Effects of estrogen receptor agonists on regulation of the inflammatory response in astrocytes from young adult and middle-aged female rats - PubMed (original) (raw)

Effects of estrogen receptor agonists on regulation of the inflammatory response in astrocytes from young adult and middle-aged female rats

Danielle K Lewis et al. J Neuroimmunol. 2008 Mar.

Abstract

Estrogen has been shown to attenuate the inflammatory response following injury or lipopolysaccharide treatment in several organ systems. Estrogen's actions are transduced through two estrogen receptor sub-types, estrogen receptor (ER) -alpha and estrogen receptor-beta, whose actions may be overlapping or independent of each other. The present study examined the effects of ERalpha- and ERbeta-specific ligands in regulating the inflammatory response in primary astrocyte cultures. Pre-treatment with 17beta-estradiol (ERalpha/ERbeta agonist), HPTE (ERalpha agonist/ERbeta antagonist) and DPN (ERbeta agonist) led to attenuation of IL-1beta, TNFalpha, and MMP-9 in astrocyte media derived from young adult (3-4 mos.) and reproductive senescent female (9-11 mos., acyclic) astrocyte cultures, while pretreatment with PPT (ERalpha agonist) attenuated IL-1beta (but not MMP-9) in both young and senescent-derived astrocyte cultures. Our previous work determined that 17beta-estradiol was unable to attenuate the LPS-induced increase in IL-1beta in olfactory bulb primary microglial cultures derived from either young adult or reproductive senescent females. In young adult-derived microglial cultures, the LPS-induced increase in IL-1beta was not attenuated by pre-treatment with 17beta-estradiol, PPT or HPTE. Interestingly, the ERbeta agonist, DPN significantly decreased IL-1beta following LPS treatment in young adult-derived microglia. Thus while both microglia and astrocytes synthesize and release inflammatory mediators, the present data shows that compounds which bind ERbeta are more effective in attenuating proinflammatory cytokines in both cell types and may therefore be a more effective agent for future therapeutic use.

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Figures

Figure 1

Figure 1

Glial fibrillary acidic protein (GFAP) mRNA expression levels increase in the olfactory bulb following an excitotoxic injury. Ovariectomized, placebo-replaced or estrogen-replaced female rats were subjected to a 24 hour NMDA lesion directed to the olfactory bulb. RNA was extracted and subjected to real time PCR. The relative expression of each mRNA was calculated by the comparative CT method using the following formula 2-ΔΔCt [where the amount of the target (GFAP) was normalized to the endogenous reference (Cyclophilin) and relative to a calibrator constant; ABI methods]. Main effect of hormone (a), main effect of injury (b), interaction effect (c) was significant at p≤0.05. OS: ovariectomized, placebo-replaced, sham-lesioned; OL: ovariectomized, NMDA-lesioned; ES: ovariectomized, estrogen-replaced, sham-lesioned; EL: ovariectomized, estrogen-replaced, NMDA-lesioned.

Figure 2

Figure 2

Characterization of primary astrocyte cultures derived from the olfactory bulb of young adult and reproductive senescent female rats. Primary astrocytes cultures were immunoreactive for GFAP (red: a,b) and were counter-stained with the nuclear dye, Hoechst (blue). Primary astrocyte cultures derived from young adult (c) and reproductive senescent females (d) were not immunoreactive for the neuronal marker, microtubule-associated protein-2 (MAP2). A small proportion (5-8%) of cells in these primary cultures derived from young adults (e,g) and reproductive senescent females (f,h) were immunopositive for the microglial markers, ionized-calcium binding adapter molecule 1 (Iba1) and the cell surface glycoprotein Mac-1 (CD11b). Iba-1 and CD11b immunopositive cells and their associated nuclei stained with Hoechst are indicated by white arrows. Immunohistochemistry with the nuclear dye Hoechst is shown in the bottom panel (c-h). Bar: 50 μm.

Figure 3

Figure 3

Gene expression of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in primary astrocyte cultures derived from young adult and reproductive senescent females as determined by RT-PCR. The ERα-specific primers span Exon 4 to Exon 6 while the ERβ-specific primers span from Exon 4 to Exon 5. Two gene-specific products approximately 300 base pairs long (ERα) and 200 bases long (ERβ) were present in both young and senescent astrocyte cultures. Cyclophilin was used as a loading control (cy) for the transcription of each gene. The bands were visualized on a 1.5% agarose gel and verified by sequencing.

Figure 4

Figure 4

Primary astrocyte cultures derived from young adult and reproductive senescent females express the toll like recepter-4 (TLR-4). TLR4 (a, red) was observed in astrocytes derived from young adult and reproductive senescent females. Cells were counter-stained with the nuclear dye Hoechst (a, blue). Bar: 50 μm. IL-1β protein expression in astrocyte media from young adults (left panel, b) and reproductive senescent females (right panel,b) was determined following a 4-hour pre-treatment with 17β-estradiol (17bE, 20 nM), PPT (2 nM), HPTE (1μM) and DPN (10 nM) followed by a 24 hour treatment with LPS (10 μg/mL) and appropriate agonist. Asterisk (*) indicates significance at p≤0.05 relative to the saline/LPS-treated condition. Zero (0) indicates no detectable levels of expression in saline controls.

Figure 5

Figure 5

TNF-α protein expression in astrocyte media from young adults (left panel) and reproductive senescent females (right panel) was determined following a 4-hour pretreatment with 17β-estradiol (17bE, 20 nM), PPT (2 nM), HPTE (1μM) and DPN (10 nM) followed by a 24 hour treatment with LPS (10 μg/mL) and appropriate agonist. Asterisk (*) indicates significance at p≤0.05 relative to the saline/LPS-treated condition. Zero (0) indicates no detectable levels of expression in saline controls.

Figure 6

Figure 6

MMP-9 activity was measured by zymography in astrocyte media derived from young adults and reproductive senescent females following a 4-hour pretreatment with 17β-estradiol (17bE, 20 nM), PPT (2 nM), HPTE (1μM) and DPN (10 nM) followed by a 24 hour treatment with LPS (10 μg/mL) and appropriate agonist. Asterisk (*) indicates significance at p≤0.05 relative to the saline/LPS-treated condition. Zero (0) indicates no detectable levels of expression in saline controls.

Figure 7

Figure 7

IL-1β protein expression in microglial media from young adults was determined following a 4-hour pretreatment with 17β-estradiol (17bE, 2 nM), PPT (2 nM), HPTE (2μM) and DPN (10 nM) followed by a 24 hour treatment with LPS (10 μg/mL) and appropriate agonist. Asterisk (*) indicates significance at p≤0.05 relative to the saline/LPS-treated condition. Zero (0) indicates no detectable levels of expression in saline controls.

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