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Activation of the inflammatory transcription factor nuclear factor interleukin-6 during inflammatory and psychological stress in the brain.

Fuchs F, Damm J, Gerstberger R, Roth J, Rummel C - J Neuroinflammation (2013)

Bottom Line: In vitro NF-IL6-activation was associated with induction and secretion of TNFα in folliculostellate cells, which could be antagonized by the JAK-STAT-inhibitor AG490.Both stressors were associated with time-dependent activation of NF-IL6 in different cell types of the brain and the pituitary.Moreover, while NF-IL6-IR was partially linked to STAT3 and NFκB activation, TNFα production, and ACTH-IR after LPS stimulation; this was not the case after exposure to a novel environment, suggesting distinct underlying signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary-Physiology and -Biochemistry, Justus-Liebig-University Giessen, Frankfurter Strasse 100, Giessen D-35392, Germany. Christoph.D.Rummel@vetmed.uni-giessen.de.

ABSTRACT

Background: The transcription factor nuclear factor interleukin 6 (NF-IL6) is known to be activated by various inflammatory stimuli in the brain. Interestingly, we recently detected NF-IL6-activation within the hypothalamus-pituitary-adrenal (HPA)-axis of rats after systemic lipopolysaccharide (LPS)-injection. Thus, the aim of the present study was to investigate whether NF-IL6 is activated during either, inflammatory, or psychological stress in the rat brain.

Methods: Rats were challenged with either the inflammatory stimulus LPS (100 μg/kg, i.p.) or exposed to a novel environment. Core body temperature (Tb) and motor activity were monitored using telemetry, animals were killed at different time points, brains and blood removed, and primary cell cultures of the anterior pituitary lobe (AL) were investigated. Analyses were performed using immunohistochemistry, RT-PCR, and cytokine-specific bioassays.

Results: Stress stimulation by a novel environment increased NF-IL6-immunoreactivity (IR) in the pituitary's perivascular macrophages and hypothalamic paraventricular cells and a rise in Tb lasting approximately 2 h. LPS stimulation lead to NF-IL6-IR in several additional cell types including ACTH-IR-positive corticotrope cells in vivo and in vitro. Two other proinflammatory transcription factors, namely signal transducer and activator of transcription (STAT)3 and NFκB, were significantly activated and partially colocalized with NF-IL6-IR in cells of the AL only after LPS-stimulation, but not following psychological stress. In vitro NF-IL6-activation was associated with induction and secretion of TNFα in folliculostellate cells, which could be antagonized by the JAK-STAT-inhibitor AG490.

Conclusions: We revealed, for the first time, that NF-IL6 activation occurs not only during inflammatory LPS stimulation, but also during psychological stress, that is, a novel environment. Both stressors were associated with time-dependent activation of NF-IL6 in different cell types of the brain and the pituitary. Moreover, while NF-IL6-IR was partially linked to STAT3 and NFκB activation, TNFα production, and ACTH-IR after LPS stimulation; this was not the case after exposure to a novel environment, suggesting distinct underlying signaling pathways. Overall, NF-IL6 can be used as a broad activation marker in the brain and might be of interest for therapeutic approaches not only during inflammatory but also psychological stress.

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Cellular phenotypes showing lipopolysaccharide (LPS)-induced NF-IL6-IR in the rat pituitary 8 h after stimulation. NF-IL6-IR (red) was co-localized with specific cell marker proteins (green) after LPS (100 μg/kg i.p.) or PBS stimulation. (A-L) LPS-induced NF-IL6-IR occurred in corticotrope cells (ACTH, B), pituicytes (GFAP, D), endothelial cells (VWF, F), neuronal nitric oxide synthase-expressing neurons (nNOS, H), perivascular macrophages (CD163, J), and activated macrophages (CD68, L) in the rat pituitary. PBS-treated controls also exhibited NF-IL6-IR in pituicytes (C), nNOS-expressing neurons (G), perivascular macrophages (I), and activated macrophages (K) to a certain extent. Please note that representative microphotographs depict only very few NF-IL6-IR cells in the intermediate lobe (IL), including endothelial cells (b). Insets (a-c) show high magnifications of the PL, IL, and AL, respectively. Cell nuclei were labeled with DAPI (blue). Brightness, contrast, and color balance were adjusted for better representation of the actual data. The scale bars in A and C represent 100 μm (applies to A-L) and 10 μm in all insets (applies to a-c). AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe.
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Figure 5: Cellular phenotypes showing lipopolysaccharide (LPS)-induced NF-IL6-IR in the rat pituitary 8 h after stimulation. NF-IL6-IR (red) was co-localized with specific cell marker proteins (green) after LPS (100 μg/kg i.p.) or PBS stimulation. (A-L) LPS-induced NF-IL6-IR occurred in corticotrope cells (ACTH, B), pituicytes (GFAP, D), endothelial cells (VWF, F), neuronal nitric oxide synthase-expressing neurons (nNOS, H), perivascular macrophages (CD163, J), and activated macrophages (CD68, L) in the rat pituitary. PBS-treated controls also exhibited NF-IL6-IR in pituicytes (C), nNOS-expressing neurons (G), perivascular macrophages (I), and activated macrophages (K) to a certain extent. Please note that representative microphotographs depict only very few NF-IL6-IR cells in the intermediate lobe (IL), including endothelial cells (b). Insets (a-c) show high magnifications of the PL, IL, and AL, respectively. Cell nuclei were labeled with DAPI (blue). Brightness, contrast, and color balance were adjusted for better representation of the actual data. The scale bars in A and C represent 100 μm (applies to A-L) and 10 μm in all insets (applies to a-c). AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe.

