2and C6-ceramide. The incubation of astrocytes prepared from pair-fed controls and rats prenatally exposed to alcohol with dihydro-C2-ceramide, an inactive ceramide analog, did not affect cell viability (Fig. 3D).
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
67
Fig. 4. MTT dye-reduction analysis of the effect of sphingosine-1phosphate (SPP) on TNFa-induced cell death in cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines), or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with SPP for 4 h and then stimulated with TNFa (50 ng/ml) in combination with SPP in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control. Fig. 3. MTT dye-reduction analysis of the effect of prenatal alcohol exposure on cell viability. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines), or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with SMase (A), C2-ceramide (B), C6-ceramide (C), or DihydroC2-ceramide (D) in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control.
decreased cell viability by 90%. Pre-incubation of cells with SPP block TNFa-induced cell death, but required a higher concentration of SPP (75 nM) to maintain cell viability at control levels. DMS is a potent competitive inhibitor of sphingosine kinase resulting in decreased cellular SPP levels and
3.3. Effect of sphingosine-1-phosphate (SPP), DMS and Noleoylethanolamine on TNFa-induced cell death in cultured astrocytes The current models of TNFa-induced cell death suggest that the growth factor-induced activation of SPP, a breakdown product of sphingolipid, protects cells from the cytotoxic effects of TNFa (Cuvillier et al., 1996). Accordingly, we examined the effect of SPP on TNFa-induced cell death in astrocytes prepared from pair-fed subjects and rats prenatally exposed to alcohol (Fig. 4). Astrocytes were incubated with SPP for 4 h and then stimulated with TNFa in combination with SPP. The incubation of astrocytes prepared from pair-fed controls with TNFa alone, or in combination with SPP, did not decrease astrocyte viability. In astrocytes prepared from rats prenatally exposed to alcohol, or exposed to ethanol in vitro, TNFa markedly decreased astrocyte viability. In astrocytes prepared from pair-fed rats and rats exposed to alcohol in utero, SPP resulted in a dose-dependent inhibition of TNFa-induced cell death, with 50 nM SPP maintaining cell viability at control levels. In astrocytes prenatally exposed to alcohol, and incubated in the presence of ethanol in vitro, TNFa
Fig. 5. MTT dye-reduction analysis of the effect of N,N-dimethylsphingosine (DMS) on TNFa-induced cell death in cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines) or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with DMS for 4 h and then incubated with TNFa (5 ng/ml) in combination with DMS in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control.
68
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
increased ceramide levels (Edsall et al., 1998). Accordingly, we examined the effect of DMS on TNFa-induced cell death (Fig. 5). In astrocytes prepared from pair-fed controls, there was no effect of low concentrations of DMS on TNFa-induced cell death. At higher concentrations, DMS (50 ±75 uM) alone decreased cell viability in astrocytes prepared from pair fed rats. In astrocytes prepared for rats prenatally exposed to alcohol lower concentrations of DMS (25 uM) decreased cell viability in quiescent astrocytes (data not presented). In contrast, in astrocytes prepared from rats prenatally exposed to alcohol, low concentrations of DMS markedly increased TNFa-induced cell death. Similarly, in astrocytes prepared from pair-fed controls and exposed to alcohol in vitro, DMS increased the cytotoxic effect of TNFa. Next, we examined the effect of N-oleoylethanolamine, an inhibitor of acid ceramidases (Bielawska et al., 1996) on TNFainduced cell death in astrocytes prepared from pair fedcontrols and rats prenatally exposed to alcohol. Astrocytes were incubated with N-oleoylethanolamine for 2 h and then with TNFa for 18 h. In astrocytes prepared from pair-fed controls, the low concentrations of N-oleoylethanolamine did not effect TNFa-induced cell death (Fig. 6). In contrast, in astrocytes prepared from rats prenatally exposed to alcohol, N-oleoylethanolamine increased the cytotoxic effect of TNFa in a dose dependent manner. Similarly, in astrocytes prepared from pair-fed controls and exposed to alcohol in vitro, N-oleoylethanolamine increased the cytotoxic effect of TNFa in a dose-dependent manner.
Fig. 6. MTT dye-reduction analysis of the effect of N-oleoylethanolamine on TNFa-induced cell death in cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (Solid Lines), or presence (Stippled Lined) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with N-oleoylethanolamine for 2 h and then incubated with TNFa (5 ng/ml) in combination with N-oleoylethanolamine in the absence, or presence of ethanol (50 mM) for 18 h. Each value represents the mean SEM, N = 5. *p < 0.05 vs. Control.
4. Discussion In this study, two indices of cell death (DNA content and the MTT cytotoxicity assay) were used to examined the effect of PAE on cell viability in cultured astrocytes. In apoptotic cells, the induction of endonuclease(s) leads to DNA fragmentation and DNA loss. In cell cultures, the induction of apoptosis can be examined by flow cytometric analysis of DNA content by using intercalating DNA dyes, such as propidium iodide. Here, we show that the incubation of cultured astrocytes prepared from chow-fed or pair-fed controls with a low concentration of TNFa did not induce DNA fragmentation. In contrast, in astrocytes prepared from rats prenatally exposed to alcohol, the same concentration of TNFa induced a marked increase in DNA fragmentation. That is, in chow-fed and pair-fed controls, the flow cytometric analysis of DNA profiles revealed that less than 10% of the cells had DNA contents less than those cells in G1. In primary astrocytes prepared from rats prenatally exposed to alcohol and grown for 14 days in the absence of alcohol, the same concentration of TNFa induced a marked increase in DNA fragmentation with 85% having DNA contents less than those cells in G1. To further examine the effect of PAE on astrocyte viability, we used the MTT cytotoxicity assay, which is a sensitive index of the onset of cell death. Analysis of cell viability by MTT dye-reduction analysis confirmed that in utero alcohol exposure resulted in a marked increase of the cytotoxic effect of TNFa in cultured astrocytes. Similarly, in vitro, the pre-incubation of primary astrocytes prepared from pair-fed controls with ethanol increased the susceptibility of astrocytes to the cytotoxic effect of TNFa. This is consistent with our recent observation using a homogenous population of cultured astrocytes, which showed that the pre-incubation of astrocytes with 5 mM ethanol was sufficient to increase the susceptibility of astrocytes to the cytotoxic effect of TNFa (DeVito et al., 2000). Furthermore, our studies are consistent with the effect of in vivo alcohol exposures on hepatocytes, which shows that hepatocytes from ethanol-fed animals have an increased susceptibility to TNFa, resulting in inappropriate responses to mitogenic signals and increased production of hydrogen peroxide that is inversely correlated with cell survival (Colell et al., 1998; Diehl, 1999). Together, these studies suggest that in utero alcohol exposure increases the sensitivity of astrocytes and other cell types to the cytotoxic effect of TNFa, which persists in the absence of ethanol. TNFa was isolated based on its ability to kill tumor cells in vitro and to cause hemorrhagic necrosis of a transplantable tumor (Carswell et al., 1975). It is now clear, however, that TNFa is an important inflammatory cytokine and is involved in the regulation of cell growth and death (Natoli et al., 1998). Studies by a number of investigators have identified multiple signaling systems, and clearly show that a change in a cell's response to TNFa has a major impact on cell viability (Wiegmann et al., 1994; Schulze-Osthoff et al., 1998). The effects of TNFa are mediated by two distinct
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
