TNF-alpha pretreatment prevents subsequent activation of cultured brain cells with TNF-alpha and hypoxia via ceramide

Irene Ginis, Ulrich Schweizer, Michael Brenner, Jie Liu, Nabil Azzam, Maria Spatz, and John M. Hallenbeck

Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have developed a cellular model in which cultured astrocytes and brain capillary endothelial cells preconditioned with tumor necrosis factor-alpha (TNF-alpha ) fail to upregulate intercellular adhesion molecule-1 (ICAM-1) protein (80% inhibition) and mRNA (30% inhibition) when challenged with TNF-alpha or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-alpha , its levels were measured at various times after the TNF-alpha preconditioning. We present evidence for the first time that, in normal brain cells, TNF-alpha pretreatment causes a biphasic increase of ceramide levels: an early peak at 15-20 min, when ceramide levels increased 1.9-fold in astrocytes and 2.7-fold in rat brain capillary endothelial cells, and a delayed 2- to 3-fold ceramide increase that occurs 18-24 h after addition of TNF-alpha . The following findings indicate that the delayed ceramide accumulation results in cell unresponsiveness to TNF-alpha : 1) coincident timing of the ceramide peak and the tolerance period, 2) mimicking of preconditioning by addition of exogenous ceramide, and 3) attenuation of preconditioning by fumonisin B1, an inhibitor of ceramide synthesis. In contrast to observations in transformed cell lines, the delayed ceramide increase was transient and did not induce apoptosis in brain cells.

ceramide; intercellular adhesion molecule-1; astrocytes; brain endothelial cells; ischemic tolerance; tumor necrosis factor-alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VARIOUS SUBLETHAL STRESS conditions such as oxidative stress (43), radiation (48), or heat (36) induce an adaptive response that results in cell tolerance to a subsequent challenge that would otherwise be lethal. The best-studied stress adaptation phenomenon in mammals, ischemic preconditioning, is well documented in animal models of ischemic injury of brain and heart (for reviews see Refs. 6 and 51, respectively) and of other organs as well (21, 35).

Adaptation to one type of stress can be achieved even by preconditioning with a different type of stress. For example, heat stress preconditioning (23) or pretreatment with chemicals causing oxidative stress (55) has been found to confer protection against ischemia-reperfusion injury of the myocardium. Preconditioning exposure to global brain ischemia can protect against focal ischemia and vice versa (39). These observations suggest a common denominator underlying protective mechanisms.

One candidate mediator of the stress adaptation reaction is a pleiotropic cytokine, tumor necrosis factor-alpha (TNF-alpha ). TNF-alpha is known as a key contributor to cell dysfunction and death in many pathological conditions including brain ischemia (8). In brain, TNF-alpha is released early after trauma and ischemia-reperfusion injury (1) and triggers expression of adhesion molecules, activation of inducible nitric oxide synthase, and release of cytokines, thus contributing to leukocyte extravasation and free radical production, key elements of the inflammatory reaction (18). Under certain conditions, however, TNF-alpha has been shown to protect animals from radiation (33), hyperoxia (47), endotoxic shock (25), oxidative stress (50), and stroke (30). Recent studies in murine and rat models of brain ischemia demonstrate that TNF-alpha can substitute for ischemic preconditioning when applied intracisternally (32) or elicited systemically via injection of lipopolysaccharide (42) 48-72 h before ischemic insult. However, signal transduction pathways that regulate TNF-alpha preconditioning have not been elucidated.

The goal of this work was to study molecular events triggered by TNF-alpha preconditioning of brain capillary endothelial cells and astrocytes. One of the best-studied effects of TNF-alpha in endothelial cells (41) and astrocytes (2) is upregulation of expression of adhesion molecules. Among the many cell adhesion ligands, intercellular adhesion molecule-1 (ICAM-1) is an important mediator of critical steps in leukocyte transmigration through the endothelium and accumulation at the site of injury. Animal models demonstrate a substantial increase of ICAM-1 mRNA at the site of infarction as early as 3 h after onset of permanent ischemia (46). Injection of monoclonal antibodies directed against ICAM-1 has been shown to decrease infarct size in models of transient focal ischemia (7). On the basis of these observations, ICAM-1 expression was chosen as the biological readout of TNF-alpha -induced activation of cultured astrocytes and capillary endothelium as we developed conditions in which TNF-alpha pretreatment causes unresponsiveness to its own action in these cells.

Ceramide, a product of sphingomyelin hydrolysis, has been implicated as a second messenger in many of the multiple signaling pathways initiated on TNF-alpha binding to its p55 receptor (15). Although most of the studies of ceramide focus on its role in TNF-alpha -triggered apoptosis and cell cycle control (for recent reviews see Refs. 34 and 40), there is also evidence that ceramide can cause cytoprotection (17). These observations suggested that TNF-alpha -induced preconditioning of brain cells could be mediated via ceramide. To test this hypothesis, we measured intracellular ceramide levels after TNF-alpha pretreatment in cultured astrocytes and rat brain capillary endothelial cells (RBEC). We present evidence for the first time that in these differentiated cells TNF-alpha causes a biphasic increase of ceramide levels similar to that observed in TNF-alpha -sensitive tumor cell lines, but in contrast to tumor cells, the delayed ceramide release in astrocytes and RBEC is transient and subsides after 4-6 h, causing no apoptosis but resulting in cell unresponsiveness to subsequent activation by TNF-alpha .


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. RBEC cultures were prepared from adult Wistar-Kyoto rat brains, as previously described for similar human cultures (41), except fetal bovine serum was substituted for human serum and heparin (90 µg/ml) was added to the medium. The purity of the RBEC was >95%, as determined by positive immunostaining for von Willebrand factor (factor VIII) and angiotensin-converting enzyme, incorporation of acetylated low-density lipoprotein, and negative staining for glial cells (glial fibrillary acidic protein, galactocerebroside, ED-2), muscle cells (alpha -actin), and pericytes (tropomyosin). Four cell cultures at passages 7-12 derived from four different brains were used.

