Cytokine production, glutamate release and cell death in rat cultured astrocytes treated with unconjugated bilirubin and LPS

Journal of Neuroimmunology 153 (2004) 64 – 75 www.elsevier.com/locate/jneuroim

Cytokine production, glutamate release and cell death in rat cultured astrocytes treated with unconjugated bilirubin and LPS Adelaide Fernandes, Rui F.M. Silva, Ana S. Falca˜o, Maria A. Brito, Dora Brites * Centro de Patoge´nese Molecular (UBMBE), Faculdade de Farma´cia, University of Lisbon, Av. Forcßas Armadas, 1600-083 Lisbon, Portugal Received 7 January 2004; received in revised form 21 April 2004; accepted 21 April 2004

Abstract In hyperbilirubinemic newborns, sepsis is considered a risk factor for kernicterus. Evidence shows that injury to astrocytes triggers cytokine release. We examined the effects of unconjugated bilirubin (UCB) alone, or in combination with LPS, on the release of glutamate and cytokines from astrocytes in conditions inducing less than 10% of cell death. UCB leads to an increase of extracellular glutamate and highly enhances the release of TNF-a and IL-1h, while inhibiting the production of IL-6. LPS potentiates immunostimulatory properties of UCB. These results point out the role of cytokines and provide a basis for the significance of sepsis in UCB encephalopathy. D 2004 Elsevier B.V. All rights reserved. Keywords: Astrocytes; Cell death; Glutamate release; Lipopolysaccharide (LPS); Proinflammatory cytokines; Unconjugated bilirubin

1. Introduction Increased levels of unconjugated bilirubin (UCB), the main product of heme catabolism, are responsible for the clinical manifestation of jaundice, and are potentially toxic, particularly in premature newborns (Oh et al., 2003). Hyperbilirubinemia is a common condition in the neonatal period as a result of decreased erythrocyte survival and defective hepatic clearance of UCB (Dennery et al., 2001). While modest concentrations of UCB were indicated as being neuroprotective against oxidative stress (Dore´ and Snyder, 1999; Dore´ et al., 1999), moderate to elevated levels of UCB may impart deposition in the central nervous system originating UCB encephalopathy and kernicterus (Seidman et al., 1991; Hansen, 2002; Porter and Dennis, 2002; Croitoru-Lamoury et al., 2003). The duration of exposure to exaggerated hyperbilirubinemia and sepsis is believed to represent increased risks for neurologic sequelae (Dennery et al., 2001; Hansen, 2002) and is one of the most common factors related with the readmission of term and near-term infants (Brown et al., 1999). In addition, the emergence of new technologies in the

* Corresponding author. Tel.: +351-21-7946450; fax: +351-217946491. E-mail address: [email protected] (D. Brites). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.04.007

management of smaller and more premature neonates has increased the incidence of neurodevelopmental abnormalities due to UCB (Oh et al., 2003). A combination of early discharge and less aggressive clinical approaches has also contributed to the resurgence of kernicterus, and there is now evidence that even moderate degrees of hyperbilirubinemia may be responsible for a significant increase in minor neurologic dysfunction throughout the first year of life (Ahlfors and Herbsman, 2003; Ebbesen, 2000; Newman and Maisels, 2000; Soorani-Lunsing et al., 2001). Uncertainty and concern have brought this unresolved problem to daylight again, since clinicians can neither recognize newborns at risk of UCB encephalopathy nor agree on preventive recommendations. Thus, there is an increasing need to clarify the underlying mechanisms of UCB neurotoxicity and to identify the role of sepsis as a risk factor. It is generally accepted that sepsis-related complications in neonates are crucially mediated by the action of proinflammatory cytokines produced by cells of the central nervous system (Quan et al., 1999; Schultz et al., 2002). Interleukin (IL-)-1h, tumor necrosis factor-a (TNF-a) and IL 6 are among the best characterized early response cytokines (Saliba and Henrot, 2001). TNF-a and IL-1h contribute to acute neurodegeneration during hypoxia or ischemia and to the progression of perinatal brain injury (Allan and Rothwell, 2001). If the role of TNF-a can either be detrimental or neuroprotective, depending on the signal-