cell surface receptors that are expressed on almost all cells: p55TNF-R1 and p75TNF-R2. Studies indicate that TNFa binding to the p55TNF-receptor initiates two signals; one that activates the cell death pathway, and a second pathway that protects the cell from the cytotoxic effects of TNFa. As a rule, the second pathway overcomes the death signal. One pathway involved in the regulation of TNFa-induced cell growth or death is the activation of sphingolipid metabolism (Schutze et al., 1992; Merrill et al., 1997). Studies clearly show that sphingolipid metabolitesÐsuch as ceramide, sphingosine, and SPPÐare the second messengers involved in the regulation of diverse cellular processes (Spiegel et al., 1996; Mathias et al., 1998). TNFa and Fas ligand, which are members of the TNFa superfamily, stimulate SMase leading to the formation of ceramide, an important regulator of programmed cell death (apoptosis) in a number of tissues (Cifone et al., 1994; Hannun, 1994; Heller & Kronke, 1994; Mathias et al., 1998). In contrast, SPP mediates mitogenesis in several cell lines (Zhang et al., 1991; Gomez-Munoz et al., 1995; Pyne & Pyne, 1996; Verheij et al., 1996; Spiegel et al., 1998). Together, current studies suggest that the TNFa-induced activation of SMase results in the hydrolysis of sphingomyelin to ceramide, which can activate the Jun Kinase/stress-activated protein kinase (JNK/SAPK) pathway resulting in cell death and the inhibition of extracellular-signal-regulated kinase (ERK), that is involved in the regulation of cell growth. In contrast, the increased activation of sphingosine kinase by TNFa or growth factors, such as platelet derived growth factor, results in increased cellular SPP levels. SPP in turn, activates other signal transduction systems, such as the ERK signalling pathway resulting in cell growth and the inhibition of JNK/SAPK. In this study, we examined the effect of sphingolipid metabolites on cell death in astrocytes prepared from rats prenatally exposed to alcohol. To determine if PAE increased the susceptibility of astrocytes to the cytotoxic effect of ceramide, astrocytes from pair-fed controls and animals that were prenatally exposed to alcohol were stimulated with exogenous SMase, or cell-permeable ceramide analogs, C2- and C6-ceramide. We found that the primary astrocytes prepared from pair-fed controls were resistant to the cytotoxic effect of low concentrations of SMase, C2-, and C6-ceramide. In contrast, in primary astrocytes prepared from rats exposed to alcohol in utero cell, viability was markedly decreased when exposed to concentrations of SMase, C2-, and C6-ceramide, which were not cytotoxic to control cells. These studies suggest that in utero alcohol exposure alters the cells' response to TNFa, possibly by increasing ceramide production, and/or, that in utero alcohol exposure alters the cells' response to ceramide resulting in cell death. Studies show that the TNFa-induced activation of ceramide activates the JNK/SAPK pathway, resulting in cell death and the inhibition of ERK (Cuvillier et al., 1996). Consistent with these studies, we have recently found that in passaged rat astrocytes, in vitro alcohol exposure increases JNK/SAPK phosphorylation and
69
TNFa-induced activation of the JNK/SAPK pathway (DeVito et al., 2000). In contrast, in hepatocytes, ethanol decreases the activation of JNK and the subsequent phosphorylation of cJun in response to partial hepatectomy (Diehl, 1999). The reason for this discrepancy is unclear, but suggests that the effect of alcohol on TNFa-induced signal transduction pathways may be cell type-specific. Studies conducted on several cell lines show that increased SPP production stimulates mitogenesis, protecting the cell from TNFa-induced cell death (Zhang et al., 1991; Gomez-Munoz et al., 1995; Pyne & Pyne, 1996; Verheij et al., 1996; Spiegel et al., 1998). Here, we show that in primary astrocytes prepared from rats exposed to alcohol in utero, SPP blocked the cytotoxic effect of TNFa. Similarly, in astrocytes prepared from pair-fed rats and exposed to alcohol in vitro, SPP blocked TNFa-induced cell death. In primary astrocytes prepared from rats exposed to alcohol in utero and stimulated with TNFa in the presence of ethanol, SPP blocked the cytotoxic effect of TNFa, but higher concentrations of SPP were required to maintain cell viability at control levels. This suggests that in utero alcohol exposure has a pronounced effect on the cellular response to SPP and decreases the ability of SPP to play a role in decreasing cell death. To further determine the role of SPP in the regulation of cell death, we examined the effect of DMS, a potent competitive inhibitor of sphingosine kinase that decreases cellular SPP levels and increases ceramide levels (Edsall et al., 1998) on TNFa-induced cell death in astrocytes prenatally exposed to alcohol. Here, we show that pretreatment of astrocytes prepared from animals exposed to alcohol in utero or in the presence of alcohol in vitro with DMS enhanced the cytotoxic effect of TNFa. Furthermore, N-oleoylethanolamine, an inhibitor of acid ceramidases that increases cellular ceramide levels, enhanced the cytotoxic effect of TNFa in astrocytes prepared from rats exposed to alcohol in utero. From these studies, we can conclude that in utero alcohol exposure may result in the increased activation of the ceramide pathway, resulting in decreased astrocyte viability. Together, our studies suggest that one of the mechanisms through which in utero alcohol exposure increases the susceptibility of astrocytes to TNFa-induced cell death is through a shift in sphingolipid metabolism, favoring the production of ceramide and the inhibition of SPP production. The mechanisms involved, however, remain to be determined. Studies indicate that astrocytes and microglial cells can be activated to function as antigen-presenting cells (APC). In vitro studies show that astrocytes express MHC II in response to IFNg and TNFa (Shrikant & Benveniste, 1996). The expression of adhesion moleculesÐsuch as ICAM-1, VCAM-1, and E-selectinÐare increased in astrocytes in response to TNFa, interleukin-1 (IL-1), IFNg, LPS, or viral proteins (Shrikant et al., 1994; Rosenman et al., 1995). In addition, an antigenic challenge can stimulate astrocytes to produce and secrete several inflammatory cytokines, such as TNFa and IL-1. We have shown that
70
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
in vitro exposure of astrocytes to low concentrations of ethanol blocks cytokine-induced mitogenesis and the synthesis and release of TNFa and the activation of the JAK/ STAT pathway, a primary signaling system for a number of cytokines and growth factors (DeVito & Stone, 1999; DeVito et al., 1997). In human astrocytoma cell, we have found that ethanol blocks the cell surface expression of ICAM-1 (DeVito et al., 2000). Further, studies in primary astrocytes prepared from rats exposed to alcohol in utero show that PAEs blocks growth factor-induced mitogenesis (DeVito et al., 1997), and as shown here, markedly increases the susceptibility of astrocytes to the cytotoxic effect of TNFa. Accordingly, these studies suggest that in utero alcohol exposure could alter the CNS response to injury or infection resulting in an inappropriate neuroimmune response and worsened neurological outcome. Glial cells are one of the most abundant cell types in the CNS, and they are essential for normal CNS development. Glial cells provide the structure and nutritive support for developing neurons and are excellent substrates for neurite extension and can provide a permissive substrata to support axonal regrowth and promote oligodendrite process extension by expressing adhesion molecules and neurotrophic factors (Oh & Youg, 1996). One striking effect of PAE is the disruption in the proliferation of neurons and glial cells. Animal models show that PAE-induced microencephaly is the result of decreased cell numbers in several brain regions (Bonthius & West, 1990; West et al., 1990; Pantazis et al., 1993; Miller, 1995). Consistent with the in vivo effects of alcohol, in vitro studies clearly show that alcohol exposure results in decreased cell proliferation (Pantazis et al., 1992; Kane et al., 1996; Klein et al., 1996; DeVito et al., 1997). In agreement with these studies, the present study suggests that alcohol-induced decrease in glial cell proliferation and increased cell death in response to cytokines could be a potentially important mechanism involved in the neurological and neurobehavioral dysfunction associated with PAE. In summary, we show that PAE markedly increases the susceptibility of astrocytes to the cytotoxic effect of TNFa. More importantly, the observation that astrocytes prepared from rats prenatally exposed to alcohol are more susceptible to the cytotoxic effect of TNFa when grown in the absence of ethanol, suggests that in utero alcohol exposure may have a long-term effect on astrocyte function. One mechanism through which prenatal alcohol exposure could increase the sensitivity of astrocytes to the cytotoxic effect of TNFa may be attributed to the altered metabolism of sphingolipids, resulting in the increased production of ceramide and a decrease in SPP levels. Acknowledgments This work was supported in part by funds by the National Institute of Alcohol Abuse and Alcoholism Grant No. AA11277.
