To investigate the expression profile of inflammation-related genes in infiltrating cells, quantitative real-time PCR (qRTCPCR) was performed on FACS-sorted CD11b+CD45med (microglia), Ly6G?Ly6C+ (Q1) and Ly6G+Ly6C+ (Q2) fractions 2 d after SE

To investigate the expression profile of inflammation-related genes in infiltrating cells, quantitative real-time PCR (qRTCPCR) was performed on FACS-sorted CD11b+CD45med (microglia), Ly6G?Ly6C+ (Q1) and Ly6G+Ly6C+ (Q2) fractions 2 d after SE. and contained Ly6G+Ly6C+ and Ly6G?Ly6C+ cells. Ly6G+Ly6C+ cells expressed higher levels of proinflammatory cytokines such as IL-1 and TNF suggesting that these cells were inflammatory neutrophils. Depletion of peripheral Ly6G+Ly6C+ cells prior to SE by anti-Ly6G antibody (NIMP-R14) treatment completely suppressed the infiltration of Ly6G+Ly6C+ cells into the brain. Proteome analysis revealed the downregulation of a variety of inflammatory cytokines, which exhibited increased expression in the post-SE hippocampus. These results suggest that Ly6G+Ly6C+ neutrophils are involved in the induction of acute brain inflammation after SE. The proteome expression profile of the hippocampus treated with LEV after SE was similar to that Impurity of Doxercalciferol after NIMP-R14 treatment. Therefore, LEV may prevent acute brain inflammation after SE by suppressing inflammatory neutrophil infiltration. indicates the number of independent experiments. Data are means SEMs. The data were analyzed using ANOVA followed by Dunnetts test. ** 0.01 vs. Pre-SE. (C) Mice were killed 2 days after pilocarpine-induced SE. mRNA was isolated from FACS-sorted CD11b+CD45med and CD11b+CD45high cells and measured by qRTCPCR. The expression of Tmem119 was normalized to that of Actb. indicates the number of independent experiments. Data are means SEMs. * 0.05, unpaired test. 2.2. Characterization of CD11b+CD45high Infiltrating Cells To explore in detail the characterization of CD11b+CD45high infiltrating cells, we investigated the expression of Ly6G and Ly6C in Impurity of Doxercalciferol the CD11b+CD45high fraction by flow cytometry. Ly6G and Ly6C express granulocytic and monocytic subsets, respectively. Flow cytometry analysis demonstrated that the CD11b+CD45high fraction included three subsets (Q1; Ly6G?Ly6C+, Q2; Ly6G+Ly6C+, Q3; Ly6G?Ly6C-) (Figure 2A). Ly6G+Ly6C+ cells, which are considered neutrophils, accounted for most of the infiltrating cells 1 d after SE, and 2 to 3 3 d after SE, the number Impurity of Doxercalciferol of Ly6G?Ly6C+ cells, which are considered monocytes, was the highest (Figure 2B). To investigate the expression profile of inflammation-related genes in infiltrating cells, quantitative real-time PCR (qRTCPCR) was performed on FACS-sorted CD11b+CD45med (microglia), Ly6G?Ly6C+ (Q1) and Ly6G+Ly6C+ (Q2) Impurity of Doxercalciferol fractions 2 d after SE. The mRNA levels Rabbit polyclonal to AnnexinVI of proinflammatory cytokines IL-1 and TNF in the Ly6G+Ly6C+ fraction were 4-fold and 6-fold higher than those in the Ly6G?Ly6C+ fraction, respectively. In contrast, the mRNA levels of the anti-inflammatory cytokines IL-6 and IL-10 in the Ly6G?Ly6C+ fraction were higher than those in the Ly6G+Ly6C+ fraction (Figure 2C). The Ly6G?Ly6C+ fraction also showed high expression of CCR2, which is an important chemokine receptor for monocytes to infiltrate the brain after SE [11]. These findings suggested that Ly6G+Ly6C+ neutrophils that infiltrate the brain early after SE and express proinflammatory cytokines might contribute to acute neuroinflammation, while Ly6G?Ly6C+ monocytes expressing anti-inflammatory cytokines may contribute to the end of neuroinflammation. Open in a separate window Figure 2 Characterization of CD11b+CD45high brain-infiltrating leukocytes. (A) Flow cytometry analysis of CD11b+CD45high fractions 2 d after SE. As shown in Figure 1, debris, doublets and dead cells were excluded. Live cells were subgated on CD11b+CD45high cells. Ly6C/Ly6G immunoreactivity was used to distinguish neutrophils and monocytes. The results are representative of at least 3 independent experiments. (B) Changes as a function of time in the percentage of cells in the Q1, Q2, Q3 and Q4 fractions within the PI? population defined by flow cytometry analysis. indicates the number of independent experiments. Data are means SEMs. * 0.05, unpaired test. (C) Mice were killed 2 days after pilocarpine-induced SE. mRNA was isolated from FACS-sorted CD11b+CD45med (microglia), CD11b+CD45high Q1 (monocytes) and CD11b+CD45high Q2 (neutrophils) cells and measured by qRTCPCR. The expression of each gene was normalized to the expression of Actb. Number of samples: microglia, = 5; Q1, = 4; Q2, = 4. Data are means SEMs. N.D., not detected. 2.3. LEV Suppresses the Infiltration of CD11b+CD45high Cells into the Brain and the Expression of Inflammatory Cytokines after SE We have previously reported that LEV suppressed the increased expression of proinflammatory cytokines such as IL-1 and TNF in the hippocampus 2 d after SE [17]. However, it remains unclear how LEV Impurity of Doxercalciferol suppresses the expression of these genes. It was reported that infiltrating leukocytes contribute to neuroinflammation and pathogenesis after status epilepticus [10,11,24]. Therefore, we investigated the effect of LEV on infiltrating cells in the brain after SE. Interestingly, LEV-treated mice showed little infiltration of CD11b+CD45high cells 2 d after SE (Figure 1A,B and Figure 2A,B). To further investigate the effects of LEV on the hippocampus after SE, we used a Proteome Profiler Mouse XL Cytokine Array that can simultaneously detect 111 soluble mouse.