Cortical astrocyte cultures were established from 3-day-old Sprague-Dawley rats according to the method adapted from Ballestas and Benveniste (2). Cells were grown to confluency for 2 wk (medium was changed twice a week), and contaminating microglia and oligodendrocytes were dislodged by shaking the cultures for 48 h at 200 rpm. The cultures were then replated at 1:3 dilution onto gelatin-coated culture dishes or microtiter plates, allowed to become confluent for 12-14 days, and used in the experiments. When stained for glial fibrillary acidic protein, >= 98% of cells were positive.

TNF-alpha preconditioning and treatment. Astrocytes and RBEC cultures were incubated for 4 h with 50 and 20 ng/ml rat recombinant TNF-alpha (Chemicon International, Temecula, CA), respectively, in their culture media, washed, allowed to rest in respective media without TNF-alpha for 20 h, and then activated again with the same doses of TNF-alpha for 24 h.

Hypoxic treatment. RBEC placed in modular incubator chambers (Billups Rothenberg, Del Mar, CA) were first flushed with 5% CO2-95% N2 for 15 min, then sealed and incubated at 37°C for 20 h and reoxygenated for 24 h. O2 tension at the end of hypoxic treatment was ~30 Torr (3%). For preconditioning, RBEC cultures were incubated for 4 h with TNF-alpha (20 ng/ml), and TNF-alpha was washed out immediately before the beginning of hypoxic treatment.

Ceramide studies. Astrocytes (100-mm culture dish; 2 × 106) or RBEC (60-mm culture dish; 2 × 106) were treated with TNF-alpha for indicated periods of time. At the end of each incubation, cells were washed twice with cold PBS, scraped off into Eppendorf tubes, and microcentrifuged. Cell pellets were stored at -70°C. For lipid extraction, cells were transferred to 13 × 100-mm borosilicate screw-cap tubes, and lipids were extracted with 3 ml of 2:1 MeOH-CHCl3, as described by Dbaibo et al. (9). The samples were centrifuged at 480 g for 10 min, the organic phase was collected and evaporated under N2, and the lipids were dissolved in 1 ml of CHCl3. An aliquot of 100 µl was set aside for lipid phosphate measurements.

Ceramide quantitation was performed by means of reverse-phase HPLC according to Santana et al. (38). Briefly, lipids were dried under N2 and hydrolyzed in 0.5 ml of 1 N KOH in methanol at 95°C for 1.5 h, then dried again, dissolved in 1 ml of chloroform, briefly centrifuged, dried, and resuspended in 50 µl of methanol in conical borosilicate tubes (catalog no. 66022-278, VWR). For derivatization with the fluorescent reagent o-phthaldialdehyde (OPA; Sigma Chemical, St. Louis, MO), samples were incubated with 50 µl of 0.5 mg/ml OPA solution in 3% boric acid (pH 10.5), 0.5% 2-mercaptoethanol for 10 min at room temperature, and 100 µl of HPLC eluant (90% MeOH-10% 5 mM KOH) for 50 min. OPA derivatives of deacylated ceramide were stable for 2-3 days, as previously described by Merrill et al. (31). Fluorescent lipids (15-µl aliquots) were injected into an Alltech Nucleosil C18 5-µm column (150 mm × 4.6 mm) and eluted isocratically at a flow rate of 2 ml/min. Fluorescence was measured at excitation/emission wavelengths of 340/455 nm. Type III ceramide from bovine brain sphingomyelin (Sigma Chemical), processed in the same way as the samples, was used as an internal standard. The standard curve for ceramide standards remained linear in the range of 0.5-100 ng. Elution profiles of fluorescent lipids were highly reproducible; the sphingoid base peak was resolved with a retention time of 4 min in cell extracts and ceramide standards, although in some samples the peak was split into two, probably reflecting formation of sphingosine and sphinganine derivatives. Because purified ceramide represents a family of ceramide compounds with different fatty acid chain lengths, a 1:1 mixture of synthetic D-erythrosphingosine and dihydrosphingosine (Sigma Chemical) provided a more precise quantitation of fluorescently labeled sphingoid bases derived from cellular ceramide. This sphingosine standard showed exactly the same two-peak elution pattern as was observed in cellular samples. Comparison of peak heights for ceramide and sphingosine standards demonstrated that 75.9 ± 18.5% (mean ± SD, n = 3) of ceramide could be recovered as an OPA derivative after the experimental procedures described above.

Ceramide values were normalized per lipid phosphate. Aliquots of extracted lipids were dried under N2, dissolved in 150 µl of 60% perchloric acid, and incubated for 1.5 h at 160°C. After addition of 800 µl of HPLC water and 150 µl of 10% ascorbic acid and 2.5% ammonium molybdate, samples were incubated at 55°C for 15 min. Absorbance was measured at 820 nm. Ten to 40 nM dilutions of potassium phosphate were used for the standard curve.