References Benveniste, E. N. (1992). Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action. Am J Physiol 263, C1 ± C16. Bielawska, A., Greenberg, M. S., Perry, D., Jayadev, S., Shayman, J. A., McKay, C., & Hannun, Y. A. (1996). (1S, 2R)-D-erthro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor a ceramidase. J Biol Chem 271, 12646 ± 12654. Bonthius, D. J., & West, J. R. (1990). Alcohol-induced neuronal loss in developing rats: increased brain damage with binge exposure. Alcohol Clin Exp Res 14, 107 ± 118. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., & Williamson, B. (1975). An endotoxin-induced factor that causes necrosis of tumors. Proc Natl Acad Sci USA 72, 3666 ± 3670. Cavani, M., Ghirelli, D., Fortuna, C., Lalli, F., & Marcolini, P. (1985). La sindrome feto-alcolica: follow-up clinico ± metabolico ± immunitario di 14 casi. Minerva Pediatr 37, 77 ± 88. Chung, I. Y., Norris, J. G., & Benveniste, E. N. (1991). Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and resistant rat strains. J Exp Med 173, 801 ± 811. Cifone, M. G., DeMaria, R., Roncaioli, P., Rippo, M. R., Azuma, M., Lanier, L. L., Santoni, A., & Testi, R. (1994). Apoptotic signaling through CD95 (Fas/Apo-1) activates an acid sphingomyelinase. J Exp Med 180, 1547 ± 1552. Colell, A., Garcia-Ruiz, C., Miranda, M., Ardite, E., Mari, M., Morales, A., Corrales, F., Kaplowitz, N., & Fernandez-Checa, J. C. (1998). Selective glutathione depletion of microchondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 115, 1541 ± 1551. Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., & Spiegel, S. (1996). Suppression of ceramidemediated programmed cell death by sphingosine-1-phosphate. Nature 381, 800 ± 803. Davies, D. L., & Vernadakis, A. (1984). Effects of ethanol on cultured glial cells: proliferation and glutamine synthetase activity. Brain Res 318, 27 ± 35. DeVito, W. J., & Stone, S. (1999). Ethanol inhibits prolactin-induced activation of the JAK/STAT pathway in cultured Astrocytes. J Cell Biochem 74, 278 ± 291. DeVito, W. J., Okulicz, W. C., Stone, S., & Avakian, C. (1992). Prolactin-stimulated mitogenesis of cultured astrocytes. Endocrinology 130, 2549 ± 2556. DeVito, W. J., Stone, S., & Mori, K. (1997). Low concentrations of ethanol inhibits prolactin-induced mitogenesis and cytokine expression in cultured astrocytes. Endocrinology 138, 922 ± 928. DeVito, W. J., Stone, S., Mori, K., & Shamgochian, M. (2000). Ethanol inhibits prolactin and tumor necrosis factor a-, but not gamma interferon-induced expression of intercellular adhesion molecule-1 in human astrocytoma cells. J Cellular Biochem 77, 455 ± 464. Diehl, A. M. (1999). Cytokines and the molecular mechanisms of alcoholic liver disease. Alcohol Clin Exp Res 23, 1419 ± 1424. Edsall, L. C., Van Brocklyn, J. R., Cuvillier, O., Kleuser, B., & Spiegel, S. (1998). N,N-dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphinosine 1-phosphate and ceramide. Biochemistry 37, 12892 ± 12898. Eng, L. F., Yu, A. C. H., & Lee, Y. L. (1992). Astrocytic response to injury. Prog Brain Res 94, 353 ± 365. Ewald, S. J. (1989). T lymphocyte populations in fetal alcohol syndrome. Alcohol Clin Exp Res 13, 485 ± 489. Ewald, S. J., & Frost, W. W. (1987). Effect of prenatal exposure to ethanol on development of the thymus. Thymus 9, 211 ± 215. Ewald, S. J., Huang, C., & Bray, L. (1991). Effect of prenatal alcohol exposure on lymphocyte population in mice. Adv Exp Med Biol 288, 237 ± 244. Gomez-Munoz, A., Waggoner, D. W., O'Brien, L., & Brindley, D. N.
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71 (1995). Interaction of ceraminds, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and phospholipase D activity. J Biol Chem 270, 26318 ± 26325. Gottesfeld, Z., Christie, R., Felten, D., & LeGrue, S. (1990). Prenatal ethanol exposure alters immune capacity and noradrenergic synaptic transmission in lymphoid organs of the adult mouse. Neuroscience 35, 185 ± 194. Guerri, C., Saez, R., Sancho-Tello, M., de Aquilera, E. M., & Renau-piqueras, J. (1990). Ethanol alters astrocyte development: a study of critical periods using primary cultures. Neuochem Res 15, 559 ± 565. Hannun, Y. A. (1994). The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem 269, 3125 ± 3128. Hatten, M. H., Liem, R. K. H., Shelanski, M. L., & Mason, C. A. (1991). Astroglia in CNS injury. Glia 4, 233 ± 243. Heller, R. A., & Kronke, M. (1994). Tumor necrosis factor receptormediated signaling pathways. J Cell Biol 126, 5 ± 9. Hickey, W. F., Osborn, J. P., & Kirby, W. M. (1985). Expression of Ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat. Cell Immunol 91, 528 ± 535. Johnson, S., Knight, R., Marmer, D. J., & Steele, R. W. (1981). Immune deficiency in fetal alcohol syndrome. Pediatr Res 15, 908 ± 911. Kane, C. J., Berry, A., Boop, F. A., & Davis, D. L. (1996). Proliferation of astroglia from the adult cerebrum is inhibited by ethanol in vitro. Brain Res 731, 39 ± 44. Klein, R. F., Fausti, K. A., & Carlos, A. S. (1996). Ethanol inhibits human osteoblastic cell proliferation. Alcohol Clin Exp Res 20, 572 ± 578. Martin, R., McFarland, H. F., & McFarlin, D. E. (1992). Immunological aspects of demyelinating diseases. Annu Rev Immunol 10, 153 ± 187. Mathias, S., Pene, L. A., & Kolesnick, R. N. (1998). Signal transduction of stress via ceramide. Biochem J 335, 465 ± 480. Merrill, A. H. J., Schemelz, E.-M., Dillehay, D. L., Spiegel, S., Shayman, J. J., Schroeder, J. J., Riley, R. T., Voss, K. A., & Wang, E. (1997). Shingolipids Ð the enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 142, 208 ± 225. Miller, M. W. (1995). Effect of pre- or postnatal exposure to ethanol on the total number of neurons in the principal sensory nucleus of the trigeminal nerve: cell proliferation and neuronal death. Alcohol Clin Exp Res 19, 1359 ± 1363. Natoli, G., Costanzo, A., Guido, F., Moretti, F., & Levrero, M. (1998). Apoptotic, non-apoptotic, and anti-apoptotic pathways of tumor necrosis factor signalling. Biochem Pharmacol 56, 915 ± 920. Normon, D. C., Chang, M. P., Wong, C. M., Branch, B. J., Castle, S., & Taylor, A. N. (1991). Changes with age in the proliferative response of splenic T cells from rats exposed to ethanol in utero. Alcohol Clin Exp Res 15, 423 ± 432. Oh, Y. S., & Youg, V. W. (1996). Astrocytes promote progress outgrowth by adult human oligodendrocytes in vitro through interaction between bFGF and astrocyte extracellular matrix. Glia 17, 237 ± 253. Pantazis, N. J., Dohrman, D. P., Goodlett, C. R., Cook, R. T., & West, J. R. (1993). Vulnerability of cerebellar granule cells to alcohol-induced cell death diminishes with time in culture. Alcohol Clin Exp Res 17, 1014 ± 1021. Pantazis, N. J., Dohrman, D. P., Luo, J., Goodlett, C. R., & West, J. R. (1992). Alcohol reduces the number of pheochromocytoma (PC12) cells in culture. Alcohol 9, 171 ± 180. Pyne, S., & Pyne, N. J. (1996). The differential regulation of cyclic AMP by sphingomylin-derived lipids and the modulation of sphingolipid-stimulated extracellular signal regulated kinase-2 in airway smooth muscle. Biochem J 315, 917 ± 923. Redei, E., Clark, W. R., & McGivern, R. F. (1989). Alcohol exposure in utero results in diminished T-cell function and alterations in brain corticotropin-releasing factor and ACTH content. Alcohol Clin Exp Res 13, 439 ± 443.
71
Renau-piqueras, J., Sancho-Tello, M., Zaragoza, R., & Guerri, C. (1988). Effects of ethanol on the development of astrocytes in primary culture. Adv Biosci 71, 269 ± 273. Renau-piqueras, J., Zaragoza, R., De Paz, P., Baguena-Cervellera, R., Megias, L., & Guerri, C. (1989). Effects of prolonged ethanol exposure on glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study. J HIstochem Cytochem 37, 229 ± 240. Rosenman, S. T., Shrikant, P., Dubb, L., Benveniste, E. N., & Ransohoff, R. M. (1995). Cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-) by astrocytes and astrocytoma cell lines. J Immunol 154, 1888 ± 1899. Saez, R., Burgal, M., Renau-piqueras, J., Marques, A., & Guerri, C. (1991). Evolution of several cytoskeletal proteins of astrocytes in primary culture: effect of prenatal ethanol exposure. Neurochem Res 16, 737 ± 747. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., & Peter, M. E. (1998). Apoptosis signaling by death receptors. Eur J Biochem 254, 439 ± 459. Schutze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K., & Kronke, M. (1992). TNF activates NK-kB by phosphatidylcholine-specific phospholipase C-induced ``acidic'' sphingomyelin breakdown. Cell 71, 765 ± 776. Shrikant, P., & Benveniste, E. N. (1996). The central nervous system as an Immunocompetent organ: role of glial cells in antigen presentation. J Immunol 157, 1819 ± 1822. Shrikant, P., Chung, I. Y., Ballestas, M., & Benveniste, E. N. (1994). Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-a, interleukin-b, and interferon-g in astrocytes. J Neuroimmunol 51, 209. Spiegel, S., Cuvillier, O., Edsall, L. C., Kohama, T., Menzeleev, R., Olah, A., Olivera, A., Pirianov, G., Thomas, D. M., Tu, Z., Van Brocklyn, J. R., & Wang, F. (1998). Sphingosine-1-phosphate in cell growth and cell death. Ann N Y Acad Sci 845, 11 ± 18. Spiegel, S., Foster, D., & Kolesnick, R. N. (1996). Signal transduction through lipid second messengers. Curr Opin Cell Biol 8, 159 ± 167. Verheij, M., Bose, R., Lin, Z. H., Yoa, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., & Kolesnick, R. N. (1996). Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380, 75 ± 79. Weinberg, J., & Jerrells, T. R. (1991). Suppression of immune responsiveness: sex differences in prenatal ethanol effects. Alcohol Clin Exp Res 15, 525 ± 531. West, J. R., Goodlett, C. R., Bonthius, D. J., Hamre, K. M., & Marcussen, B. L. (1990). Cell population depletion associated with fetal alcohol brain damage: mechanisms of BAC-dependent cell loss. Alcohol Clin Exp Res 14, 813 ± 818. Wiegmann, K., Schutze, S., Machleidt, T., Witte, D., & Kronke, M. (1994). Functional dichotomy of neutral and acidic sphingomylinases in tumor necrosis factor signaling. Cell 78, 1005 ± 1015. Wolcott, R. M., Jennings, S. R., & Chervenak, R. (1995). In utero exposure to ethanol affects postnatal development of T- and B-lymphocytes, but not natural killer cells. Alcohol Clin Exp Res 19, 170 ± 176. Wong, C. M., Chiappelli, F., Chang, M. P., Norman, D. C., Cooper, E. L., Branch, B. J., & Taylor, A. N. (1992). Prenatal exposure to alcohol enhances thymocyte mitogenic responses postnatally. Int J Immunopharmacol 14, 303 ± 309. Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., & Spiegel, S. (1991). Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation. J Cell Biol 114, 155 ± 167.
