Treatment Of Microglial Cultures Research Articles

Abstract 160: Angiotensin-(1-7) Decreases the Pro-inflammatory Effects of Prorenin in Hypothalamic Microglia

The chronic hypertensive action of angiotensin II (Ang II) mediated within central cardiovascular control centers includes a pro-inflammatory effect and induction of microglial activation in the paraventricular nucleus (PVN) of the hypothalamus. The objective here was to investigate whether angiotensin-(1-7), a protective arm of the renin-angiotensin system (RAS), can exert anti-inflammatory effects in hypothalamic microglia. The presence of Mas, the receptor for Ang-(1-7), within the PVN and other cardiovascular control centers of normotensive (SD; Sprague Dawley) rats was demonstrated by quantitative real time PCR analyses. Mas was maintained in microglial cultures prepared from the hypothalamus of SD rat pups based on immunostaining and PCR. Treatment of microglial cultures with Ang-(1-7) (100nM, 3 -12h) elicited time-dependent significant (P<0.05) decreases in the basal levels of mRNAs for the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor-necrosis factor α (TNFα), and in the microglial-macrophage marker CD11b. For example, the levels of IL-1β mRNA (normalized to GAPDH) within control and Ang-(1-7) treated cells were 1.0±0.09, 0.70±0.06, 0.40±0.04, and 0.48±0.07 (n=6). There was no significant effect of Ang-(1-7) on basal IL-1β mRNA at 10 nM (6h), and 1000 nM Ang-(1-7) produced no greater effect than 100 nM of this peptide. Treatment of cultures with the RAS component prorenin (PRO; 10, 20 or 50 nM; 6h) elicited highly significant (P<0.001) increases in IL-1β mRNA (4.2±0.10, 7.4±0.35, 16.7±0.46, respectively, n=6) when compared with controls (0.78±0.06), effects that far exceeded the ~2.0 fold increase in IL-1β mRNA elicited by Ang II (100 nM). Similar stimulatory effects of PRO on TNFα, IL-6 and CD11b mRNAs were also observed. The stimulatory action of PRO (10 nM) on IL-1β and TNFα mRNAs, but not CD11b mRNA, was attenuated by co-treatment with Ang-(1-7) (100 nM). For example, levels of IL-1β mRNA in PRO and Ang-(1-7)/PRO treated cells were 34.1±1.3 and 24.4±1.1 respectively (n=6), a reduction of ~29% (P<0.001). Collectively, these data suggest that Ang-(1-7) exerts a direct anti-inflammatory action at hypothalamic microglia, and inhibits PRO-induced pro-inflammatory effects.

Transient early neurotrophin release and delayed inflammatory cytokine release by microglia in response to PAR-2 stimulation

Activated microglia exerts both beneficial and deleterious effects on neurons, but the signaling mechanism controlling these distinct responses remain unclear. We demonstrated that treatment of microglial cultures with the PAR-2 agonist, 2-Furoyl-LIGRLO-NH2, evoked early transient release of BDNF, while sustained PAR-2 stimulation evoked the delayed release of inflammatory cytokines (IL-1β and TNF-α) and nitric oxide. Culture medium harvested during the early phase (at 1 h) of microglial activation induced by 2-Furoyl-LIGRLO-NH2 (microglial conditioned medium, MCM) had no deleterious effects on cultured neurons, while MCM harvested during the late phase (at 72 h) promoted DNA fragmentation and apoptosis as indicated by TUNEL and annexin/PI staining. Blockade of PAR-1 during the early phase of PAR-2 stimulation enhanced BDNF release (by 11%, small but significant) while a PAR-1 agonist added during the late phase (24 h after 2-Furoyl-LIGRLO-NH2 addition) suppressed the release of cytokines and NO. The neuroprotective and neurotoxic effects of activated microglial exhibit distinct temporal profiles that are regulated by PAR-1 and PAR-2 stimulation. It may be possible to facilitate neuronal recovery and repair by appropriately timed stimulation and inhibition of microglial PAR-1 and PAR-2 receptors.

IL-5 induces proliferation and activation of microglia via an unknown receptor.

While the effects of interleukin-3 (IL-3) and granulocyte macrophage-colony stimulating factor (GM-CSF) on microglia are well documented, very little is known about the effects of a related cytokine, interleukin-5 (IL-5). We therefore undertook studies to determine how IL-5 alters various aspects of microglial functioning. Treatment of microglia with IL-5 resulted in the induction of proliferation at levels similar to those induced by GM-CSF. IL-5 also increased cellular metabolism of microglial cells. To determine whether increased metabolism correlated with activation of microglia, we measured levels of nitrite, a breakdown product of nitric oxide. Treatment of microglial cultures with IL-5 increased nitrite levels, while GM-CSF treatment had no effect. Treatment of microglia with IL-5 did not cause activation of the signal transduction pathways linked to the classical IL-5 receptor, STAT5A/5B and ERK1 and ERK2. It is therefore likely that the effects of IL-5 on microglia are not mediated via the classical IL-5 receptor, but rather via a novel receptor.