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ing receptor that is activated (Gupta, 2002), IL-1h has been pointed as a mediator of neuronal and glial damage during excitotoxic and traumatic brain injury (Rothwell, 1999). Both these cytokines are able to induce the expression of the pleiotropic interleukin IL-6 in the brain (Van Wagoner et al., 1999). IL-6 is involved in the regulation of inflammatory and immunological responses within the central nervous system, but can also be detrimental as a mediator of inflammation, demyelination, and astrogliosis (Van Wagoner and Benveniste, 1999). UCB exerts various cellular effects and changes, at various levels of the immune system, have long been reported (Rola-Plezczynski et al., 1975; Sima et al., 1980; Vetvicka et al., 1991). Nevertheless, the immune response of brain cells to UCB was never investigated. In addition, although a net accumulation of UCB in rat brain was observed after intravenous administration of bacterial endotoxin lipopolysaccharide (LPS) (Hansen et al., 1993), the causal mechanism for this association remains unknown. Recent data indicate that both LPS and pro-inflammatory cytokines have additive effects on the UCB-induced loss of viable fibroblasts (Ngai and Yeung, 1999) and endothelial cells (Yeung and Ngai, 2001). Decreased cell viability, following exposure to UCB, was also observed in astrocytes (Amit and Brenner, 1993; Silva et al., 2001), neurons (Grojean et al., 2001; Silva et al., 2002) and oligodendrocytes (Genc et al., 2003). There is now experimental evidence that apoptosis of neural cells may play a crucial role in UCB neurotoxicity (Grojean et al., 2000; Rodrigues et al., 2000) and that UCB induces apoptosis, at least in part, via the mitochondrial pathway (Grojean et al., 2000; Rodrigues et al., 2002). Interestingly, the level of apoptosis in cultured astrocytes exposed to UCB is similar to that of necrosis, reinforcing the concept that UCB-induced cell death is not linked to a single pathway (Silva et al., 2001; Ostrow et al., 2003). Recently, in vitro studies, have shown that exposure of astrocytes to UCB decreases the uptake of glutamate (Silva et al., 1999, 2002) engendering overstimulation of NMDA receptors (Grojean et al., 2000, 2001). Overstimulation of glutamate receptors may originate cell swelling and trigger cell death both by apoptosis and necrosis (Mattson, 2003). Interestingly, sepsis is associated with a decrease in uptake and increased secretion of glutamate by astroglial cultures (Korcok et al., 2002; McNaught and Jenner, 2000). TNF-a was also reported to regulate the release of glutamate to the extracellular space (Bezzi et al., 2001). In this study, we examined the effects of UCB alone, or in combination with LPS, on the survival of rat astrocytes in primary cultures, as well as on the release of glutamate and cytokines TNF-a, IL-1h and IL-6. We demonstrate that UCB leads to an increase of extracellular level of glutamate and that LPS treatment additionally affects the decrease of astrocyte viability by UCB. We further show, for the first time, that UCB highly enhances the release of TNF-a and IL-1h. While immunostimulatory properties are aggravated

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by LPS, suppression of cytokine IL-6 by UCB remains unchanged. These findings implicate a novel role for proinflammatory cytokines in UCB-induced neurotoxicity.

2. Materials and methods 2.1. Animals The Institutional Animal Ethics Committee approved the study protocol and all procedures complied with international standards of humane care in animal experimentation. Wistar rats were maintained on a 12 h light/dark cycle under conditions of constant temperature and humidity. Animals were supplied with standard laboratory chow and water ad libitum. 2.2. Chemicals Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were purchased from Biochrom AG (Berlin, Germany). Antibiotic antimycotic solution (20
), human serum albumin (HSA) (fraction V, fatty acid free), Hoechst dye 33258, rabbit antibody anti-glial fibrillary acidic protein (GFAP) and goat antibody antirabbit labeled with TRITC were acquired from Sigma (St. Louis, MO, USA). The monoclonal antibody against the CR3 complement receptor of microglia (OX-42) was obtained from Serotec (Raleigh, NC, USA) and horse antibody anti-mouse labeled with FICT from Vector (Burlingame, CA, USA). Recombinant TNF-a was purchased from R&D Systems (Minneapolis, MN, USA). Unconjugated bilirubin (UCB), also from Sigma, was purified according to the method of McDonagh and Assisi (1972). Escherichia coli O111:B4 (LPS) was purchased from Calbiochem (La Jolla, CA, USA), dissolved at 1 mg/ ml in phosphate buffered saline (PBS) (pH 7.4) and kept at 4 jC. All the experiments with UCB were performed under light protection (tin foil wrapping of the vials and dim light) to avoid photodegradation. 2.3. Cell culture Astrocytes were isolated from 2-day-old rats as previously described (Blondeau et al., 1993), with minor modifications (Silva et al., 1999). In brief, rats were decapitated and the brains collected in DMEM containing 11 mM sodium bicarbonate, 71 mM glucose and 1% antibiotic antimycotic solution. Following the removal of meninges, blood vessels and white matter, the neocortices were homogenized by mechanical fragmentation and the cells collected by centrifugation at 700
g for 10 min. Finally, cells were resuspended in culture medium supplemented with 10% FCS, plated (2.0
105 cells/cm2) on 12-well tissue culture plates (Corning Costar, Cambridge, MA, USA) and cultured for 10 days. Microglial contamination