Prenatal alcohol exposure increases TNFa-induced cytotoxicity in primary astrocytes William J. De Vito*, Krisanthi Xhaja, Scott Stone Division of Endocrinology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA, 01655, USA Received 10 December 1999; received in revised form 26 January 2000; accepted 28 January 2000
Abstract We examined the effect of prenatal alcohol exposure (PAE) on tumor necrosis factor-a-(TNFa) induced cell death in primary astrocyte cultures. Flow cytometry revealed that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFa when compared to astrocytes prepared from pair-fed and chow-fed controls. In a number of cell types, TNFa regulates cell growth or death, in part, by the hydrolysis of sphingomyelin to ceramide and sphingosine-1-phosphate (SPP). Using a 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxic assay we found that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFa, sphingomyelinase (SMase), and C2- and C6-ceramide. The increasing cellular concentrations of SPP, a sphingolipid metabolic that induces cell growth, protected the cells from TNFa-induced cell death. N,N-dimethylsphingosine (DMS), which inhibits SPP production, and Noleoylethanolamine, which inhibits acid ceramidases, increased TNFa-induced cytotoxicity in astrocytes prepared from PAE rats. These studies suggest that PAE shifts the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNFa. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Prenatal alcohol exposure; TNFa; Astrocytes; Ceramide
1. Introduction 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. Prenatal alcohol syndrome is associated with growth retardation, central nervous system dysfunction, and is one of the leading causes of mental retardation. Children exposed to ethanol in utero also have defects in host defense and increased incidence and severity of infection with some children with FAS showing long lasting deficiencies in both humoral and cell mediated immunity (Johnson et al., 1981; Cavani et al., 1985). Animal studies have substantiated the immunoteratogenic effect of in utero alcohol exposure. Rodents prenatally exposed to alcohol have been shown to have decreased thymic cellularity, decreased lymphocyte numbers and de-
* Corresponding author. Tel.: +1-508-856-6244; fax: +1-508-8566950. E-mail address: [email protected] (W.J. De Vito).
fects in thymic and splenic proliferation in response to T-cell mitogens (Ewald, 1989; Ewald & Frost, 1987; Ewald et al., 1991). Studies further show that splenic lymphocytes prepared from neonatal and adult animals, which were exposed to alcohol in utero display long-lasting defects in T-cell proliferation (Redei et al., 1989; Normon et al., 1991; Weinberg & Jerrells, 1991; Wong et al., 1992). In utero ethanol exposure also has a marked impact on B-cell development. Among neonatal mice exposed to alcohol in utero there is a marked deficiency in B-cell development and response to lipopolysaccharide (LPS), pokeweed mitogen, phytohemagglutinin, and concanavalin A, which extends passed weaning (Wolcott et al., 1995). Further, in the utero alcohol exposure disrupts the developmental pathway of B-lineage intermediates, thereby leaving the animal immunocompromized at birth (Gottesfeld et al., 1990). Studies clearly show that alcohol is an immunosuppressive drug and that prenatal alcohol exposure (PAE) has marked effects on T- and B-cell development, which compromises the ability of neonatal animals to mount an effective immune response. Little is known, however, about the effects of alcohol and PAE on the neuroimmune response. In the CNS, the response to injury and disease involves
0741-8329/00/$ ± see front matter D 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 0 ) 0 0 0 7 8 - 1
64
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
interactions among various cell types (neurons, astrocytes, oligodendroglia, microglia, and infiltrating inflammatory cells), cytokines, growth factors, and membrane-associated proteins (Hatten et al., 1991; Eng et al., 1992). Astrocytes comprise as much as 25% of the cells in the CNS; however, until recently, astrocytes have received little attention. Unlike neurons, astrocytes retain the ability to divide and multiply. In the CNS, astrocytes can function as immunocompetent cells by secreting cytokines, and by expressing MHC class I and II antigens and adhesion molecules (Chung et al., 1991). In response to injury or infection, astrocytes undergo hypertrophy and proliferation and are transformed into reactive astrocytes. 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 (MS), acquired immune deficiency syndrome dementia complex, Alzheimer's disease, and the animal model for MS, experimental allergic encephalomyelitis (Hickey et al., 1985; Benveniste, 1992; Martin et al., 1992). The in vitro exposure of primary astrocyte cultures to ethanol have many of the same morphological and biochemical changes found in primary cultures prepared from animals that were prenatally exposed to ethanol (Davies & Vernadakis, 1984; Renau-piqueras et al., 1988, 1989; Guerri et al., 1990; Saez et al., 1991). This has allowed for the examination of the direct effects of ethanol on glial cells. We have shown that ethanol decreases cytokine- and hormoneinduced astrocyte proliferation, as well as the expression and secretion of tumor necrosis factor-alpha (TNFa) (DeVito et al., 1997), the cell surface expression of ICAM-1 in human astrocytoma cells (DeVito et al., 2000), and blocks the prolactin-induced activation of the JAK/STAT pathway in cultured astrocytes (DeVito & Stone, 1999). Further, we have recently shown that the pre-exposure of astrocytes to low concentrations of ethanol increases their susceptibility to the cytotoxic effect of TNFa (DeVito et al., 2000). In this study, we examined the effect of PAE on the cytotoxic effect of TNFa in primary astrocyte cultures. Here, we show that primary astrocytes, which were prepared from rats prenatally exposed to alcohol and grown in the absence of ethanol, are sensitized to the cytotoxic effect of TNFa. Further, our data suggest that TNFa-induced metabolism of sphingolipids is shifted to favor the production of ceramide resulting in cell death. 2. Materials and methods 2.1. Materials Recombinant rat TNFa was obtained from the Genzyme (Cambridge, MA). Other materials were purchased from the following sources: bacterial sphingomyelinase (SMase; Staphylococcus aureus), C2-ceramide, C6-ceramide, dihydroC2-ceramide N,N-Dimethylsphingosine (DMS), D,L-dithio-
threitol (DTT), N-oleoylethanolamine, from Sigma (St. Louis, MO); calf serum from Gibco BRL (Grand Island, NY). All other chemicals and reagents were obtained from commercial sources and were of reagent or molecular biology grade. 2.2. Cell culture The astrocytes were prepared from cerebral hemispheres of 1-day-old rat pups as previously described (DeVito et al., 1992). Cells were seeded at 2 105 cell/cm2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum and cultured at 37°C, under an atmosphere of 5% CO2, 95% air. Cells were grown to confluence and used after 14 days in culture. 2.3. 3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay Cell viability was determined by the MTT assay. Astrocytes were seeded in 96-well plates and treated as described in the figure legends. MTT was dissolved in PBS at a concentration of 5 mg/ml. From this stock solution, 20 ul per 100 ul of medium was added to each well, and the plates were incubated at 37°C for 4 h. Acid ±isopropanol (100 ul of 0.04 M HCl in isopropanol) was added to lyse the cells. After 15 min at room temperature, the plates were read on a Labsystems Multiskan MS plate reader at a test wavelength of 595 nm and a reference wavelength of 650 nm. 2.4. Prenatal alcohol exposure Nulliparous females (150 ± 210 g) were individually housed each evening with a male until a vaginal smear, which was indicated as day 1 of pregnancy. They were then housed individually under controlled lighting, lights on between 0600 and 1900 h. Prospective mothers were fed a chow diet (Purina 5008) until gestational (G) day 7. At this time, the rats were weight-matched and separated into the three dietary groups: a chow (Ch) diet, a control (Ct) diet or a ethanol diet (Et). The control and ethanol diets were highprotein, liquid diets (Lieber ± DeCarli), which are nutritionally balanced to meet the specific needs of pregnant rats (Bioserve, Frenchtown, NJ). Both diets were fortified with Vitamin Diet Fortification Mixture and Salt Mixture. Commencing on day 7 of gestation, the dams were fed an alcohol-containing liquid diet (No 1265, Bio-Serv, Frenchtown, NY) containing 2.2% (v/v) ethanol at day 7 and 4.5% v/v ethanol at day 14. In the control diet, an isocaloric amount of dextrin-maltose was substituted for the ethanol. Tap water was available ad libitum. The chow diet consisted of standard rat chow and water ad libitum. Liquid diets were provided in 120-ml graduated feeding tubes. All ethanol-fed rats were paired by weight with a rat fed with control diet, and the two were pair-fed. That is, each control-fed rat received the same volume of liquid diet as that consumed
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