Mentions: Systemic LPS stimulation (100 μg/kg) induced increased NF-IL6-IR in the posterior and anterior pituitary lobe of rats (Figure 4A-C and E-G) compared to saline injected controls (Figure 4D). This IR started to increase at 4 h (Figure 4C) and peaked in its intensity and cell density 8 h after the LPS challenge (Figure 4A and E) followed by a decline after the 10 h (Figure 4F,G) to basal NF-IL6-IR (Table 3). Interestingly, the magnitude of this response was not altered, at 8 and 24 h, when a 10× higher dose of LPS was injected (1 mg/kg, Figure 4H,I). While the intermediate pituitary lobe did not show any NF-IL6-IR, particular strong NF-IL6-IR was observed lining its junctions with both the anterior and the posterior lobe, as can be observed in the overview and higher magnifications of Figures 4 and 5. Semi-quantitative five-point scale evaluation of two to four sections per rat for three animals per group confirmed these qualitative observations (Table 3). Figure 5 shows co-localization of several cell marker proteins with NF-IL6-IR in pituitary sections of LPS- compared to saline-stimulated animals 8 h after injection. We revealed some ACTH-IR positive corticotropes (Figure 5Bc) in the anterior and GFAP-IR positive pituicytes (Figure 5Ca and Da) in the posterior lobe co-localized with nuclear NF-IL6-IR. Endothelial cells (von Willebrand factor, VWF-positive cells) showed NF-IL6-IR-positive nuclei in all parts of the pituitary (Figure 5Fa-c), whereas nNOS positive cells co-localized with NF-IL6-IR only in the posterior lobe (Figure 5Ha). Moreover, CD163 or CD68 positive cells, indicative of the appearance of perivascular macrophages or activated macrophages, respectively, showed nuclear NF-IL6-IR in the anterior and posterior lobe (Figure 5I-L, a and c for each).


Activation of the inflammatory transcription factor nuclear factor interleukin-6 during inflammatory and psychological stress in the brain.

Fuchs F, Damm J, Gerstberger R, Roth J, Rummel C - J Neuroinflammation (2013)

Cellular phenotypes showing lipopolysaccharide (LPS)-induced NF-IL6-IR in the rat pituitary 8 h after stimulation. NF-IL6-IR (red) was co-localized with specific cell marker proteins (green) after LPS (100 μg/kg i.p.) or PBS stimulation. (A-L) LPS-induced NF-IL6-IR occurred in corticotrope cells (ACTH, B), pituicytes (GFAP, D), endothelial cells (VWF, F), neuronal nitric oxide synthase-expressing neurons (nNOS, H), perivascular macrophages (CD163, J), and activated macrophages (CD68, L) in the rat pituitary. PBS-treated controls also exhibited NF-IL6-IR in pituicytes (C), nNOS-expressing neurons (G), perivascular macrophages (I), and activated macrophages (K) to a certain extent. Please note that representative microphotographs depict only very few NF-IL6-IR cells in the intermediate lobe (IL), including endothelial cells (b). Insets (a-c) show high magnifications of the PL, IL, and AL, respectively. Cell nuclei were labeled with DAPI (blue). Brightness, contrast, and color balance were adjusted for better representation of the actual data. The scale bars in A and C represent 100 μm (applies to A-L) and 10 μm in all insets (applies to a-c). AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Cellular phenotypes showing lipopolysaccharide (LPS)-induced NF-IL6-IR in the rat pituitary 8 h after stimulation. NF-IL6-IR (red) was co-localized with specific cell marker proteins (green) after LPS (100 μg/kg i.p.) or PBS stimulation. (A-L) LPS-induced NF-IL6-IR occurred in corticotrope cells (ACTH, B), pituicytes (GFAP, D), endothelial cells (VWF, F), neuronal nitric oxide synthase-expressing neurons (nNOS, H), perivascular macrophages (CD163, J), and activated macrophages (CD68, L) in the rat pituitary. PBS-treated controls also exhibited NF-IL6-IR in pituicytes (C), nNOS-expressing neurons (G), perivascular macrophages (I), and activated macrophages (K) to a certain extent. Please note that representative microphotographs depict only very few NF-IL6-IR cells in the intermediate lobe (IL), including endothelial cells (b). Insets (a-c) show high magnifications of the PL, IL, and AL, respectively. Cell nuclei were labeled with DAPI (blue). Brightness, contrast, and color balance were adjusted for better representation of the actual data. The scale bars in A and C represent 100 μm (applies to A-L) and 10 μm in all insets (applies to a-c). AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe.
Mentions: Systemic LPS stimulation (100 μg/kg) induced increased NF-IL6-IR in the posterior and anterior pituitary lobe of rats (Figure 4A-C and E-G) compared to saline injected controls (Figure 4D). This IR started to increase at 4 h (Figure 4C) and peaked in its intensity and cell density 8 h after the LPS challenge (Figure 4A and E) followed by a decline after the 10 h (Figure 4F,G) to basal NF-IL6-IR (Table 3). Interestingly, the magnitude of this response was not altered, at 8 and 24 h, when a 10× higher dose of LPS was injected (1 mg/kg, Figure 4H,I). While the intermediate pituitary lobe did not show any NF-IL6-IR, particular strong NF-IL6-IR was observed lining its junctions with both the anterior and the posterior lobe, as can be observed in the overview and higher magnifications of Figures 4 and 5. Semi-quantitative five-point scale evaluation of two to four sections per rat for three animals per group confirmed these qualitative observations (Table 3). Figure 5 shows co-localization of several cell marker proteins with NF-IL6-IR in pituitary sections of LPS- compared to saline-stimulated animals 8 h after injection. We revealed some ACTH-IR positive corticotropes (Figure 5Bc) in the anterior and GFAP-IR positive pituicytes (Figure 5Ca and Da) in the posterior lobe co-localized with nuclear NF-IL6-IR. Endothelial cells (von Willebrand factor, VWF-positive cells) showed NF-IL6-IR-positive nuclei in all parts of the pituitary (Figure 5Fa-c), whereas nNOS positive cells co-localized with NF-IL6-IR only in the posterior lobe (Figure 5Ha). Moreover, CD163 or CD68 positive cells, indicative of the appearance of perivascular macrophages or activated macrophages, respectively, showed nuclear NF-IL6-IR in the anterior and posterior lobe (Figure 5I-L, a and c for each).