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was assessed by immunocytochemical staining using primary antibodies raised against OX-42 (mouse) (Liu et al., 2002) and GFAP (rabbit), followed by a species-specific fluorescent-labeled secondary antibody, and was inferior to 2.5%. Thus, the high purity level of astrocyte cultures excludes interference of contaminant microglial cells that would account for the released cytokines or glutamate (Hanisch, 2002; Nakamura et al., 2003). 2.4. Astrocyte treatment Cells were stimulated with LPS in concentrations ranging from 1 to 1000 ng/ml, for 2– 24 h periods, at 37 jC, in order to select the concentrations that lead to an immunological response without significantly reducing cell survival. In another set of experiments, cultures were exposed to 50, 100 and 150 AM UCB in the presence of 100 AM HSA (molar ratios of 0.5, 1.0 and 1.5, respectively), for 4 h, at 37 jC. For co-incubation studies, astrocytes were exposed to combinations of UCB (50 or 100 AM) and LPS (1 or 10 ng/ ml), which were added simultaneously to the incubation medium. In selected experiments, cells were also incubated with 1 and 10 ng/ml of recombinant TNF-a for 4 h, at 37 jC. At the end of the incubation periods, cell-free medium was collected for LDH, cytokine and glutamate determinations and attached cells were fixed for 30 min with freshly prepared 4% paraformaldehyde in PBS for apoptosis evaluation. Controls were performed in the absence of UCB and LPS. All UCB solutions were prepared from a 10 mM stock solution in 0.1 N NaOH and used immediately after preparation. The restoration of the pH value to 7.4 was achieved by the addition of 0.1 N HCl. 2.5. Cell death Cell death was estimated by evaluating activity of lactate dehydrogenase (LDH) released by nonviable cells, as well as by the number of apoptotic cells, after exposure to toxic stimuli. LDH was determined in the incubation medium using the Cytotoxicity Detection kit, LDH (Roche Molecular Biochemicals, Mannheim, Germany). The reaction was performed in a 96-well microplate and the absorbance measured at 490 nm. All readings were corrected for the possible interference of UCB absorption and the results expressed as percent of the maximum amount of releasable LDH, obtained by lysing non-incubated cells with 2.0% Triton X-100 in DMEM for 5 min. Apoptosis was evaluated by assessment of nuclear morphology in cells plated on glass coverslips previously placed in the 12-well tissue culture plates. In brief, astrocytes were incubated with Hoechst dye 33258 at 5 Ag/ml in PBS for 2 min at room temperature, washed with PBS, and mounted using PBS/glycerol (3:1, v/v). Fluorescence was visualized using an AxioskopR microscope (Zeiss, Germany). Stained nuclei were scored according to the condensation and staining characteristics of chromatin.

Apoptotic nuclei were identified by condensed chromatin, as well as nuclear fragmentation, and were counted in at least five random microscopic fields (
400) per sample. The mean values were expressed as the percentage of apoptotic nuclei. 2.6. Cytokine determinations Aliquots of the cell culture supernatants, collected at the end of the incubations, were placed in a 96-well microplate and assessed in duplicate for TNF-a, IL-1h and IL-6 with specific DuoSetR ELISA Development kits from R&D Systems, according to the manufacturer’s instructions. Results were expressed as pg/ml for isolated toxicants or as percentage of control in combination assays. 2.7. Measurement of glutamate Release of glutamate to culture medium was determined by an adaptation of the L-glutamic acid kit (Roche), using a 10-fold volume reduction. The reaction was performed in a 96-well microplate and the absorbance measured at 490 nm. A calibration curve was used for each assay. All samples and standards were analyzed in duplicate and the mean value was used. 2.8. Statistical analysis Results of at least three different experiments, performed in duplicate, are expressed as mean F SEM. Significant differences between two groups were determined by the two-tailed t-test performed on the basis of equal and unequal variance as appropriate. Comparison of more than two groups was done by ANOVA using Instat 3.05 (GraphPad Software, San Diego, CA, USA). Mean values were considered statistically significant when P values where lower than 0.05.

3. Results 3.1. Effect of increasing LPS concentrations on cell death at different time intervals First, we evaluated the time- and dose-dependent survival of astrocytes when exposed to LPS. Cell viability was assessed by the determination of LDH activity in the incubation medium after incubation with LPS at concentrations from 1 to 1000 ng/ml, for 2, 4, 6 or 24 h. As demonstrated in Fig. 1A, LPS concentrations of up to 100 ng/ml did not affect cell survival and only the highest concentration (1000 ng/ml) significantly increased ( P < 0.05) the release of intracellular LDH at 4, 6 and 24 h. Treatment of cells for 24 h greatly increased necrosis, even in the controls. Therefore, in the ensuing experiments we decided to exclude this time point, to avoid excessive

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stimulation was bell-shaped. However, the maximum production of TNF-a shifted backward from 100 to 1 ng/ml when astrocyte exposure to LPS changed from 4 to 6 h. In addition, at the 4 h time point a linear relationship between 1 and 100 ng/ml LPS was obtained. To avoid a higher activation that might mask the response of astrocytes to additive effects of UCB and LPS in experiments described subsequent to this, we therefore chose to use 1 and 10 ng/ml LPS and 4 h incubation. Because LPS-induced activation of culture astrocytes was shown to increase extracellular levels of glutamate (McNaught and Jenner, 2000), we tested whether LPS was able, by itself, to induce the secretion of glutamate in our experimental model. This was of particular interest since we had demonstrated that UCB impairs the uptake of this neurotransmitter by astrocytes (Silva et al., 1999), and the UCB-induced release of glutamate was determined in a later experiment in the presence of LPS. However, we ruled out any interference of LPS on the