Quantitation of cell apoptosis. Apoptosis was measured by means of a fluorescent, cell-permeable, DNA-binding dye, Hoechst-33342. Hoechst fluorescence on binding to DNA is inversely proportional to the degree of DNA degradation by DNase. On the basis of these characteristics of Hoechst-33342, a novel microtiter plate fluorescence assay for DNA fragmentation in the cells undergoing apoptosis has been developed and described elsewhere (16). In addition, we used the ethidium homodimer fluorescence exclusion test to confirm plasma membrane integrity and to identify necrotic cells, inasmuch as a prominent feature of apoptosis is cell shrinkage, without development of a leaky cellular membrane, whereas DNA fragmentation alone could result from nonapoptotic DNA damage. The third dye, calcein, was employed to estimate a possible cell loss resulting from the detachment of dead cells. Briefly, sister cultures of astrocytes or RBEC, plated in 96-well microtiter plates, were incubated with either 25 µM Hoechst-33342, 4 µM ethidium homodimer, or 1 µM calcein solutions in Hanks' balanced salt solution (all 3 dyes were from Molecular Probes, Eugene, OR), which were added at 100 µl/well for 45 min at 37°C. Cell fluorescence was measured using a CytoFluor 4000 fluorescent plate reader (PerSeptive Biosystems, Framingham, MA) at excitation/emission wavelengths of 360/460, 530/620, and 485/530 nm for Hoechst, ethidium homodimer, and calcein, respectively. Background fluorescence was measured on each plate and subtracted. The fluorescence signal measured on Hoechst- and ethidium iodide-labeled cultures was normalized to that of calcein-stained sister cultures. The percentage of apoptotic cells was calculated from Hoechst fluorescence by means of the following formula
% apoptotic cells = <FR><NU>F<SUB>max</SUB> − F</NU><DE>F<SUB>max</SUB> − F<SUB>min</SUB></DE></FR> × 100%
where Fmax is fluorescence of untreated healthy control cultures, Fmin is fluorescence of cells treated with the cytotoxic alkylating agent methyl iodide, and F is fluorescence of the unknown sample.

The percentage of dead cells was calculated from ethidium homodimer fluorescence by means of the following formula
% dead cells = <FR><NU>F − F<SUB>min</SUB></NU><DE>F<SUB>max</SUB> − F<SUB>min</SUB></DE></FR> × 100%
where Fmin is fluorescence of untreated healthy control cultures, Fmax is fluorescence of cultures treated with the mild detergent digitonin at 1 mg/ml to disrupt plasma membranes, and F is fluorescence of the unknown sample.

Flow fluorocytometric analysis. Naive and TNF-alpha -pretreated cell cultures of RBEC and astrocytes were activated with TNF-alpha for 24 h, trypsinized, and incubated with 10 µg/ml anti-rat ICAM-1 monoclonal antibody (Endogen, Woburn, MA) for 1 h at 4°C, washed, and stained with 4 µg/ml fluorescein-labeled fluorescein-conjugated goat anti-mouse IgG secondary antibody (Accurate Chemicals and Scientific, Westbury, NY). Cells were fixed with 1% paraformaldehyde and analyzed by means of a flow cytometer (FACScan, Becton-Dickinson, San Jose, CA).

Fluorescent cell ELISA. ICAM-1 expression was quantitated as previously described (4). Cells were fixed with 1% paraformaldehyde, and then the following incubations were performed at room temperature for 1 h each: first with anti-rat ICAM-1 monoclonal antibody at 1 µg/ml, then with biotinylated horse anti-mouse IgG, rat adsorbed (Vector, Burlingame, CA), at 10 µg/ml, then with streptavidin beta -galactosidase (Molecular Probes) at 10 µg/ml, and finally with fluorescein-di-beta -D-galactopyranoside (Molecular Probes). Fluorescence was measured in a CytoFluor 4000 fluorescent plate reader at an excitation/emission wavelength of 485/530 nm. Background fluorescence of the cells stained without anti-ICAM-1 antibody or with the isotype-matched control was measured and subtracted in each experiment. The results are presented as the average of 10 wells.

RNA isolation and Northern blotting. Total RNA was extracted with STAT-60 (Tel-Test "B," Friendswood, TX) according to the manufacturer's instructions. RNA samples (15-µg aliquots) were electrophoresed through a 1.2% (wt/vol) agarose gel containing 5.4% (vol/vol) formaldehyde, transferred to a nitrocellulose membrane (GIBCO), and baked in a vacuum oven for 2 h at 80°C. Prehybridization was performed at 42°C with Digene Fast Pair Reagent. Hybridization was carried out at 42°C for 24 h by adding to the prehybridization solution [32P]DNA probes for ICAM-1 (cDNA probe template for rat ICAM-1 mRNA was a generous gift from Dr. Donald Anderson, Pharmacia). Cyclophilin probe was obtained by random priming. The blots were then washed (0.1× saline-sodium phosphate-EDTA-0.1% SDS at 45°C), dried, and quantitated using a PhosphorImager. Values for ICAM-1 mRNA were adjusted for background and normalized to cyclophilin mRNA. For nuclear runoff analysis, cells were activated with TNF-alpha for 2 h, then transcription was stopped by addition of 5 µg/ml actinomycin D and RNA was purified at different times.

Statistical analysis. Statistical analysis was carried out by two-factor ANOVA with replication and by paired t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
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TNF-alpha pretreatment makes cells unresponsive to subsequent activation by TNF-alpha and hypoxia-reoxygenation. FACS analysis showed that exposure of RBEC and astrocytes to TNF-alpha for 24 h caused a significant, twofold increase of surface ICAM-1 expression (Fig. 1). However, when cells were pretreated with TNF-alpha for 4 h, washed, and 20 h later challenged again with TNF-alpha , 83% and 92% inhibition of ICAM-1 induction was demonstrated in RBEC and astrocytes, respectively (Fig. 1, B and D). Similar results were obtained using ELISA (Table 1, experiment A), demonstrating that TNF-alpha pretreatment caused 81% and 74% inhibition of ICAM-1 induction in RBEC and astrocytes, respectively. The 4-h pretreatment of astrocytes and RBEC with TNF-alpha did not cause upregulation of ICAM-1 expression (data not shown).