Bottom Line: In vitro NF-IL6-activation was associated with induction and secretion of TNFα in folliculostellate cells, which could be antagonized by the JAK-STAT-inhibitor AG490.Both stressors were associated with time-dependent activation of NF-IL6 in different cell types of the brain and the pituitary.Moreover, while NF-IL6-IR was partially linked to STAT3 and NFκB activation, TNFα production, and ACTH-IR after LPS stimulation; this was not the case after exposure to a novel environment, suggesting distinct underlying signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary-Physiology and -Biochemistry, Justus-Liebig-University Giessen, Frankfurter Strasse 100, Giessen D-35392, Germany. Christoph.D.Rummel@vetmed.uni-giessen.de.

ABSTRACT

Background: The transcription factor nuclear factor interleukin 6 (NF-IL6) is known to be activated by various inflammatory stimuli in the brain. Interestingly, we recently detected NF-IL6-activation within the hypothalamus-pituitary-adrenal (HPA)-axis of rats after systemic lipopolysaccharide (LPS)-injection. Thus, the aim of the present study was to investigate whether NF-IL6 is activated during either, inflammatory, or psychological stress in the rat brain.

Methods: Rats were challenged with either the inflammatory stimulus LPS (100 μg/kg, i.p.) or exposed to a novel environment. Core body temperature (Tb) and motor activity were monitored using telemetry, animals were killed at different time points, brains and blood removed, and primary cell cultures of the anterior pituitary lobe (AL) were investigated. Analyses were performed using immunohistochemistry, RT-PCR, and cytokine-specific bioassays.

Results: Stress stimulation by a novel environment increased NF-IL6-immunoreactivity (IR) in the pituitary's perivascular macrophages and hypothalamic paraventricular cells and a rise in Tb lasting approximately 2 h. LPS stimulation lead to NF-IL6-IR in several additional cell types including ACTH-IR-positive corticotrope cells in vivo and in vitro. Two other proinflammatory transcription factors, namely signal transducer and activator of transcription (STAT)3 and NFκB, were significantly activated and partially colocalized with NF-IL6-IR in cells of the AL only after LPS-stimulation, but not following psychological stress. In vitro NF-IL6-activation was associated with induction and secretion of TNFα in folliculostellate cells, which could be antagonized by the JAK-STAT-inhibitor AG490.

Conclusions: We revealed, for the first time, that NF-IL6 activation occurs not only during inflammatory LPS stimulation, but also during psychological stress, that is, a novel environment. Both stressors were associated with time-dependent activation of NF-IL6 in different cell types of the brain and the pituitary. Moreover, while NF-IL6-IR was partially linked to STAT3 and NFκB activation, TNFα production, and ACTH-IR after LPS stimulation; this was not the case after exposure to a novel environment, suggesting distinct underlying signaling pathways. Overall, NF-IL6 can be used as a broad activation marker in the brain and might be of interest for therapeutic approaches not only during inflammatory but also psychological stress.

Show MeSH
Related in: MedlinePlus