Fig. 1. Effect of LPS on astrocyte viability (A) and apoptosis (B). Cells were incubated with 1 – 1000 ng/ml LPS or no addition (control) as described in Materials and methods. The samples were collected from the culture medium at 2, 4, 6 and 24 h after LPS stimulation for determination of LDH activity (A) and at 2, 4 and 6 h after LPS stimulation for morphological evaluation of apoptosis (B). Results are mean F SEM from at least three independent experiments performed in duplicate. *P < 0.05 from control levels; apoptosis induced by LPS was increased in all data series compared to respective controls (at least P < 0.05).

loss of cell survival. Since apoptosis often coexists with necrosis, we investigated if LPS also induces astrocyte apoptotic death at the selected periods of 2, 4 and 6 h (Fig. 1B). Although LPS significantly increased apoptosis in all experimental conditions ( P < 0.05), it only achieved a 2 – 4% elevation over control values for each time point, and never reached 8%. 3.2. Effect of increasing LPS concentrations on the release of TNF-a and glutamate at different time points To characterize the inflammatory response of our culture model, astrocytes were stimulated with LPS at concentrations ranging from 1 to 1000 ng/ml. The level of the proinflammatory cytokine TNF-a secreted to the incubation medium was determined at 2, 4 and 6 h after LPS treatment (Fig. 2A). The dose – response curves showed that for up to 2 h stimulation the release of TNF-a by astrocytes was insensitive to the immune stimulant concentration. In contrast, the profile of TNF-a production at 4 and 6 h LPS

Fig. 2. Time course and dose-dependent effect of LPS on astrocyte TNF-a secretion (A) and glutamate release (B). Cells were incubated with 1 – 1000 ng/ml LPS or no addition (control) as described in Materials and methods. The samples were collected from the culture medium at 2, 4 and 6 h after LPS stimulation for determination of TNF-a and glutamate secretion. Results are mean F SEM from at least three independent experiments performed in duplicate. *P < 0.05 and **P < 0.01 from control levels.

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extracellular level of glutamate in the selected conditions (Fig. 2B). 3.3. Effect of increasing UCB concentrations on cell death Based on our previous studies indicating that UCB induces mixed features of cell death in cultured astrocytes (Silva et al., 2001, 2002), we next examined the level and the mode of death exhibited by astrocytes after 4 h exposure to 50, 100 and 150 AM UCB. The assays demonstrated that UCB led to a concentration-dependent release of intracellular LDH (Fig. 3A), with the highest concentration producing a f 5-fold increase over control values. Lower UCB concentrations, corresponding to UCB/HSA molar ratios found in moderate to severe hyperbilirubinemia, although significant ( P < 0.01), only caused an additional f 4% loss in viability. Therefore, later experiments were designed to use 50 and 100 AM UCB in order to assure that evaluation of cytokine and glutamate release by astrocytes

Fig. 4. Analysis of the additive effects of UCB and LPS on astrocyte viability (A) and apoptosis (B). Cells were incubated with 50 and 100 AM UCB, 1 and 10 ng/ml LPS, UCB plus LPS, or no addition, in the presence of 100 AM albumin as described in Materials and methods. Results are mean F SEM from at least three independent experiments performed in duplicate. *P < 0.05 and **P < 0.01 from control levels; yP < 0.05 and yy P < 0.01 from UCB alone; §P < 0.05 and §§P < 0.01 from LPS alone.

would not be compromised by excessive loss of cell viability. As expected, the level of cell death by apoptosis was similar to that observed for necrosis (Fig. 3B). In fact, treatment with 50 and 100 AM UCB, although inducing a two-fold increase in apoptotic cell death ( P < 0.01), only produced a f 4% impairment in astrocyte survival over basal level of death. Thus, in our model, the percentage of injured astrocytes by apoptosis was maintained lower than 10%. 3.4. LPS evidenced to have additive effects on UCB-induced cell death, namely on astrocyte necrosis Fig. 3. Effect of UCB on astrocyte viability (A) and apoptosis (B). Cells were incubated with 50, 100 and 150 AM UCB or no addition (0) in the presence of 100 AM albumin for determination of LDH activity (A), and with 50 and 100 AM UCB or no addition (0) in the presence of 100 AM albumin for morphological evaluation of apoptosis (B), as described in Materials and methods. Results are mean F SEM from at least three independent experiments performed in duplicate. **P < 0.01 from control levels.

It has been reported that bacterial infection is a major etiologic co-factor in the development of kernicterus (Pearlman et al., 1980; Yeung and Ngai, 2001). To investigate the combined effects of UCB and LPS on the survival of astrocytes, we evaluated necrosis and apoptosis in cultures exposed for 4 h to 50 or 100 AM UCB, and to 1 or 10 ng/ml LPS, alone or in association (Fig. 4).