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Fig. 1.   Tumor necrosis factor-alpha (TNF-alpha ) preconditioning inhibits TNF-alpha -induced upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on surface of brain cells. Rat brain capillary endothelial cells (RBEC, A and B) and astrocytes (C and D) were activated with TNF-alpha for 24 h. Cells were fluorescently labeled with anti-intercellular adhesion molecule-1 (ICAM-1) antibody, and FACS analysis of ICAM-1 surface expression was performed. A and C: representative experiments. B and D: summary graphs (means ± SD, n = 7 for RBEC and n = 4 for astrocytes). TNF-alpha treatment (thick lines in A and C and "TNF 24 hours" in B and D) resulted in significant shift of mean fluorescence (x-axis, logarithmic scale) over control values (thin lines in A and C and control in B and D). ICAM-1 expression in cells preconditioned for 4 h with TNF-alpha and 20 h later treated again with TNF-alpha (dotted lines in A and C and "TNF 4/24 hours" in B and D) was not different from that of controls. * Significantly different from control (P < 0.002 and P < 0.03 for RBEC and astrocytes, respectively); ** significantly different from TNF 24 hours (P < 0.001 and P < 0.01 for RBEC and astrocytes, respectively).


                              
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Table 1.   TNF-alpha preconditioning and ceramide prevent upregulation of ICAM-1 by TNF-alpha and hypoxia-reoxygenation

We next investigated whether cell unresponsiveness to TNF-alpha induced by short TNF-alpha pretreatment would attenuate the reported upregulation of ICAM-1 caused by hypoxia-reoxygenation in cultured brain endothelial cells (20). Using cell ELISA, we first found that in RBEC subjected to 20 h of hypoxia and 24 h of reoxygenation the amount of ICAM-1 protein on their surface was indeed significantly increased (Table 1, experiment B). Incubation of RBEC with TNF-alpha for 4 h immediately before the beginning of the hypoxic treatment prevented hypoxia-reoxygenation-induced ICAM-1 upregulation (Table 1, experiment B).

TNF-alpha pretreatment inhibits TNF-alpha -induced ICAM-1 transcription. The effect of TNF-alpha pretreatment was also studied at the level of transcription. RBEC and astrocytes were pretreated with TNF-alpha for 4 h, and 20 h later they were treated again with TNF-alpha , as described for the ICAM-1 protein quantitation. RNA was purified 2 h after the second TNF-alpha addition, because preliminary experiments demonstrated that maximum accumulation of ICAM-1 mRNA in naive and TNF-alpha -pretreated RBEC and astrocytes occurred at this time. ICAM-1 mRNA levels in TNF-alpha -pretreated RBEC (Fig. 2, A and C) and astrocytes (Fig. 2, B and D) were inhibited by 31.4% and 31.6%, respectively (the level of inhibition of the message was significantly less than the inhibition of protein synthesis, suggesting that, in addition to transcription, preconditioning affects postranscriptional events). Comparison of the kinetics of ICAM-1 mRNA degradation in control and preconditioned RBEC activated for 2 h with TNF-alpha and then treated with the transcription inhibitor actinomycin D demonstrated that TNF-alpha preconditioning did not affect ICAM-1 mRNA stability (Fig. 2E).


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Fig. 2.   TNF-alpha preconditioning inhibits TNF-alpha -induced ICAM-1 mRNA. RBEC (A and C) and astrocytes (B and D) were activated with TNF-alpha for 2 h, and ICAM-1 mRNA accumulation was measured. A and B: representative Northern blots: untreated cells (lane 1), naive cells activated with TNF-alpha for 2 h (lane 2), and preconditioned cells activated with TNF-alpha for 2 h (lane 3). C and D: summaries of mRNA quantitation by PhosphorImager. Values for ICAM-1 mRNA were adjusted for background and normalized to cyclophilin mRNA. Open bars, control cells; filled bars, cells preconditioned with TNF-alpha 24 h before TNF-alpha activation. Each bar represents mean fold induction over basal levels; error bars, SD; n = 3 and n = 5 for RBEC and astrocytes, respectively. * Difference is statistically significant. E: ICAM-1 mRNA measured in control and preconditioned RBEC activated for 2 h with TNF-alpha and then treated with actinomycin D for 7, 14, and 24 h. A representative result of 3 independent experiments is shown.

Effect of exogenous ceramide on brain cell response to TNF-alpha . Because ceramide has been implicated as a second messenger in many of the TNF-alpha signaling pathways, we sought to investigate whether inhibition of ICAM-1 expression in TNF-alpha -pretreated cells was mediated by ceramide. Astrocyte and RBEC cultures were treated with the cell-permeable ceramide analog N-acetylsphingosine (C-2 ceramide). Substitution of C-2 ceramide pretreatment (10 µM for 4 h, 20 h before TNF-alpha activation) for TNF-alpha preconditioning neither produced tolerance to subsequent TNF-alpha treatment nor upregulated ICAM-1 expression in control cultures of astrocytes and RBEC (data not shown). When exogenous C-2 ceramide was added to RBEC 30 min before or even 60 min after TNF-alpha activation, however, an induction of ICAM-1 mRNA was inhibited in RBEC (Fig. 3, A and C) and astrocytes (Fig. 3, B and D). The inhibitory effect of C-2 ceramide on ICAM-1 induction in RBEC and astrocytes has been confirmed on the protein level as well (Table 1, experiment C). The extent of inhibition of ICAM-1 achieved by C-2 ceramide was comparable to that produced by TNF-alpha preconditioning, as documented by Northern blotting and cell ELISA. Addition of C-2 ceramide 60 min after TNF-alpha inhibited transcription of ICAM-1 mRNA by 39.5% and 49.3% in RBEC and astrocytes, respectively (Fig. 3, C and D), and inhibited ICAM-1 protein induction by 73.9% and 75.5% in RBEC and astrocytes, respectively (Table 1, experiment C). Biologically inactive C-2 dihydroceramide had no effect on ICAM-1 protein expression (tested in FACS assay and ELISA) or ICAM-1 mRNA synthesis induced by TNF-alpha (data not shown). C-2 ceramide had a dose-dependent inhibitory effect on ICAM-1 transcription, which reached a maximum at 5-10 µM (Fig. 3E). As was noted with TNF-alpha preconditioning, C-2 ceramide had no effect on ICAM-1 mRNA stability (Fig. 3F).


