Analysis of Protein Levels of 24 Cytokines in Scrapie Agent-Infected Brain and Glial Cell Cultures from Mice Differing in Prion Protein Expression Levels

Abstract

Activation of microglia and astroglia is seen in many neurodegenerative diseases including prion diseases. Activated glial cells produce cytokines as a protective response against certain pathogens and as part of the host inflammatory response to brain damage. In addition, cytokines might also exacerbate tissue damage initiated by other processes. In the present work using multiplex assays to analyze protein levels of 24 cytokines in scrapie agent-infected C57BL/10 mouse brains, we observed elevation of CCL2, CCL5, CXCL1, CXCL10, granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-␥), interleukin 1␣ (IL-1␣), IL-1␤, IL-6, and IL-12p40. Scrapie agent-infected wild-type mice and transgenic mice expressing anchorless prion protein (PrP) had similar cytokine responses in spite of extensive differences in neuropathology. Therefore, these responses may be primarily a reaction to brain damage induced by prion infection rather than specific inducers of a particular type of pathology. To study the roles of astroglia and microglia in these cytokine responses, primary glial cultures were exposed to scrapie agent-infected brain homogenates. Microglia produced only IL-12p40 and CXCL10, whereas astroglia produced these cytokines plus CCL2, CCL3, CCL5, CXCL1, G-CSF, IL-1␤, IL-6, IL-12p70, and IL-13. Glial cytokine responses from wild-type mice and transgenic mice expressing anchorless PrP differed only slightly, but glia from PrP-null mice produced only IL-12p40, indicating that PrP expression was required for scrapie agent induction of other cytokines detected. The difference in cytokine response between microglia and astroglia correlated with 20-fold-higher levels of PrP expression in astroglia versus microglia, suggesting that high-level PrP expression on astroglia might be important for induction of certain cytokines.

Role of Cyclophilin A from Brains of Prion-infected Mice in Stimulation of Cytokine Release by Microglia and Astroglia in Vitro

“…1A). The analysis of the stimulatory effect of the different fractions was performed by induction of IL-12p40 release by microglia and CCL2 release by astroglia; these were the strongest cytokine responses seen in previous experiments (22). Three points were noted as shown in Fig.…”

“…Wild-type mice were euthanized at the time of clinical signs (around 135-155 days postinoculation (dpi)) unless otherwise indicated. Infected and uninfected brains were homogenized at a 20% (w/v) concentration using a Mini Bead Beater (BioSpec Products) as described previously (22) in sterile PBS with 1ϫ Complete protease inhibitor mixture (Roche Applied Science). Brain homogenates were sonicated for 1 min, vortexed aggressively for 30 s, and frozen in aliquots at Ϫ80°C for future use.…”

“…Glial Cell Cultures-Glial cells from cerebral cortices of 1-3-day-old mice were cultured as a primary passage in T-75 flasks for 10 -14 days as described previously (22). Twenty-four hours after secondary passage into 96-well plates at 5 ϫ 10 4 cells/well, cells were overlaid with fresh medium containing various materials to be tested for the ability to stimulate cytokine release by the glial cells.…”

“…Cytokine Quantification-23-cytokine multiplex kits (BioRad) were used to determine the concentration of 23 cytokines (IL-1␣, IL-1␤, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, IFN-␥, CCL11 (Eotaxin), G-CSF, GM-CSF, CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1␣), CCL4 (MIP-1␤), CCL5 (RANTES), and TNF) in glial culture supernatant fluids as described previously (22). On some occasions, Single-Plex kits were also used to analyze IL-6, CCL2, and IL-12p40 in glial culture supernatants.…”

“…Contrary to bacterial or viral infections, which induce high levels of many cytokines (19 -21), only a few cytokines were found in vivo in scrapie-infected brains and also released in vitro by microglia or astroglia after exposure to scrapie-infected brain homogenates (22). In the present work, our goal was to identify molecules present in scrapie-infected brain that are responsible for stimulation of cytokine release by microglia and astroglia.…”