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Release of LDH by UCB was significantly aggravated by co-incubation with 1 ng/ml LPS ( P < 0.05 for 50 AM and P < 0.01 for 100 AM UCB) or 10 ng/ml LPS ( P < 0.05 for 50 and 100 AM UCB), while no changes were noticed for LPS treatment alone (Fig. 4A). In addition, the highest concentration of LPS did not further enhance necrosis of astrocytes due to UCB plus 1 ng/ml LPS, suggesting that the activation reached the maximum level. Although UCB and LPS co-treatment did not significantly increase the level of apoptotic cells induced by UCB alone (Fig. 4B), significance was found for 100 AM UCB plus 1 and 10 ng/ml LPS relatively to astrocytes treated alone with LPS ( P < 0.05). These results point out that not only does sepsis increase cell death during unconjugated hyperbilirubinemia, but that endotoxin-induced apoptosis will also be further enhanced by jaundice. 3.5. Stimulation of TNF-a release by UCB in astrocytes increased upon co-activation with LPS As previously established, stimulation with 1 and 10 ng/ ml LPS led to the release of f 400 and f 550 pg/ml TNFa by cultured astrocytes, respectively. To date, the immunostimulant effect of UCB on nerve cells has only been reported by us, in preliminary form (Falca˜o et al., 2003). Additional data have indicated that both TNF-a and endotoxin decrease cell viability in cultured mouse fibroblasts (Ngai and Yeung, 1999) and in human endothelial cells (Yeung and Ngai, 2001) in the presence of UCB. Therefore, we then examined the responsiveness of astrocytes to UCB and LPS alone or in combination. Similarly to the LPS activation mentioned above, cultured astrocytes were extremely responsive to UCB, producing comparable amounts of TNF-a that represented more than 40% increase over control values ( P < 0.01) (Fig. 5A). On the other hand, simultaneous exposure to both stimuli further increased TNF-a secretion in more than 40% ( P < 0.05) (Fig. 5B). Again, co-treatment with 10 ng/ ml LPS did not additionally raise the cytokine production observed with 1 ng/ml in association with both UCB concentrations. Moreover, co-treatment with UCB and LPS significantly increased TNF-a secretion produced by 1 ng/ml LPS ( P < 0.05, 50 AM UCB; P < 0.01, 100 AM UCB). Our results clearly indicate that UCB has the potency equivalent to LPS in simulating the secretion of the proinflammatory cytokine TNF-a and that both LPS and UCB mutually aggravate the effects produced by either one alone. 3.6. Stimulation of IL-1b release by UCB in astrocytes increased upon co-activation with LPS To assess whether UCB also induced the release of IL1h, another early-response cytokine normally associated with the TNF-a production, we tested the IL-1h level in

Fig. 5. Effect of UCB on TNF-a secretion (A) and analysis of the additive effects of LPS (B). Cells were incubated with no addition (0), 50 and 100 AM UCB in the presence of 100 AM albumin (A), and with or without UCB (50 and 100 AM) and LPS (1 and 10 ng/ml), alone or in combination, in the presence of 100 AM albumin (B), as described in Materials and methods. Results are mean F SEM from at least three independent experiments performed in duplicate. *P < 0.05 and **P < 0.01 from control levels; yP < 0.05 from UCB alone; §P < 0.05 and §§P < 0.01 from LPS alone.

cultured supernatants after treating the cells with 50 and 100 AM UCB for 4 h, and found increases by 60% ( P < 0.05) and 100% ( P < 0.01), when compared with control (Fig. 6A). It should be noted that the response was consistent with the one indicated above for TNF-a. Likewise, we verified a further 60% increase in the secretion of IL-1h by cells incubated with UCB plus LPS ( P < 0.05 from UCB alone, both concentrations) (Fig. 6B) that was maintained when LPS concentration was increased from 1 to 10 ng/ml. Nevertheless, contrasting with LPS-induced TNF-a secretion, no IL-1h stimulation was triggered by LPS alone. First line cytokines such as TNF-a and IL-1h are known to give stimuli for the expression of IL-6, a second line cytokine (Bencsath et al., 2003). IL-6 occupies a central role in inflammatory response to brain injury because it has pro- and antiinflammatory actions, and has a negative feedback effect on the production of IL-1h and TNF-a (McKeating and Andrews, 1998). Therefore, we next checked whether interaction of UCB with astrocytes also resulted in increased levels of IL-6 in cellular supernatants.

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levels of glutamate. D’Arcangelo et al. (2000) demonstrated that IL-6 has inhibitory effects on glutamate release in the rat cerebral cortex and it has been reported that toxicity of glutamate to neurons may be inhibited by the release of IL-6 (Yamada and Hatanaka, 1994). Indeed, as shown in Fig. 8A, UCB was able to markedly enhance the extracellular accumulation of glutamate. This effect was concentration-dependent, duplicating from 50 to 100 AM UCB and representing a two- ( P < 0.05) and four-fold ( P < 0.01) increase over control values, respectively. By contrast, glutamate release by UCB was insensitive to treatment with 1 or 10 ng/ml LPS (Fig. 8B), indicating that this efflux is independent from the immunostimulant effects of UCB. In addition, UCB may also induce an elevation of glutamate in culture supernatants through the secretion of TNF-a, which was suggested to reduce glutamate uptake and activate the neurotransmitter release from astrocytes (Bezzi et al., 2001). Nevertheless, incubation of astrocytes with 1 or 10 ng/ml TNF-a did not change the extracellular levels

Fig. 6. Effect of UCB on IL-1h secretion (A) and analysis of the additive effects of LPS (B). Cells were incubated with no addition (0), 50 and 100 AM UCB in the presence of 100 AM albumin (A), and with or without UCB (50 and 100 AM) and LPS (1 and 10 ng/ml), alone or in combination, in the presence of 100 AM albumin (B), as described in Materials and methods. Results are mean F SEM from at least three independent experiments performed in duplicate. *P < 0.05 and **P < 0.01 from control levels; y P < 0.05 from UCB alone; §§P < 0.01 from LPS alone.