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Fig. 3.   C-2 ceramide inhibits TNF-alpha -induced ICAM-1 mRNA. RBEC (A and C) and astrocytes (B and D) were activated with TNF-alpha for 2 h, and ICAM-1 mRNA accumulation was measured. A and B: representative Northern blots: untreated cells (lane 1), naive cells activated with TNF-alpha for 2 h (lane 2), cells pretreated with 10 µM C-2 ceramide 30 min before TNF-alpha addition (lane 3), and cells pretreated with 10 µM C-2 ceramide 60 min after TNF-alpha addition (lane 4). C and D: summaries of mRNA quantitation by PhosphorImager. Values for ICAM-1 mRNA were adjusted for background and normalized to cyclophilin mRNA. Open bars, control cells; filled bars, cells pretreated with 10 µM C-2 ceramide 60 min after TNF-alpha activation. Each bar represents mean fold induction over basal levels; error bars, SD; n = 2 and n = 5 for RBEC and astrocytes, respectively. * Difference is statistically significant. E: ceramide dose-dependent inhibition of TNF-alpha -induced ICAM-1 mRNA in astrocytes (C-2 ceramide was added 30 min before TNF-alpha ). F: effect of C-2 ceramide on ICAM-1 mRNA stability in astrocytes. A representative result of 2 independent experiments is shown.

TNF-alpha induces biphasic ceramide release in primary cultures of astrocytes and RBEC. The observation that C-2 ceramide exerted its inhibitory effect only when added at about the same time as TNF-alpha suggested that if ceramide mediates the preconditioning effect of TNF-alpha , its levels should be elevated near the time of the second TNF-alpha stimulation, i.e., ~24 h after the start of the preconditioning treatment. Such late ceramide release has been previously described in tumor cell lines (22, 43). To test this possibility, ceramide concentrations were measured at different times out to 30 h after TNF-alpha addition. In both types of cells, two peaks of ceramide concentration were observed. The first peak occurred between 15 and 20 min, reaching a level 2.7-fold over baseline in the RBEC and 1.9-fold over baseline in the astrocytes (Fig. 4A). The second peak occurred much later, between 18 and 24 h after addition of TNF-alpha , and then quickly subsided (Fig. 4, B and C). The exact time of this second ceramide peak varied from culture to culture, but at least a twofold increase in ceramide levels was observed in all experiments. In three of five experiments in RBEC and in four of seven experiments in astrocytes, TNF-alpha was washed out after 4 h of treatment, as for TNF-alpha preconditioning, but its withdrawal at this time had no effect on the delayed generation of ceramide. The average time of the peak in RBEC calculated on the basis of ceramide measurements in five different batches of RBEC derived from four animals was ~21 h (P < 0.025 vs. baseline values). A representative experiment is also shown for comparison (Fig. 4B). In astrocyte cultures, delayed ceramide accumulation peaked between 21 h (P < 0.02 vs. baseline levels) and 24 h (P < 0.002 vs. baseline levels and P < 0.005 vs. 27-h levels; Fig. 4C).


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Fig. 4.   TNF-alpha pretreatment induces biphasic ceramide response in brain cells. A: ceramide levels (means ± SD) were measured by means of reverse-phase HPLC in RBEC (n = 3, peak P < 0.01) and astrocytes (n = 4, peak P < 0.025) at 5-min intervals for first 30 min after TNF-alpha addition. Results are presented as percent increase over concentrations in unstimulated cells (1.7 ± 0.7 and 1.1 ± 0.3 pmol/nmol lipid phosphate for RBEC and astrocytes, respectively). B: ceramide levels measured in RBEC at 4, 7, 15, 18, 21, 24, 27, and 30 h after addition of TNF-alpha . * Statistically significant peak was observed at 21 h (black-diamond ; means ± SE, n = 5); a representative experiment is also included (star ). C: ceramide levels measured in astrocytes at 4, 7, 15, 18, 21, 24, 27, and 30 h after addition of TNF-alpha . * Statistically significant peak was observed between 21 and 24 h (; mean ± SE, n = 7); a representative experiment is also included (star ).

Time of cell unresponsiveness to TNF-alpha correlates with the time of the ceramide peak. Because C-2 ceramide exerted the inhibitory effect only if added shortly before or after TNF-alpha addition and because the increase of endogenous ceramide levels was transient, we hypothesized that if ceramide is a mediator of tolerance, TNF-alpha -preconditioned cells should remain unresponsive to the second TNF-alpha addition only during the short time when ceramide levels are elevated. Extending or shortening the time between the two TNF-alpha stimuli should result in the loss of preconditioning effect. To test this prediction, astrocytes or RBEC preconditioned for 4 h with TNF-alpha were given the second TNF-alpha stimulus 18, 21, 24, or 27 h later, and ICAM-1 mRNA (Fig. 5, A and B) or surface expression of ICAM-1 protein (Fig. 5C) was measured. In RBEC (Fig. 5A) and astrocytes (Fig. 5B) the degree of inhibition of ICAM-1 mRNA synthesis was dependent on the time between preconditioning and the subsequent TNF-alpha challenge. When cells were activated with TNF-alpha at 21 h after addition of the preconditioning TNF-alpha stimulus, maximum inhibition of ICAM-1 mRNA synthesis was observed in RBEC (n = 4, P < 0.04) and astrocytes (n = 3, P < 0.05). This temporal optimum (21 h after the first TNF-alpha addition) for cell unresponsiveness to TNF-alpha activation corresponds to the time when ceramide release caused by TNF-alpha reaches peak levels.