the previous day by the weight-matched ethanol-fed rat. Thus, during their pregnancies, both the control- and ethanol-fed rats consumed diets of identical caloric content. The amount of liquid ingested by the control- and ethanol-fed females was determined at 0900 h each morning and at 1700 h each evening. If an ethanol-fed rat required more than the estimated 100 ml of diet, additional diet was added at 1700 h to insure that each ethanol-fed rat received an adequate supply of diet to meet an individual animals needs. After recording the past day's food consumption, the ethanol-fed and control-fed rats received their daily food allotments. Throughout the gestation period, chow-fed rats were fed chow and water ad libitum. All pregnant rats were weighed on alternative days, and only those weight-matched controlfed and ethanol-fed rats that maintained a weight within 10% of the chow-fed rats were kept in the study. At birth, the pups were counted and weighed. Maternal blood samples were collected by orbital venipuncture under general anesthesia (pentobarbital 50 mg/kg, i.p.) on the morning of birth from control- and ethanol-fed rats. Pooled blood samples was collected from the pups of each litter during the preparation of the primary cultures. The concentration of ethanol in the blood samples was measured using a Sigma diagnostic kit (#332 UV), according to the manufacturer's instructions. All experimental procedures were approved by the University of Massachusetts Medical School institutional animal care and use committees. 2.5. DNA content The cells were collected by centrifugation and transferred into 1 ml of Hanks' buffered salt solution and then into tubes containing 10 ml of 70% ethanol on ice. The cell were stored in the fixative at ÿ20°C for 24 h. The cells were then centrifuged at 800 g for 5 min and the ethanol was thoroughly removed. The cells were stained with propidium iodide and the florescence of individual cells was measured by flow cytometry using a FACScan instrument (Becton Dickinson, Sunnyvale, CA). 2.5.1. Statistical analysis The effect of PAE and ethanol on cell viability were analyzed with a randomized factorial analysis of variance. The test of simple main effects was used to test for differences between two groups. The size of the region of rejection of the null hypothesis was set by an alpha error of 5%. 3. Results 3.1. Effect of PAE on DNA content in TNFa stimulated primary astrocytes The exposure of rats to ethanol during gestation resulted in blood alcohol concentrations in dams and pups ranging
65
between 52 and 78 mg/dl, and 31 and 56 mg/dl, respectively on the day of birth. To examine the effect of PAE on TNFainduced DNA fragmentation, flow cytometric analysis of cellular DNA content was preformed on primary astrocytes prepared from control, pair-fed and ethanol-fed rats. Flow cytometric analysis of cellular DNA content from astrocytes prepared from rats fed a chow diet shows a typical cell cycle DNA profile with a primary G1 DNA peak and a smaller G2M DNA peak (Fig. 1A). The flow cytometric analysis of cellular DNA content from astrocytes prepared from chowand pair-fed rats showed that incubation of astrocytes with TNFa for 18 h did not increase the number of cells having a DNA content less (M1) than those cells in G1 (Fig. 1B and C). In astrocytes prepared from rats that were prenatally exposed to alcohol, however, TNFa stimulation significantly (P < 0.05) increased the number of cells with DNA content less than those cells in G1 when compared to chowand pair-fed controls. This indicates that PAE increases the sensitivity of astrocytes to the cytotoxic effect of TNFa, resulting in increased DNA fragmentation and decreased DNA content. Next, we examined the effect of PAE on cell viability using the MTT cytotoxic assay, which is a sensitive index of decreased cell viability. In a series of preliminary studies, we found that the response of astrocytes to TNF and SMase, C6- and C2-ceramide where similar in astrocytes prepared from chow- and pair-fed rats. Therefore, to reduce the number of animals required to complete these studies and to simplify the presentation of the data, we used astrocytes from pair-fed rats as controls. We found that incubation of astrocytes prepared from pair fed-rats with low concentrations of TNFa did not decrease cell viability (Fig. 2). The incubation of astrocytes prepared from rats prenatally exposed to alcohol with low concentrations of TNFa, however, resulted in a dose-dependent decrease in cell viability. We then examined the effect of in vitro ethanol exposure on TNFa-induced cell death in astrocytes prepared from pair-fed and PAE rats (Fig. 2). Astrocytes were cultured in the presence of ethanol (50 mM) for 18 h, and then stimulated with increasing concentrations of TNFa for 18 h. The incubation of astrocytes with ethanol (50 mM) for 36 h had no effect on cell viability (data not shown). In astrocytes prepared from pair-fed controls, pre-incubation with 50 mM ethanol for 18 h increased the TNFa-induced cytotoxicity with TNFa concentrations of 5 ng/ml, or greater, decreasing cell viability. Similarly, in astrocytes prepared from rats prenatally exposed to alcohol, pre-incubation with ethanol resulted in a further increase in TNFa-induced cell death. 3.2. Effect of PAE on SMase- and ceramide-induced cell death in cultured astrocytes Recent studies have revealed that the hydrolysis of sphingomyelin to ceramide is involved in the regulation of the cytotoxic effect of TNFa in a variety of cell types (Natoli et al., 1998). Accordingly, studies were performed
66
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
Fig. 1. Effect of prenatal alcohol exposure on DNA content in TNFa-stimulated cultured rat astrocytes. Astrocytes were prepared from rats fed with a chow diet (Ch), control diet (Ct) or an ethanol diet (Et). Astrocytes were grown to confluence and stimulated with TNFa (100 ng/ml) for 18 h. Data are presented as DNA fluorescence histograms of propidium iodide-stained astrocytes. The data presented are representative of three separate experiments.
Fig. 2. MTT dye-reduction analysis of the effect of prenatal alcohol exposure on cell viability in TNFa-stimulated cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines), or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with TNFa in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control.
to determine if this pathway is involved in TNFa-induced cell death in cultured astrocytes prepared from rats prenatally exposed to alcohol. Astrocytes were incubated with SMase, which results in the hydrolysis of sphingomyelin to ceramide, or C2- or C6-ceramide, membrane-permeable ceramide analogs. In astrocytes prepared from pair-fed control rats, the incubation of cultured astrocytes with increasing concentrations of SMase did not decrease cell viability (Fig. 3A). Similarly, the incubation of astrocytes prepared from pair-fed controls with increasing concentrations of C2- or C6-ceramide did not decrease astrocyte viability (Fig. 3B and C). In contrast, the incubation of astrocytes prepared from rats prenatally exposed to alcohol with SMase, C2- or C6-ceramide resulted in a dose dependent decrease in cell viability (Fig. 3A,B,C, respectively). In astrocytes prepared from pair-fed controls, pre-incubation with ethanol for 18 h sensitized the cells to the cytotoxic effects of SMase, C2-, and C6-ceramide. In astrocytes prepared from rats prenatally exposed to alcohol, the in vitro ethanol exposure increased the cytotoxic effects of C2and C6-ceramide. The incubation of astrocytes prepared from pair-fed controls and rats prenatally exposed to alcohol with dihydro-C2-ceramide, an inactive ceramide analog, did not affect cell viability (Fig. 3D).
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
67
Fig. 4. MTT dye-reduction analysis of the effect of sphingosine-1phosphate (SPP) on TNFa-induced cell death in cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines), or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with SPP for 4 h and then stimulated with TNFa (50 ng/ml) in combination with SPP in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control. Fig. 3. MTT dye-reduction analysis of the effect of prenatal alcohol exposure on cell viability. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines), or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with SMase (A), C2-ceramide (B), C6-ceramide (C), or DihydroC2-ceramide (D) in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control.
decreased cell viability by 90%. Pre-incubation of cells with SPP block TNFa-induced cell death, but required a higher concentration of SPP (75 nM) to maintain cell viability at control levels. DMS is a potent competitive inhibitor of sphingosine kinase resulting in decreased cellular SPP levels and
3.3. Effect of sphingosine-1-phosphate (SPP), DMS and Noleoylethanolamine on TNFa-induced cell death in cultured astrocytes The current models of TNFa-induced cell death suggest that the growth factor-induced activation of SPP, a breakdown product of sphingolipid, protects cells from the cytotoxic effects of TNFa (Cuvillier et al., 1996). Accordingly, we examined the effect of SPP on TNFa-induced cell death in astrocytes prepared from pair-fed subjects and rats prenatally exposed to alcohol (Fig. 4). Astrocytes were incubated with SPP for 4 h and then stimulated with TNFa in combination with SPP. The incubation of astrocytes prepared from pair-fed controls with TNFa alone, or in combination with SPP, did not decrease astrocyte viability. In astrocytes prepared from rats prenatally exposed to alcohol, or exposed to ethanol in vitro, TNFa markedly decreased astrocyte viability. In astrocytes prepared from pair-fed rats and rats exposed to alcohol in utero, SPP resulted in a dose-dependent inhibition of TNFa-induced cell death, with 50 nM SPP maintaining cell viability at control levels. In astrocytes prenatally exposed to alcohol, and incubated in the presence of ethanol in vitro, TNFa
Fig. 5. MTT dye-reduction analysis of the effect of N,N-dimethylsphingosine (DMS) on TNFa-induced cell death in cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (solid lines) or presence (stippled lines) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with DMS for 4 h and then incubated with TNFa (5 ng/ml) in combination with DMS in the absence or presence of ethanol (50 mM) for 18 h. Each value represents the mean S.E.M., N = 5. *p < 0.05 vs. Control.