3.7. UCB suppressed IL-6 release by astrocytes and impaired LPS-stimulation Contrasting with previous results for IL-1h and TNF-a secretion, UCB greatly reduced the release of IL-6 (Fig. 7A). Upon stimulation with 50 and 100 AM UCB, IL-6 levels dropped to 55% and 46% of control values respectively ( P < 0.01). These unexpected results led us to examine the response to LPS. As we had anticipated, LPS per se was able to increase the release of IL-6 in 34% or 55% ( P < 0.05 and P < 0.01, respectively), depending on whether 1 or 10 ng/ml was used (Fig. 7B). Nevertheless, astrocytes did not regain their responsiveness to neither 1 nor 10 ng/ml LPS, once in the presence of UCB. 3.8. UCB-induced elevation of extracellular glutamate remained unchanged during co-incubation of astrocytes with LPS We hypothesized that the UCB-induced inhibition of IL-6 secretion might mediate an increase in extracellular

Fig. 7. Effect of UCB on IL-6 secretion (A) and analysis of the additive effects of LPS (B). Cells were incubated with no addition (0), 50 and 100 AM UCB in the presence of 100 AM albumin (A), and with or without UCB (50 and 100 AM) and LPS (1 and 10 ng/ml), alone or in combination, in the presence of 100 AM albumin (B), as described in Materials and methods. Results are mean F SEM from at least three independent experiments performed in duplicate. *P < 0.05 and **P < 0.01 from control levels; §§ P < 0.01 from LPS alone.

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Fig. 8. Effect of UCB on glutamate release (A) and analysis of the additive effects of LPS (B). Cells were incubated with no addition (0), 50 and 100 AM UCB in the presence of 100 AM albumin (A), and with or without UCB (50 and 100 AM) and LPS (1 and 10 ng/ml), alone or in combination, in the presence of 100 AM albumin (B), as described in Materials and methods. Results are mean F SEM from at least three independent experiments performed in duplicate. *P < 0.05 and **P < 0.01 from control levels; §§ P < 0.01 from LPS alone.

of glutamate as compared to control values (data not shown).

4. Discussion We have shown that treatment of astrocytes with UCB for 4 h leads to the release of the pro-inflammatory cytokines TNF-a and IL-1h, which were further stimulated by LPS and correlated with necrosis. Another new major finding of this study is that UCB causes a depression in the secretion of IL-6. UCB insult also leads to an extracellular accumulation of glutamate, but direct evidence between glutamate release and UCB immunostimulation was not demonstrated. Since glial cell types are virtually identical in form, chemical composition and functional properties in mammalian species (Dobbing, 1974), we believe that our results may be extrapolated to human species. Several reports and reviews that focus on UCB toxicity in the neonatal period refer to the onset of sepsis as an aggravating factor for the development of encephalopathy during hyperbilirubinemia (Odell, 1980; Dawodu et al.,

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1984; Gourley, 1997; Dennery et al., 2001). The concept that UCB interacts with the immune system was stated several years ago (Rola-Plezczynski et al., 1975; Vetvicka et al., 1991) but was first restricted to the effects on peripheral blood leucocytes (Miler et al., 1985; Vetvicka ¨ zkan et al., 1995) where ‘‘yellow’’ crystals et al., 1989; O (Sen Gupta et al., 1983) and ‘‘cytomorphological’’ alterations (Leipe et al., 1983) were found. Other described effects of UCB at pathological levels on the circulating immune cells relates to the inhibition of human T lymphocytes cytotoxic activity (Haga et al., 1996a) and proliferative responses of human peripheral blood mononuclear cells (Haga et al., 1996b). Contradictory results were obtained for IL-2 production in two different studies, one pointing out no significant change (Haga et al., 1996c), and the other revealing an inverse relationship with serum levels of total bilirubin in patients with obstructive jaundice (Haga et al., 1996b). In addition, besides several other suppressive effects, UCB was also shown to increase phagocytosis in both granulocytes and monocytes (Vetvicka et al., 1991). Therefore, these findings outline that it is impossible to formulate a unifying concept of the immunological effect of UCB and corroborate our present data evidencing stimulation on the secretion of TNF-a and IL-1h, while suppressing IL-6 with the same concentration. Earlier studies have demonstrated that exposure of astrocytes to UCB/HSA, at a molar ratio of 3, induced comparable levels of necrosis and apoptosis (f 20%) (Silva et al., 2001, 2002), excluding a single commitment point. Based on these previous observations regarding UCB-induced cell death, we have extended our initial studies to show that, although significantly elevated, less than 10% of either necrosis or apoptosis is produced by the molar ratio of 0.5 and 1.0, that we selected to more correctly simulate physiological conditions. In our view, this implies that the UCBinduced stimulation of cytokine secretion is a subroutine that precedes the execution of the death programme to the end. In addition, the fact that apoptosis did not significantly increase from 50 to 100 AM UCB corroborates previous findings in brain endothelial cells revealing that produced apoptosis is not clearly concentration-dependent (Akin et al., 2002). Expanded studies on the immunological action of UCB will contribute to clarify whether the effects result from obstruction of membrane transport signals and/or upregulation of cell execution initiators. Cultured rat glial cells are immunostimulated by LPS (Bhat et al., 1998), providing a good model for evaluating whether UCB aggravates the responsiveness of astrocytes to endotoxin. LPS is a component of the bacterial cell wall that is responsible for most of the inflammatory effects of infection from bacteria. Recent data has shown that intracerebral injection of LPS in the neonatal rat induced inflammatory responses, as indicated by the increased concentrations of TNF-a and IL-1h in the brain, and results in selective white matter injury (Cai et al., 2003). In the current study, the profile of TNF-a production by astrocytes upon