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Fig. 5.   Correlation between TNF-alpha -induced ceramide release in preconditioned cells and tolerance to TNF-alpha . Ceramide kinetics (; left y-axis) was related to degree of inhibition of ICAM-1 mRNA (; right y-axis) produced when time interval between preconditioning and subsequent TNF-alpha activation was varied in RBEC (A) and astrocytes (B). Cells were pretreated with TNF-alpha for 4 h, washed, and activated with TNF-alpha again 15, 18, 21, 24, or 27 h after first TNF-alpha addition for 2 h, and ICAM-1 mRNA was measured by Northern blotting. Zero point represents ICAM-1 mRNA induction and ceramide levels in naive cells. Each data point represents fold mRNA induction over quiescent levels (means ± SE, n = 4, P < 0.035 vs. naive cells for RBEC; n = 3, P < 0.05 vs. naive cells for astrocytes). C: ICAM-1 protein expression in astrocytes as a function of time interval between preconditioning and subsequent TNF-alpha activation. Astrocytes were pretreated with TNF-alpha for 4 h in absence of ceramide synthase inhibitor fumonisin B1 or in presence of 25 or 50 µM fumonisin B1, then TNF-alpha was washed out, and cells were allowed to rest in absence or presence of inhibitor, respectively. At 18, 21, or 24 h after first TNF-alpha addition, inhibitor was washed out (control cultures were also washed), and astrocytes were activated with TNF-alpha for second time. Surface expression of ICAM-1 was measured 24 h later by means of cell ELISA. Each treatment was performed in 10 wells. Results from 1 experiment representative of 3 independent experiments are shown.

Similar results were obtained when TNF-alpha activation was measured at the level of ICAM-1 protein expression by cell ELISA (Fig. 5C). Naive astrocytes treated with TNF-alpha for 24 h upregulated surface ICAM-1 up to 152.8 ± 6.3% of the control value (mean ± SE, n = 5). Astrocytes preconditioned with TNF-alpha for 4 h responded to the subsequent TNF-alpha treatment by upregulation of ICAM-1 to 140.0 ± 5.1%, 125.7 ± 8.0%, and 147.3 ± 1.7% when the time between the first and the second TNF-alpha stimulus was 24, 21, and 18 h, respectively. The temporal optimum for inhibition of ICAM-1 response to TNF-alpha was again ~21 h (P < 0.035 compared with 24 or 18 h), coinciding with the highest ceramide levels elicited by the preconditioning. In parallel experiments, TNF-alpha preconditioning was performed in the presence of the ceramide synthase inhibitor fumonisin B1. Fumonisin B1 attenuated TNF-alpha -induced preconditioning of astrocytes in a dose-dependent manner (Fig. 5C). Fumonisin B1 also inhibited delayed ceramide release in astrocytes: ceramide concentrations in astrocytes treated with TNF-alpha in the presence of 100 µM fumonisin B1 did not exceed the baseline values (1.32 vs. 1.14 pmol/nmol lipid phosphate). In the absence of fumonisin B1, TNF-alpha induced a threefold increase (up to 3.41 pmol/nmol lipid phosphate) of ceramide.

TNF-alpha -induced delayed ceramide accumulation does not trigger apoptosis in cultured brain cells. Studies in tumor cell lines have linked stress- or cytokine-induced prolonged and persistent elevation in ceramide levels to signaling for apoptosis (19). Delayed ceramide release in nontumor cells has not been previously reported. Thus our observation of TNF-alpha -triggered delayed accumulation of ceramide in astrocytes and RBEC prompted us to investigate whether increased ceramide levels result in cell death. A single TNF-alpha treatment of astrocyte and RBEC cultures resulted in no significant apoptosis even after 48 h of incubation (Table 2, Figs. 6E and 7E). Similarly, two TNF-alpha treatments, as was done in the above preconditioning studies (the first for 4 h and the second 20 h later for 24 h) caused no significant apoptosis in astrocytes or RBEC (Table 2). The effect of exogenous C-2 ceramide strongly depended on its dose. When treated with 10 µM C-2 ceramide, only 7% and 12% of astrocytes and RBEC, respectively, became apoptotic (Table 2, Figs. 6C and 7C). In contrast, when treated with 50 µM C-2 ceramide, 70% of astrocytes and 56% of RBEC underwent apoptosis (Table 2). Morphological changes characteristic of apoptosis similar to that observed in cultures treated with methyl iodide, an alkylating agent known to cause cell apoptosis (Figs. 6B and 7B), appeared as early as 24 h after addition of 50 µM C-2 ceramide (Figs. 6D and 7D). After 48 h of incubation with 50 µM C-2 ceramide, practically all RBEC were dead; significant cell loss occurred, which interfered with the assays. To mimic the apoptotic effect of high doses of C-2 ceramide, the ceramidase inhibitor N-oleoylethanolamine (NOE; Alexis Biomedicals, San Diego, CA), which has been shown to increase ceramide formation in tumor cells (48), was added to the cells to potentiate TNF-alpha -induced production of endogenous ceramide. As shown above, TNF-alpha treatment alone caused no apoptosis in astrocyte or RBEC cultures, even if the treatment was prolonged for 48 h; however, at 48 h of TNF-alpha -NOE treatment, morphological changes characteristic of apoptosis could be seen in both cultures (Figs. 6F and 7F). Treatment of RBEC with NOE alone had no significant effect on Hoechst fluorescence and cell morphology (data not shown).

