Differential ex vivo nitric oxide production by acutely isolated neonatal and adult microglia

Abstract

Microglia are the macrophage population residing in the parenchyma of the central nervous system (CNS), and are thought to play critical roles in CNS development, homeostasis and defense against pathogens. Microglia are capable of rapidly responding to microbial pathogens through engagement of their Toll-like receptors (TLRs). We first compared the efficiency of these responses in primary microglia acutely isolated from adult and neonatal mice. While the cytokine and chemokine responses of adult microglia were generally higher than those of neonatal cells stimulated ex vivo through TLRs, the nitric oxide response of neonatal microglia was markedly enhanced relative to the adult cells. We then went on to identify culture conditions such as exposure to M-SCF or GM-CSF that markedly enhanced the nitric oxide response of microglia, particularly those from the adult CNS. Finally, we demonstrate that the differential nitric oxide response of neonatal and adult microglia is not only limited to the mouse, but also extends to rat microglia.

Figure 1. Cytokine and chemokine mRNA induction by microglia from adult or neonatal mouse CNS

Microglia were acutely isolated from adult (black bars) or 8 day-old neonatal (striped bars) mouse CNS, and after overnight culture, stimulated for 4 hours with TLR-2, -3, or -4 agonists. TNF-α (a.), IL-1β (b.),IL-6 (c.), RANTES, (d.), MIP-1α, (e.), MIP-1β (f.),MCP-1 (g.), or KC (h.) mRNA expression was analyzed by real-time RT-PCR as detailed in Materials and Methods. The data is presented as RNA units relative to HPRT (n=3 for both adult and neonate).

Figure 2. Cytokine and chemokine protein production by microglia from adult or neonatal mouse CNS

Microglia were acutely isolated from adult or 8-day neonatal mouse CNS, and stimulated for 24 hours with TLR agonists. Supernatants were then harvested, and TNF-α (a.), IL-1β (b.), IL-6 (c.), or RANTES (d.) levels determined by ELISA (n=3 for both adult and neonate).

Figure 3. Inducible nitric oxide synthase mRNA, protein and nitric oxide production by microglia from neonatal and adult mouse CNS

(a.) Microglia were isolated from adult or 8-day neonatal mouse CNS, and stimulated with TLR agonists in the presence of exogenous IFN-γ for 24 hours. Inducible NOS mRNA expression was analyzed by real-time RT-PCR as detailed in Materials and Methods and is presented as RNA units relative to HPRT (n=3 for both adult and neonate); (b.) Microglia were isolated from 8 day-old and adult mouse CNS, and stimulated by LPS and IFN-γ or left unstimulated, for 24 hours. Supernatants were then harvested and nitrite levels, as a measure of nitric oxide, determined by Griess assay (n=3 for both adult and neonate); (c.) Microglia were isolated from 8, 12 day-old, and adult mouse CNS, and stimulated by LPS and IFN-γ or left unstimulated, for 48 hours. Supernatants were then harvested and nitrite levels, as a measure of nitric oxide, determined by Griess assay (n=3 for both adult and neonate); (d.) Microglia were isolated from 8 day-old, 12 day-old, or adult mouse CNS, and stimulated for 24 hours with LPS and IFN-γ or left unstimulated for the same time period. Cellular lysates were then generated and iNOS and β-actin protein levels determined by Western analysis.

Figure 4. Role of exogenous interferon in inducible nitric oxide response of murine microglia

Microglia were isolated from adult or 8 day-old neonatal mouse CNS and cultured in media alone, or stimulated with LPS in the presence or absence of exogenous IFN- □ (a.) After 48 hours, supernatants were removed and nitrite levels determined by Griess assay; (b.) After 24 hours, microglial lysates were harvested, and iNOS and α-tubulin protein levels determined by Western analysis.

Figure 5. Impact of culture conditions on nitric oxide production by neonatal murine microglia

(a.) Microglia that had been acutely isolated from 8-day neonatal mouse CNS and exposed to M-CSF for 5 days (striped bars), isolated from mixed glial cultures derived from 8-day cerebral cortices (white stippled bars), or acutely isolated from 8-day neonatal mouse CNS (black checkered bars), were stimulated with LPS and IFN-γ or left unstimulated for 48 hours. Supernatants were then harvested and analyzed for nitrite levels by the Griess assay. Resident macrophages isolated from adult murine peritoneal cavity were evaluated in parallel (black bars). (b.) Microglia were acutely isolated from adult mouse CNS and then cultured with M-CSF or GM-CSF for five days. After this period, cells were washed, resuspended in culture media in the absence of CSFs, and stimulated with LPS and IFN-γ or left unstimulated for 48 hours. Freshly isolated adult microglia and resident peritoneal macrophages were evaluated in parallel. Supernatants were harvested and analyzed for nitrite levels by the Greiss assay.

Figure 6. Inducible nitric oxide synthase mRNA, protein and nitric oxide production by microglia from neonatal and adult rat CNS

Microglia were isolated from adult and neonatal rat CNS, and stimulated with LPS alone, LPS and IFN-γ, or left unstimulated, for the appropriate time. Supernatants were analyzed for nitrite levels by the Griess assay at either 24 (a.) or 48 hours post-stimulation (b.) (n=3 for both adult and neonate for both time points); (c.) iNOS mRNA expression was analyzed by real-time RT-PCR as detailed in Materials and Methods. Data is represented as RNA units relative to HPRT (n=3 for both adult and neonate); (d.) Microglial lysates were analyzed for iNOS and α-tubulin protein levels by Western analysis.

Figure 7. Cytokine responses of microglia from neonatal and adult rat CNS, and relative IFN-β induction by microglia from mouse or rat CNS

Microglia from adult (solid bars) or neonatal (striped bars) rat CNS were cultured for 4 hours with or without LPS after overnight culture. TNF-α (a.) and IL-6 (b.) mRNA levels were analyzed by real-time RT-PCR as detailed in Materials and Methods. Data is presented as RNA units relative to HPRT (n=3 for both adult and neonate); (c.) Freshly isolated microglia from the adult or neonatal CNS of both rat and mouse were stimulated with or without LPS for 4 hours. IFN-β mRNA levels were analyzed by real-time RT-PCR, and data is presented as fold-induction relative to HPRT (n=3 for both adult and neonate for both species).