68
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
increased ceramide levels (Edsall et al., 1998). Accordingly, we examined the effect of DMS on TNFa-induced cell death (Fig. 5). In astrocytes prepared from pair-fed controls, there was no effect of low concentrations of DMS on TNFa-induced cell death. At higher concentrations, DMS (50 ±75 uM) alone decreased cell viability in astrocytes prepared from pair fed rats. In astrocytes prepared for rats prenatally exposed to alcohol lower concentrations of DMS (25 uM) decreased cell viability in quiescent astrocytes (data not presented). In contrast, in astrocytes prepared from rats prenatally exposed to alcohol, low concentrations of DMS markedly increased TNFa-induced cell death. Similarly, in astrocytes prepared from pair-fed controls and exposed to alcohol in vitro, DMS increased the cytotoxic effect of TNFa. Next, we examined the effect of N-oleoylethanolamine, an inhibitor of acid ceramidases (Bielawska et al., 1996) on TNFainduced cell death in astrocytes prepared from pair fedcontrols and rats prenatally exposed to alcohol. Astrocytes were incubated with N-oleoylethanolamine for 2 h and then with TNFa for 18 h. In astrocytes prepared from pair-fed controls, the low concentrations of N-oleoylethanolamine did not effect TNFa-induced cell death (Fig. 6). In contrast, in astrocytes prepared from rats prenatally exposed to alcohol, N-oleoylethanolamine increased the cytotoxic effect of TNFa in a dose dependent manner. Similarly, in astrocytes prepared from pair-fed controls and exposed to alcohol in vitro, N-oleoylethanolamine increased the cytotoxic effect of TNFa in a dose-dependent manner.
Fig. 6. MTT dye-reduction analysis of the effect of N-oleoylethanolamine on TNFa-induced cell death in cultured astrocytes. Astrocytes were prepared from pair-fed controls and rats prenatally exposed to alcohol. Astrocytes were grown to confluence and then cultured in the absence (Solid Lines), or presence (Stippled Lined) of ethanol (50 mM) for 18 h. Astrocytes were then incubated with N-oleoylethanolamine for 2 h and then incubated with TNFa (5 ng/ml) in combination with N-oleoylethanolamine in the absence, or presence of ethanol (50 mM) for 18 h. Each value represents the mean SEM, N = 5. *p < 0.05 vs. Control.
4. Discussion In this study, two indices of cell death (DNA content and the MTT cytotoxicity assay) were used to examined the effect of PAE on cell viability in cultured astrocytes. In apoptotic cells, the induction of endonuclease(s) leads to DNA fragmentation and DNA loss. In cell cultures, the induction of apoptosis can be examined by flow cytometric analysis of DNA content by using intercalating DNA dyes, such as propidium iodide. Here, we show that the incubation of cultured astrocytes prepared from chow-fed or pair-fed controls with a low concentration of TNFa did not induce DNA fragmentation. In contrast, in astrocytes prepared from rats prenatally exposed to alcohol, the same concentration of TNFa induced a marked increase in DNA fragmentation. That is, in chow-fed and pair-fed controls, the flow cytometric analysis of DNA profiles revealed that less than 10% of the cells had DNA contents less than those cells in G1. In primary astrocytes prepared from rats prenatally exposed to alcohol and grown for 14 days in the absence of alcohol, the same concentration of TNFa induced a marked increase in DNA fragmentation with 85% having DNA contents less than those cells in G1. To further examine the effect of PAE on astrocyte viability, we used the MTT cytotoxicity assay, which is a sensitive index of the onset of cell death. Analysis of cell viability by MTT dye-reduction analysis confirmed that in utero alcohol exposure resulted in a marked increase of the cytotoxic effect of TNFa in cultured astrocytes. Similarly, in vitro, the pre-incubation of primary astrocytes prepared from pair-fed controls with ethanol increased the susceptibility of astrocytes to the cytotoxic effect of TNFa. This is consistent with our recent observation using a homogenous population of cultured astrocytes, which showed that the pre-incubation of astrocytes with 5 mM ethanol was sufficient to increase the susceptibility of astrocytes to the cytotoxic effect of TNFa (DeVito et al., 2000). Furthermore, our studies are consistent with the effect of in vivo alcohol exposures on hepatocytes, which shows that hepatocytes from ethanol-fed animals have an increased susceptibility to TNFa, resulting in inappropriate responses to mitogenic signals and increased production of hydrogen peroxide that is inversely correlated with cell survival (Colell et al., 1998; Diehl, 1999). Together, these studies suggest that in utero alcohol exposure increases the sensitivity of astrocytes and other cell types to the cytotoxic effect of TNFa, which persists in the absence of ethanol. TNFa was isolated based on its ability to kill tumor cells in vitro and to cause hemorrhagic necrosis of a transplantable tumor (Carswell et al., 1975). It is now clear, however, that TNFa is an important inflammatory cytokine and is involved in the regulation of cell growth and death (Natoli et al., 1998). Studies by a number of investigators have identified multiple signaling systems, and clearly show that a change in a cell's response to TNFa has a major impact on cell viability (Wiegmann et al., 1994; Schulze-Osthoff et al., 1998). The effects of TNFa are mediated by two distinct
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
cell surface receptors that are expressed on almost all cells: p55TNF-R1 and p75TNF-R2. Studies indicate that TNFa binding to the p55TNF-receptor initiates two signals; one that activates the cell death pathway, and a second pathway that protects the cell from the cytotoxic effects of TNFa. As a rule, the second pathway overcomes the death signal. One pathway involved in the regulation of TNFa-induced cell growth or death is the activation of sphingolipid metabolism (Schutze et al., 1992; Merrill et al., 1997). Studies clearly show that sphingolipid metabolitesÐsuch as ceramide, sphingosine, and SPPÐare the second messengers involved in the regulation of diverse cellular processes (Spiegel et al., 1996; Mathias et al., 1998). TNFa and Fas ligand, which are members of the TNFa superfamily, stimulate SMase leading to the formation of ceramide, an important regulator of programmed cell death (apoptosis) in a number of tissues (Cifone et al., 1994; Hannun, 1994; Heller & Kronke, 1994; Mathias et al., 1998). In contrast, SPP mediates mitogenesis in several cell lines (Zhang et al., 1991; Gomez-Munoz et al., 1995; Pyne & Pyne, 1996; Verheij et al., 1996; Spiegel et al., 1998). Together, current studies suggest that the TNFa-induced activation of SMase results in the hydrolysis of sphingomyelin to ceramide, which can activate the Jun Kinase/stress-activated protein kinase (JNK/SAPK) pathway resulting in cell death and the inhibition of extracellular-signal-regulated kinase (ERK), that is involved in the regulation of cell growth. In contrast, the increased activation of sphingosine kinase by TNFa or growth factors, such as platelet derived growth factor, results in increased cellular SPP levels. SPP in turn, activates other signal transduction systems, such as the ERK signalling pathway resulting in cell growth and the inhibition of JNK/SAPK. In this study, we examined the effect of sphingolipid metabolites on cell death in astrocytes prepared from rats prenatally exposed to alcohol. To determine if PAE increased the susceptibility of astrocytes to the cytotoxic effect of ceramide, astrocytes from pair-fed controls and animals that were prenatally exposed to alcohol were stimulated with exogenous SMase, or cell-permeable ceramide analogs, C2- and C6-ceramide. We found that the primary astrocytes prepared from pair-fed controls were resistant to the cytotoxic effect of low concentrations of SMase, C2-, and C6-ceramide. In contrast, in primary astrocytes prepared from rats exposed to alcohol in utero cell, viability was markedly decreased when exposed to concentrations of SMase, C2-, and C6-ceramide, which were not cytotoxic to control cells. These studies suggest that in utero alcohol exposure alters the cells' response to TNFa, possibly by increasing ceramide production, and/or, that in utero alcohol exposure alters the cells' response to ceramide resulting in cell death. Studies show that the TNFa-induced activation of ceramide activates the JNK/SAPK pathway, resulting in cell death and the inhibition of ERK (Cuvillier et al., 1996). Consistent with these studies, we have recently found that in passaged rat astrocytes, in vitro alcohol exposure increases JNK/SAPK phosphorylation and
69