                              
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Table 2.   Effect of TNF-alpha and exogenous ceramide on cell viability



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Fig. 6.   Morphological analysis of astrocyte cultures treated with TNF-alpha and different doses of exogenous ceramide. Astrocytes were subjected to different treatments, fixed with 2% paraformaldehyde, and viewed and photographed with a Nikon inverted microscope (model Eclipse TE 200) equipped with a Dage MT1 3CCD digital camera with a 0.45 reducing lens. Images were captured with IPLab Spectrum software and displayed on a Power Macintosh 8600/300. Images were reduced by Power Point, and final calculated magnification was ×110. A: control astrocyte cultures form a tight monolayer of morphologically homogenous flat polyhedral cells with few processes. B: astrocytes were treated with cytotoxic alkylating agent methyl iodide for 1 h and photographed 24 h later; cells are shrinking. Plastic surface is exposed where dead cells detached. Arrows indicate dark condensed nuclei. C: cultures treated with 10 µM C-2 ceramide for 48 h have same morphological pattern as control cultures. D: astrocytes treated with 50 µM C-2 ceramide for 24 h display numerous cells with condensed hyperchromatic nuclei (arrows). E: cultures treated with TNF-alpha for 48 h show morphological patterns similar to controls. F: cultures treated with TNF-alpha in presence of 30 µM N-oleoylethanolamine for 48 h display shrunk cells with condensed nuclei (arrows).



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Fig. 7.   Morphological analysis of RBEC cultures treated with TNF-alpha and different doses of exogenous ceramide. Images of RBEC, control and treated with TNF-alpha , were captured and produced as described in Fig. 6 legend, except final calculated magnification was ×134. A: control RBEC consist of densely packed elongated cells with interdigitating processes. B: RBEC cultures were treated with methyl iodide for 1 h and photographed 24 h later; cells are dying. C: RBEC treated with 10 µM C-2 ceramide for 48 h have same morphological pattern as control cultures. D: RBEC treated with 50 µM C-2 ceramide for 24 h display numerous cells with condensed hyperchromatic nuclei (arrows). Cells spread over exposed plastic. E: cultures treated with TNF-alpha for 48 h show morphological patterns similar to controls. F: cultures treated with TNF-alpha in presence of 30 µM N-oleoylethanolamine for 48 h display shrunk cells (arrows).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented here demonstrate that TNF-alpha , a pleiotropic cytokine implicated as a major pathogenic factor in oxidative stress and in inflammatory diseases, can induce tolerance to its own action if applied for a short time to precondition cells. Two observations suggest a general role for TNF-alpha as an inducer of cell tolerance to stress: 1) TNF-alpha induced unresponsiveness in two types of brain cells, astrocytes and RBEC, with the same pretreatment schedule (4 h of pretreatment 24 h before the challenge), and 2) pretreatment with TNF-alpha rendered cells unresponsive not only against subsequent TNF-alpha challenge but also against subsequent hypoxia.

Although in vitro models of brain tolerance to hypoxia and ischemia have tended to emphasize the effect of preconditioning on neuronal survival, our data show that other brain cells such as endothelial cells and astrocytes also participate. This work addresses signaling steps in endothelial cells and astrocytes that support adaptation to TNF-alpha and hypoxia via a TNF-alpha -ceramide-dependent mechanism.

Induction of ICAM-1 protein and mRNA has been measured as a biological readout of TNF-alpha -triggered cell activation. The effect of TNF-alpha on astrocytes and endothelial cells is well studied (2, 42) and is often used as a paradigm for the proinflammatory action of TNF-alpha and other types of stress such as oxidative stress or hypoxia (20). We found that both types of TNF-alpha -preconditioned cells failed to upregulate surface expression of ICAM-1 in response to TNF-alpha or hypoxia and that this correlated with lower levels of ICAM-1 mRNA. The absence of any effect on ICAM-1 mRNA stability indicates that TNF-alpha preconditioning affected ICAM-1 transcription (and possibly that of other genes). However, inhibition of ICAM-1 protein was more pronounced than inhibition of transcription (~75% vs. 40%), which suggests that mechanisms controlling ICAM-1 translation or its transport to the surface could be also affected.

The sphingomyelin cycle has been demonstrated to participate in signal transduction pathways initiated by TNF-alpha and other types of stress (9, 24, 19, 15, 34, 40). We have investigated whether ceramide serves as a second messenger in the preconditioning of cells by TNF-alpha . When added in place of TNF-alpha for 4 h, the cell-permeable ceramide analog N-acetylceramide (C-2 ceramide) failed to precondition astrocytes and RBEC; they remained fully responsive to the TNF-alpha activation 24 h later. However, if the ceramide was added 1 h before or even 1 h after TNF-alpha , it did have an inhibitory effect on ICAM-1 mRNA and protein levels. This finding suggested that ceramide could participate in preconditioning if it were released sufficiently late in the course of TNF-alpha pretreatment that it would be elevated at the time of the second TNF-alpha stimulus. This hypothesis prompted us to systematically measure ceramide levels in astrocyte and RBEC cultures over a long period of time (30 h) after TNF-alpha treatment. We have demonstrated two peaks of ceramide response to TNF-alpha : an early 1.9-fold (astrocytes) and 2.7-fold (RBEC) ceramide increase at 15-20 min and a late 2.5- to 3-fold increase at 18-24 h after TNF-alpha addition. Our observed early ceramide kinetics in RBEC are consistent with published data on ceramide release in human umbilical vein endothelial cells (peaks at 30 min) (29) and bovine aortic endothelial cells (peaks at 30 min) (45), whereas the ceramide response of primary astrocytes to TNF-alpha has not been previously investigated. Our findings of high ceramide levels later in the course of TNF-alpha treatment are novel, inasmuch as no delayed ceramide formation has been reported in normal cells.

