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Lipopolysaccharide modulates astrocytic S100B secretion: a study in cerebrospinal fluid and astrocyte cultures from rats.

Guerra MC, Tortorelli LS, Galland F, Da Ré C, Negri E, Engelke DS, Rodrigues L, Leite MC, Gonçalves CA - J Neuroinflammation (2011)

Bottom Line: However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B.Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases.Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, Porto Alegre, Brazil.

ABSTRACT

Background: Inflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calcium-binding protein secreted by astrocytes, has properties of a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B.

Methods: In this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide.

Results: Our data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 μg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein.

Conclusions: Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

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LPS does not affect cell viability. Rat cortical astrocytes were cultured in DMEM containing 10% FCS. Confluent astrocytes were exposed to LPS (from 0.01 to 30 μg/mL), during 24 h. At the end, cells were incubated with MTT (A) or neutral red (B). Each value is the mean (± standard error) of at least 5 independent experiments performed in triplicate. Statistical analysis was performed by one way ANOVA.
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Figure 6: LPS does not affect cell viability. Rat cortical astrocytes were cultured in DMEM containing 10% FCS. Confluent astrocytes were exposed to LPS (from 0.01 to 30 μg/mL), during 24 h. At the end, cells were incubated with MTT (A) or neutral red (B). Each value is the mean (± standard error) of at least 5 independent experiments performed in triplicate. Statistical analysis was performed by one way ANOVA.

Mentions: In order to detect a possible toxic effect of LPS in our preparations, we evaluated their capacities for MTT reduction, neutral red incorporation and LDH release. No changes in MTT reduction assay (p = 0.25) (Figure 6A) or in neutral red assay (p = 0.37) (Figure 6B) were induced in astrocyte cultures exposed to LPS (from 0.01 to 30 μg/mL). In addition, no changes in LDH release were seen (data not shown). Similar assays were also carried out in slice preparations confirming cell viability and integrity (data not shown).


Lipopolysaccharide modulates astrocytic S100B secretion: a study in cerebrospinal fluid and astrocyte cultures from rats.

Guerra MC, Tortorelli LS, Galland F, Da Ré C, Negri E, Engelke DS, Rodrigues L, Leite MC, Gonçalves CA - J Neuroinflammation (2011)

LPS does not affect cell viability. Rat cortical astrocytes were cultured in DMEM containing 10% FCS. Confluent astrocytes were exposed to LPS (from 0.01 to 30 μg/mL), during 24 h. At the end, cells were incubated with MTT (A) or neutral red (B). Each value is the mean (± standard error) of at least 5 independent experiments performed in triplicate. Statistical analysis was performed by one way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198930&req=5

Figure 6: LPS does not affect cell viability. Rat cortical astrocytes were cultured in DMEM containing 10% FCS. Confluent astrocytes were exposed to LPS (from 0.01 to 30 μg/mL), during 24 h. At the end, cells were incubated with MTT (A) or neutral red (B). Each value is the mean (± standard error) of at least 5 independent experiments performed in triplicate. Statistical analysis was performed by one way ANOVA.
Mentions: In order to detect a possible toxic effect of LPS in our preparations, we evaluated their capacities for MTT reduction, neutral red incorporation and LDH release. No changes in MTT reduction assay (p = 0.25) (Figure 6A) or in neutral red assay (p = 0.37) (Figure 6B) were induced in astrocyte cultures exposed to LPS (from 0.01 to 30 μg/mL). In addition, no changes in LDH release were seen (data not shown). Similar assays were also carried out in slice preparations confirming cell viability and integrity (data not shown).

Bottom Line: However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B.Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases.Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, Porto Alegre, Brazil.

ABSTRACT

Background: Inflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calcium-binding protein secreted by astrocytes, has properties of a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B.

Methods: In this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide.

Results: Our data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 μg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein.

Conclusions: Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

Show MeSH
Related in: MedlinePlus