TNFa-induced activation of the JNK/SAPK pathway (DeVito et al., 2000). In contrast, in hepatocytes, ethanol decreases the activation of JNK and the subsequent phosphorylation of cJun in response to partial hepatectomy (Diehl, 1999). The reason for this discrepancy is unclear, but suggests that the effect of alcohol on TNFa-induced signal transduction pathways may be cell type-specific. Studies conducted on several cell lines show that increased SPP production stimulates mitogenesis, protecting the cell from TNFa-induced cell death (Zhang et al., 1991; Gomez-Munoz et al., 1995; Pyne & Pyne, 1996; Verheij et al., 1996; Spiegel et al., 1998). Here, we show that in primary astrocytes prepared from rats exposed to alcohol in utero, SPP blocked the cytotoxic effect of TNFa. Similarly, in astrocytes prepared from pair-fed rats and exposed to alcohol in vitro, SPP blocked TNFa-induced cell death. In primary astrocytes prepared from rats exposed to alcohol in utero and stimulated with TNFa in the presence of ethanol, SPP blocked the cytotoxic effect of TNFa, but higher concentrations of SPP were required to maintain cell viability at control levels. This suggests that in utero alcohol exposure has a pronounced effect on the cellular response to SPP and decreases the ability of SPP to play a role in decreasing cell death. To further determine the role of SPP in the regulation of cell death, we examined the effect of DMS, a potent competitive inhibitor of sphingosine kinase that decreases cellular SPP levels and increases ceramide levels (Edsall et al., 1998) on TNFa-induced cell death in astrocytes prenatally exposed to alcohol. Here, we show that pretreatment of astrocytes prepared from animals exposed to alcohol in utero or in the presence of alcohol in vitro with DMS enhanced the cytotoxic effect of TNFa. Furthermore, N-oleoylethanolamine, an inhibitor of acid ceramidases that increases cellular ceramide levels, enhanced the cytotoxic effect of TNFa in astrocytes prepared from rats exposed to alcohol in utero. From these studies, we can conclude that in utero alcohol exposure may result in the increased activation of the ceramide pathway, resulting in decreased astrocyte viability. Together, our studies suggest that one of the mechanisms through which in utero alcohol exposure increases the susceptibility of astrocytes to TNFa-induced cell death is through a shift in sphingolipid metabolism, favoring the production of ceramide and the inhibition of SPP production. The mechanisms involved, however, remain to be determined. Studies indicate that astrocytes and microglial cells can be activated to function as antigen-presenting cells (APC). In vitro studies show that astrocytes express MHC II in response to IFNg and TNFa (Shrikant & Benveniste, 1996). The expression of adhesion moleculesÐsuch as ICAM-1, VCAM-1, and E-selectinÐare increased in astrocytes in response to TNFa, interleukin-1 (IL-1), IFNg, LPS, or viral proteins (Shrikant et al., 1994; Rosenman et al., 1995). In addition, an antigenic challenge can stimulate astrocytes to produce and secrete several inflammatory cytokines, such as TNFa and IL-1. We have shown that
70
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71
in vitro exposure of astrocytes to low concentrations of ethanol blocks cytokine-induced mitogenesis and the synthesis and release of TNFa and the activation of the JAK/ STAT pathway, a primary signaling system for a number of cytokines and growth factors (DeVito & Stone, 1999; DeVito et al., 1997). In human astrocytoma cell, we have found that ethanol blocks the cell surface expression of ICAM-1 (DeVito et al., 2000). Further, studies in primary astrocytes prepared from rats exposed to alcohol in utero show that PAEs blocks growth factor-induced mitogenesis (DeVito et al., 1997), and as shown here, markedly increases the susceptibility of astrocytes to the cytotoxic effect of TNFa. Accordingly, these studies suggest that in utero alcohol exposure could alter the CNS response to injury or infection resulting in an inappropriate neuroimmune response and worsened neurological outcome. Glial cells are one of the most abundant cell types in the CNS, and they are essential for normal CNS development. Glial cells provide the structure and nutritive support for developing neurons and are excellent substrates for neurite extension and can provide a permissive substrata to support axonal regrowth and promote oligodendrite process extension by expressing adhesion molecules and neurotrophic factors (Oh & Youg, 1996). One striking effect of PAE is the disruption in the proliferation of neurons and glial cells. Animal models show that PAE-induced microencephaly is the result of decreased cell numbers in several brain regions (Bonthius & West, 1990; West et al., 1990; Pantazis et al., 1993; Miller, 1995). Consistent with the in vivo effects of alcohol, in vitro studies clearly show that alcohol exposure results in decreased cell proliferation (Pantazis et al., 1992; Kane et al., 1996; Klein et al., 1996; DeVito et al., 1997). In agreement with these studies, the present study suggests that alcohol-induced decrease in glial cell proliferation and increased cell death in response to cytokines could be a potentially important mechanism involved in the neurological and neurobehavioral dysfunction associated with PAE. In summary, we show that PAE markedly increases the susceptibility of astrocytes to the cytotoxic effect of TNFa. More importantly, the observation that astrocytes prepared from rats prenatally exposed to alcohol are more susceptible to the cytotoxic effect of TNFa when grown in the absence of ethanol, suggests that in utero alcohol exposure may have a long-term effect on astrocyte function. One mechanism through which prenatal alcohol exposure could increase the sensitivity of astrocytes to the cytotoxic effect of TNFa may be attributed to the altered metabolism of sphingolipids, resulting in the increased production of ceramide and a decrease in SPP levels. Acknowledgments This work was supported in part by funds by the National Institute of Alcohol Abuse and Alcoholism Grant No. AA11277.
References Benveniste, E. N. (1992). Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action. Am J Physiol 263, C1 ± C16. Bielawska, A., Greenberg, M. S., Perry, D., Jayadev, S., Shayman, J. A., McKay, C., & Hannun, Y. A. (1996). (1S, 2R)-D-erthro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor a ceramidase. J Biol Chem 271, 12646 ± 12654. Bonthius, D. J., & West, J. R. (1990). Alcohol-induced neuronal loss in developing rats: increased brain damage with binge exposure. Alcohol Clin Exp Res 14, 107 ± 118. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., & Williamson, B. (1975). An endotoxin-induced factor that causes necrosis of tumors. Proc Natl Acad Sci USA 72, 3666 ± 3670. Cavani, M., Ghirelli, D., Fortuna, C., Lalli, F., & Marcolini, P. (1985). La sindrome feto-alcolica: follow-up clinico ± metabolico ± immunitario di 14 casi. Minerva Pediatr 37, 77 ± 88. Chung, I. Y., Norris, J. G., & Benveniste, E. N. (1991). Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and resistant rat strains. J Exp Med 173, 801 ± 811. Cifone, M. G., DeMaria, R., Roncaioli, P., Rippo, M. R., Azuma, M., Lanier, L. L., Santoni, A., & Testi, R. (1994). Apoptotic signaling through CD95 (Fas/Apo-1) activates an acid sphingomyelinase. J Exp Med 180, 1547 ± 1552. Colell, A., Garcia-Ruiz, C., Miranda, M., Ardite, E., Mari, M., Morales, A., Corrales, F., Kaplowitz, N., & Fernandez-Checa, J. C. (1998). Selective glutathione depletion of microchondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 115, 1541 ± 1551. Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., & Spiegel, S. (1996). Suppression of ceramidemediated programmed cell death by sphingosine-1-phosphate. Nature 381, 800 ± 803. Davies, D. L., & Vernadakis, A. (1984). Effects of ethanol on cultured glial cells: proliferation and glutamine synthetase activity. Brain Res 318, 27 ± 35. DeVito, W. J., & Stone, S. (1999). Ethanol inhibits prolactin-induced activation of the JAK/STAT pathway in cultured Astrocytes. J Cell Biochem 74, 278 ± 291. DeVito, W. J., Okulicz, W. C., Stone, S., & Avakian, C. (1992). Prolactin-stimulated mitogenesis of cultured astrocytes. Endocrinology 130, 2549 ± 2556. DeVito, W. J., Stone, S., & Mori, K. (1997). Low concentrations of ethanol inhibits prolactin-induced mitogenesis and cytokine expression in cultured astrocytes. Endocrinology 138, 922 ± 928. DeVito, W. J., Stone, S., Mori, K., & Shamgochian, M. (2000). Ethanol inhibits prolactin and tumor necrosis factor a-, but not gamma interferon-induced expression of intercellular adhesion molecule-1 in human astrocytoma cells. J Cellular Biochem 77, 455 ± 464. Diehl, A. M. (1999). Cytokines and the molecular mechanisms of alcoholic liver disease. Alcohol Clin Exp Res 23, 1419 ± 1424. Edsall, L. C., Van Brocklyn, J. R., Cuvillier, O., Kleuser, B., & Spiegel, S. (1998). N,N-dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphinosine 1-phosphate and ceramide. Biochemistry 37, 12892 ± 12898. Eng, L. F., Yu, A. C. H., & Lee, Y. L. (1992). Astrocytic response to injury. Prog Brain Res 94, 353 ± 365. Ewald, S. J. (1989). T lymphocyte populations in fetal alcohol syndrome. Alcohol Clin Exp Res 13, 485 ± 489. Ewald, S. J., & Frost, W. W. (1987). Effect of prenatal exposure to ethanol on development of the thymus. Thymus 9, 211 ± 215. Ewald, S. J., Huang, C., & Bray, L. (1991). Effect of prenatal alcohol exposure on lymphocyte population in mice. Adv Exp Med Biol 288, 237 ± 244. Gomez-Munoz, A., Waggoner, D. W., O'Brien, L., & Brindley, D. N.