Several lines of evidence suggest that the late ceramide release observed in astrocytes and RBEC cultures is responsible for induction of unresponsiveness in TNF-alpha -preconditioned cells. 1) The degree of attenuation of ICAM-1 mRNA caused by C-2 ceramide in both types of cells was comparable to that of TNF-alpha pretreatment (30-40% reduction of the normal TNF-alpha -induced increase). 2) C-2 ceramide exposure and TNF-alpha preconditioning affected ICAM-1 transcription levels and had no effect on ICAM-1 mRNA stability. 3) TNF-alpha preconditioning resulted in inhibition of the ICAM-1 response to the subsequent TNF-alpha activation only when the time of the second TNF-alpha addition coincided with the peak of late ceramide release. 4) Fumonisin B1, a natural inhibitor of ceramide synthase, an enzyme that converts sphinganine to dihydroceramide, attenuated TNF-alpha -induced ceramide release in astrocytes and the effect of TNF-alpha preconditioning; astrocytes pretreated with TNF-alpha in the presence of fumonisin B1 upregulated surface expression of ICAM-1 in response to TNF-alpha as well as naive cells. Similarly, fumonisin B1 attenuated endothelial cell apoptosis resulting from high ceramide levels triggered by TNF-alpha plus cycloheximide (51).

On the basis of multiple observations in tumor cell lines, one would expect that the high levels of ceramide observed in our cultures of brain cells should lead to apoptosis. However, our experiments demonstrate that treatment of both cell types with TNF-alpha continuously for 48 h or with the schedule used to study tolerance (4 h for the 1st treatment, 20 h of rest, 24 h for the 2nd treatment) results in no significant apoptosis. In addition, when the time interval between the two TNF-alpha treatments was increased to 27 h, no adaptation was observed, which would not be the case if cell unresponsiveness were an artifact caused by apoptosis. Similarly, exogenous ceramide also failed to induce apoptosis at doses that inhibited TNF-alpha -induced ICAM-1 expression in brain cells. Our observations are consistent with the recent data of Casaccia-Bonnefil and co-workers (5) that showed that addition of 10 µM C-2 to neuronal and astrocyte cultures had no effect on their survival and with earlier studies (37) demonstrating resistance of cultured endothelial cells to the cytotoxic effects of TNF-alpha .

Comparison of the kinetics of delayed ceramide formation in tumor cell lines with that observed in our model reveals major differences. For example, in T cell lymphoma Jurkat cells, activation of a specific death receptor, Fas, with anti-Fas antibody resulted in a gradual increase of ceramide levels with no tendency to subside over 10 h of observation (43). TNF-alpha treatment of L929 cells caused similar continuous accumulation of ceramide over 12 h (22), and in B cell lymphoma WEHI 231 cells, high ceramide levels triggered by the anti-IgM antibody persisted for 48 h (48). In contrast, we report here that in normal cultured brain cells the ceramide increase was transient, achieving highest levels 18-24 h after addition of TNF-alpha and then quickly subsiding to the baseline levels. These observations suggest that ceramide levels in differentiated cells are under tighter metabolic control than in TNF-alpha -sensitive tumor cell lines. Indeed, it has been shown that inhibition of protein synthesis or transcription is required for TNF-alpha to produce a cytotoxic response in endothelial cells (37, 51). In our model, TNF-alpha caused rapid apoptosis of astrocytes and RBEC only if added together with the ceramidase inhibitor NOE, which has been shown to perturb ceramide metabolism (48). Thus constitutive deregulation of ceramide metabolism in transformed cells might be responsible for the disproportionate ceramide response to stress, resulting in persistently high ceramide levels that trigger cell death. This explanation fits our data and that of D'Souza and co-workers (11), who demonstrated differential sensitivity of normal and malignant astrocytes to TNF-alpha .

We hypothesize that ceramide in normal cells does not serve as a messenger for apoptosis but, rather, represents one of the feedback mechanisms inhibiting cell reaction to TNF-alpha and perhaps other types of stress. In support of this hypothesis, the ceramide has been shown to inhibit the respiratory burst and cell spreading in human neutrophils if added simultaneously with TNF-alpha (12); ceramide also mediated TNF-alpha -induced inhibition of GLUT-4 in adipocytes (28) and of sodium-myo-inositol cotransporter in endothelial cells (53).

How ceramide causes cell unresponsiveness to TNF-alpha has yet to be elucidated. The data presented here indicate that ceramide can act at the level of gene transcription, suggesting that ceramide may act by inhibiting a common transcription factor. For example, a nuclear factor-kappa B (NF-kappa B) site has been identified in the ICAM-1 promoter (26), and ceramide has been shown to inhibit NF-kappa B activation triggered by phorbol 12-myristate 13-acetate (14). In a recent study, inhibition of NF-kappa B by ceramide was associated with excessive production of p50 homodimers (3). Interestingly, the same mechanism was reported for lipopolysaccharide-induced tolerance in a monocytic cell line (55). The transient nature of the TNF-alpha -induced accumulation of ceramide, which parallels the short duration of cell desensitization, argues that ceramide-induced inhibition of ICAM-1 expression depends on a fast-acting mechanism such as phosphorylation or dephosphorylation. Recently identified ceramide-activated kinases, protein kinase Czeta (13), a proline-directed serine-threonine kinase (27), and a serine-threonine phosphatase (10) could play a role.


    ACKNOWLEDGEMENTS

The authors thank Dr. Richard Kolesnick (Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center) for expert advice on ceramide assay, Joliet Bembry for preparation of rat brain endothelial cell cultures, and Nancy Merkel for technical assistance in ceramide measurements.


    FOOTNOTES

U. Schweizer was supported by Siemens Med GT, Erlangen and Hans-Krüger-Stiftung, Berlin, Germany.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: I. Ginis, Stroke Branch, NINDS, Bldg. 36, Rm. 4A03, National Institutes of Health, Bethesda, MD 20892-4128 (E-mail: ginis{at}codon.nih.gov).

Received 11 December 1998; accepted in final form 12 February 1999.


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