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Fig. 2 | Journal of Neuroinflammation

Fig. 2

From: Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

Fig. 2

IL-36γ is selectively expressed by neutrophils, while IL-36R is expressed by different leukocytes such as monocytic cells. a Quantification of IL-36γ or IL36R mRNA by qRT-PCR in different cells purified from the spinal cord or spleen of EAE mice by FACS using the following gating strategies: neutrophils, Ly6G+CD11b+CD45+CD3CD19; microglia, CD11b+CD45lowLy6GCD3CD19; monocyte-derived cells (MDC; comprising macrophages and CD11c+ dendritic cells), CD11b+CD45+CD11c+/−Ly6GCD3CD19; other intraspinal leukocytes, CD45+CD3+/−CD19+/−CD11bLy6GCD11c; B cells, CD19+CD45+CD11bLy6GCD3; splenic macrophages, CD11b+CD45+CD11cLy6GCD3CD19; other splenic leukocytes, CD45+CD3+/−CD11b+/−CD11c+/−Ly6GCD19. Sample size: spinal cord, one pooled sample of sorted cells from four mice; spleen, five non-pooled samples from individual mice. b Western blotting showing the full-length form of IL-36γ (~22 kDa) in splenic neutrophils from a wild-type EAE mouse (IL-36γ+/+), but not from an IL-36γ-deficient EAE mouse (IL-36γ−/−). Data are representative of at least four mice per group. The recombinant (truncated) form of IL-36γ was used as a control (right lane). β-actin (lower panels) was used to control for protein loading. Asterisks indicate non-specific bands. c Autoradiograms showing in situ hybridization signals (arrows) for IL-36γ or IL-36R mRNA in the spinal cord of mice killed on day 12 after active EAE induction, but not in naïve mice. Note the submeningeal distribution of the signals (representative of at least five mice). Scale bar = 500 μm. d Double labeling for IL-36γ or IL-36R mRNA (black grains, in situ hybridization) and cell type-specific markers (red brown, immunohistochemistry) in CNS sections from EAE mice. Arrows show examples of double-labeled cells. Scale bar = 20 μm

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