W.J. De Vito et al. / Alcohol 21 (2000) 63± 71 (1995). Interaction of ceraminds, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and phospholipase D activity. J Biol Chem 270, 26318 ± 26325. Gottesfeld, Z., Christie, R., Felten, D., & LeGrue, S. (1990). Prenatal ethanol exposure alters immune capacity and noradrenergic synaptic transmission in lymphoid organs of the adult mouse. Neuroscience 35, 185 ± 194. Guerri, C., Saez, R., Sancho-Tello, M., de Aquilera, E. M., & Renau-piqueras, J. (1990). Ethanol alters astrocyte development: a study of critical periods using primary cultures. Neuochem Res 15, 559 ± 565. Hannun, Y. A. (1994). The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem 269, 3125 ± 3128. Hatten, M. H., Liem, R. K. H., Shelanski, M. L., & Mason, C. A. (1991). Astroglia in CNS injury. Glia 4, 233 ± 243. Heller, R. A., & Kronke, M. (1994). Tumor necrosis factor receptormediated signaling pathways. J Cell Biol 126, 5 ± 9. Hickey, W. F., Osborn, J. P., & Kirby, W. M. (1985). Expression of Ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat. Cell Immunol 91, 528 ± 535. Johnson, S., Knight, R., Marmer, D. J., & Steele, R. W. (1981). Immune deficiency in fetal alcohol syndrome. Pediatr Res 15, 908 ± 911. Kane, C. J., Berry, A., Boop, F. A., & Davis, D. L. (1996). Proliferation of astroglia from the adult cerebrum is inhibited by ethanol in vitro. Brain Res 731, 39 ± 44. Klein, R. F., Fausti, K. A., & Carlos, A. S. (1996). Ethanol inhibits human osteoblastic cell proliferation. Alcohol Clin Exp Res 20, 572 ± 578. Martin, R., McFarland, H. F., & McFarlin, D. E. (1992). Immunological aspects of demyelinating diseases. Annu Rev Immunol 10, 153 ± 187. Mathias, S., Pene, L. A., & Kolesnick, R. N. (1998). Signal transduction of stress via ceramide. Biochem J 335, 465 ± 480. Merrill, A. H. J., Schemelz, E.-M., Dillehay, D. L., Spiegel, S., Shayman, J. J., Schroeder, J. J., Riley, R. T., Voss, K. A., & Wang, E. (1997). Shingolipids Ð the enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 142, 208 ± 225. Miller, M. W. (1995). Effect of pre- or postnatal exposure to ethanol on the total number of neurons in the principal sensory nucleus of the trigeminal nerve: cell proliferation and neuronal death. Alcohol Clin Exp Res 19, 1359 ± 1363. Natoli, G., Costanzo, A., Guido, F., Moretti, F., & Levrero, M. (1998). Apoptotic, non-apoptotic, and anti-apoptotic pathways of tumor necrosis factor signalling. Biochem Pharmacol 56, 915 ± 920. Normon, D. C., Chang, M. P., Wong, C. M., Branch, B. J., Castle, S., & Taylor, A. N. (1991). Changes with age in the proliferative response of splenic T cells from rats exposed to ethanol in utero. Alcohol Clin Exp Res 15, 423 ± 432. Oh, Y. S., & Youg, V. W. (1996). Astrocytes promote progress outgrowth by adult human oligodendrocytes in vitro through interaction between bFGF and astrocyte extracellular matrix. Glia 17, 237 ± 253. Pantazis, N. J., Dohrman, D. P., Goodlett, C. R., Cook, R. T., & West, J. R. (1993). Vulnerability of cerebellar granule cells to alcohol-induced cell death diminishes with time in culture. Alcohol Clin Exp Res 17, 1014 ± 1021. Pantazis, N. J., Dohrman, D. P., Luo, J., Goodlett, C. R., & West, J. R. (1992). Alcohol reduces the number of pheochromocytoma (PC12) cells in culture. Alcohol 9, 171 ± 180. Pyne, S., & Pyne, N. J. (1996). The differential regulation of cyclic AMP by sphingomylin-derived lipids and the modulation of sphingolipid-stimulated extracellular signal regulated kinase-2 in airway smooth muscle. Biochem J 315, 917 ± 923. Redei, E., Clark, W. R., & McGivern, R. F. (1989). Alcohol exposure in utero results in diminished T-cell function and alterations in brain corticotropin-releasing factor and ACTH content. Alcohol Clin Exp Res 13, 439 ± 443.
71
Renau-piqueras, J., Sancho-Tello, M., Zaragoza, R., & Guerri, C. (1988). Effects of ethanol on the development of astrocytes in primary culture. Adv Biosci 71, 269 ± 273. Renau-piqueras, J., Zaragoza, R., De Paz, P., Baguena-Cervellera, R., Megias, L., & Guerri, C. (1989). Effects of prolonged ethanol exposure on glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study. J HIstochem Cytochem 37, 229 ± 240. Rosenman, S. T., Shrikant, P., Dubb, L., Benveniste, E. N., & Ransohoff, R. M. (1995). Cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-) by astrocytes and astrocytoma cell lines. J Immunol 154, 1888 ± 1899. Saez, R., Burgal, M., Renau-piqueras, J., Marques, A., & Guerri, C. (1991). Evolution of several cytoskeletal proteins of astrocytes in primary culture: effect of prenatal ethanol exposure. Neurochem Res 16, 737 ± 747. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., & Peter, M. E. (1998). Apoptosis signaling by death receptors. Eur J Biochem 254, 439 ± 459. Schutze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K., & Kronke, M. (1992). TNF activates NK-kB by phosphatidylcholine-specific phospholipase C-induced ``acidic'' sphingomyelin breakdown. Cell 71, 765 ± 776. Shrikant, P., & Benveniste, E. N. (1996). The central nervous system as an Immunocompetent organ: role of glial cells in antigen presentation. J Immunol 157, 1819 ± 1822. Shrikant, P., Chung, I. Y., Ballestas, M., & Benveniste, E. N. (1994). Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-a, interleukin-b, and interferon-g in astrocytes. J Neuroimmunol 51, 209. Spiegel, S., Cuvillier, O., Edsall, L. C., Kohama, T., Menzeleev, R., Olah, A., Olivera, A., Pirianov, G., Thomas, D. M., Tu, Z., Van Brocklyn, J. R., & Wang, F. (1998). Sphingosine-1-phosphate in cell growth and cell death. Ann N Y Acad Sci 845, 11 ± 18. Spiegel, S., Foster, D., & Kolesnick, R. N. (1996). Signal transduction through lipid second messengers. Curr Opin Cell Biol 8, 159 ± 167. Verheij, M., Bose, R., Lin, Z. H., Yoa, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., & Kolesnick, R. N. (1996). Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380, 75 ± 79. Weinberg, J., & Jerrells, T. R. (1991). Suppression of immune responsiveness: sex differences in prenatal ethanol effects. Alcohol Clin Exp Res 15, 525 ± 531. West, J. R., Goodlett, C. R., Bonthius, D. J., Hamre, K. M., & Marcussen, B. L. (1990). Cell population depletion associated with fetal alcohol brain damage: mechanisms of BAC-dependent cell loss. Alcohol Clin Exp Res 14, 813 ± 818. Wiegmann, K., Schutze, S., Machleidt, T., Witte, D., & Kronke, M. (1994). Functional dichotomy of neutral and acidic sphingomylinases in tumor necrosis factor signaling. Cell 78, 1005 ± 1015. Wolcott, R. M., Jennings, S. R., & Chervenak, R. (1995). In utero exposure to ethanol affects postnatal development of T- and B-lymphocytes, but not natural killer cells. Alcohol Clin Exp Res 19, 170 ± 176. Wong, C. M., Chiappelli, F., Chang, M. P., Norman, D. C., Cooper, E. L., Branch, B. J., & Taylor, A. N. (1992). Prenatal exposure to alcohol enhances thymocyte mitogenic responses postnatally. Int J Immunopharmacol 14, 303 ± 309. Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., & Spiegel, S. (1991). Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation. J Cell Biol 114, 155 ± 167.