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LPS stimulation was similar to that found by others (Chang et al., 2001), showing a bell-shaped curve at 6 h after activation, with the highest level of f 1000 pg/ml produced by 1 ng/ml LPS. The reduction in the secretion of TNF-a by increasing LPS concentrations from 10 to 1000 ng/ml was also noticed in LPS-activated microglia (Liu et al., 2001). LDH and glutamate release were not affected by LPS treatment and the increase of apoptosis was not more than 2%. The results of this study provide supportive evidence that endotoxin enhances the UCB-induced necrosis of astrocytes, as well as the release of TNF-a and IL-1h. Amplification of UCB cytotoxicity by TNF-a and endotoxin was previously observed in fibroblasts and endothelial cells at a UCB/HSA molar ratio of 1.0, while co-incubation with IL-1h and IL-6 did not worsen the loss in cell viability (Ngai and Yeung, 1999). Among the different inflammatory factors, TNF-a and IL-1h are secreted in the brain in response to many detrimental situations, namely traumatic injury (Stover et al., 2000) and excitotoxicity (Rostworowski et al., 1997; Acarin et al., 2000). Although controversial, it is generally accepted that increasing levels of those two cytokines may contribute directly to neurodegeneration, as well as to vasogenic edema (Rothwell, 1999; Holmin and Mathiesen, 2000; Allan and Rothwell, 2001). Therefore, overactivation by UCB and LPS, resulting in the increasing release of TNF-a and IL-1h, may actually be injurious to astrocytes as judged by loss of cell viability. Likewise, we speculate that the unexplained net accumulation of UCB in rat brain after intravenous administration of LPS (Hansen et al., 1993) can be due to blood – brain barrier breakdown mediated by those cytokines (Didier et al., 2003). However, it appears that TNF-a and IL-1h exert their function through independent pathways. Actually, IL-1h is involved in apoptotic cell death (Holmin and Mathiesen, 2000) and hypomyelination (Cai et al., 2003), while TNF-a has adverse or beneficial effects. The dual effects of TNF-a may be explained by the existence of two distinguishable pathways. In fact, TNF-a can act either on TNF receptor (TNFR) 1 that is believed to mediate cell death or in TNFR2 that serves to enhance cell death or promote cell survival (Gupta, 2002). Although it would be interesting to evaluate which signaling pathway is activated in our work model, the question is behind the scope of this study. On the other hand, activation of glial cells by cytokines is believed to induce the production of substantial amounts of nitric oxide (Chao et al., 1995, 1996). This generation of nitric oxide is known to involve the activation of the nuclear factor kappa B and the expression of inducible nitric oxide synthase leading to apoptosis (Neu et al., 2003). Nevertheless, we did not find any detectable amount of nitrite, the stable metabolite of nitric oxide, after 4 h exposure of astrocytes to UCB or LPS, either alone or in combination (results not shown). This is supported by studies with amyloid h-stimulated astrocytes showing that the expression of inducible nitric-oxide synthase results

from an initial induction of IL-1h and TNF-a with levels only detected at 6 h and steadily increasing in the next 36 h (Akama and Van Eldik, 2000). Actually, induction of nitric oxide production by UCB may be secondary to other signaling events. This is consistent with the report of Genc et al. (2003) showing expression of inducible nitric-oxide synthase in rat oligodendrocytes exposed to UCB for 24 h. Nevertheless, absence of nitrite production in our work model may also be due to UCB ability to act as a physiological scavenger of nitric oxide (Kaur et al., 2003; Mancuso et al., 2003). Astrocytes are also the major source of IL-6 in the central nervous system (Van Wagoner and Benveniste, 1999). IL-6 has been reported to diminish excitotoxic neuronal death in vitro (Yamada and Hatanaka, 1994; Carlson et al., 1999) and in vivo following cerebral ischemia (Loddick et al., 1998). In contrast, overexpression of IL-6 in transgenic mice was associated with neuronal cell loss and reactive gliosis (Campbell et al., 1993; Chiang et al., 1994). Of particular interest, synergistic induction of IL-6 is observed after treatment with TNF-a and IL-1h and increased secretion of IL-6 occurs after stimulation with LPS (Grimaldi et al., 1998). In our study, LPS also increased significantly and in a concentration-dependent manner IL-6 release from rat cortical astrocytes at very low concentrations (in the ng/ml range). On the contrary, we show that UCB strongly inhibits IL-6 production and suppresses LPS-induced IL-6 release by astrocytes. Inhibition of IL-6 secretion from endotoxinactivated murine macrophages by the antioxidant resveratrol (Wang et al., 2001; Feng et al., 2002), or from rat cortical astrocytes by the steroidal anti-inflammatory drug dexamethasone that was shown to block regulation of IL6 expression by TNF-a and IL-1h (Grimaldi et al., 1998; Van Wagoner and Benveniste, 1999), was previously reported as well. Besides the neurotoxic effects, IL-6 has been shown as a neuroroprotective cytokine. Thus, another critical action of UCB at brain level may be related with a decreased regeneration of neurons, as it was demonstrated in IL-6-deficient mice (Penkowa et al., 2000). In addition, UCB by blocking IL-6 negative regulatory loop on the production of TNF-a and IL-1h (McKeating and Andrews, 1998) may further contribute to secondary brain injury by these inflammatory cytokines. Nevertheless, the exact mechanisms by which pro-inflammatory cytokines exert beneficial or deleterious effects remain to be elucidated. Thus, present data will serve as a starting point to determine whether the immunostimulant and/or immunossupressive effects of UCB are neuroprotective or neurotoxic. Nevertheless, since the release of TNF-a by UCB is of the same order of magnitude of that produced by LPS stimulation, we speculate that it may be included in the wide spectrum of UCB neurotoxic effects. Moreover, additive effects of UCB and LPS on cell death and on secretion of pro-inflammatory cytokines support the association of infection with an increased risk of

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UCB-induced brain damage. The reverse is apparently also true. A great body of evidence suggests that accumulation of extracellular glutamate is toxic to neurons and that astrocytes protect neurons by removing glutamate from the extracellular space. It has been well documented that UCB interferes with glutamate uptake in synaptic vesicles (Roseth et al., 1998), astrocytes and neurons (Silva et al., 1999, 2002). The novel data reported here demonstrate an increase in the extracellular concentration of glutamate, compatible with either a decreased uptake or an enhanced release. Large amounts of glutamate and the excessive activation of its receptors trigger excitotoxicity (Olney, 1971), astrogliosis (Martinez-Contreras et al., 2002) and cell death, particularly when the cells are coincidentally subjected to adverse conditions such as hypoxia, ischemia, increased levels of oxidative stress, exposure to toxins or other pathogenic agents (Mattson, 2003). Several lines of evidence point out that brain damage by UCB requires the participation of glutamate toxicity. In this respect, activation of NMDA receptors and loss of protein kinase C by UCB in neurons was shown to facilitate glutamate-mediated apoptosis (Grojean et al., 2000). Interestingly, treatment of brain cell cultures with IL-1h plus TNF-a was found to inhibit glutamate uptake (Chao et al., 1995). Increased levels of extracellular glutamate were also observed after septic insult (McNaught and Jenner, 2000) and related with impairment of glutamate uptake as well (Korcok et al., 2002). Nevertheless, our results point out that glutamate release by UCB is insensitive to treatment with LPS or TNF-a. It remains to be clarified if there is any link between the suppression of IL-6 by UCB and the elevation in the amount of extracellular glutamate that occurs after incubation of astrocytes with UCB. This study has several potentially important implications. Firstly, although a multitude of different actions are mediated by cytokines, UCB-induced release of IL1h and TNF-a by astrocytes may impart neurodegeneration and vasogenic edema, thus playing an important role in mediating brain injury during hyperbilirubinemia. Since IL-6 occupies a central role in inflammatory response to brain injury, namely through its negative feedback effect on the production of IL-1h and TNF-a, suppression of astrocyte IL-6 secretion by UCB strengthens the dysregulating effects of UCB in the immune system. Secondly, extracellular accumulation of glutamate following astrocyte exposure to UCB might act cooperatively by further decreasing cell viability. More importantly, our newest results provide supportive evidence that sepsis increases the risk of cell damage by UCB. Thus, hyperbilirubinemia should be looked at more cautiously if infection by bacteria is anticipated. Ultimately, elucidation of the mechanisms of cellular injury during hyperbilirubinemia and sepsis will contribute to developing new therapies that might reduce the risk of brain injury and disabilities.

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Acknowledgements This work was supported in part by grant FCT-POCTI/ 39906/FCB/2001 from Fundacß a˜o para a Cieˆncia e a Tecnologia (to D.B.) and by a Research Award Program (Proc 1056693-S) of the Fundacß a˜o Calouste Gulbenkian (to A.F.), Lisbon, Portugal.

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