Leonid Tarassishin; Diana Casper; Sunhee C. Lee

“ We began to explore the underlying mechanism first by asking whether human astrocytes have a mechanism that actively suppresses IL-1 expression. Human fetal astrocytes or HEK293 cells were transfected with an IL-1 expression plasmid (IL-1β/GFP) or a control GFP plasmid and GFP and IL-1β expression was examined 24 h later by fluorescence microscopy and western blot analysis, respectively (Figure 5) . These experiments showed that while IL-1 and GFP (fusion protein) were readily expressed in HEK293 cells transfected with the IL-1β/GFP plasmid, neither was expressed in human astrocytes. ”

“ In human astrocytes, IL-1β mRNA was not translated to protein. Plasmid transfection also failed to produce IL-1 protein, suggesting active repression. Suppression of microRNAs that can target IL-1α/β did not induce IL-1 protein. Glioblastoma IL-1β processing occurred by the NLRP3 inflammasome, and ATP and nigericin increased IL-1β processing by upregulating NLRP3 expression, similar to macrophages. RNAi of annexin A2, a protein strongly implicated in glioma progression, prevented IL-1 induction, demonstrating its new role in innate immune activation. ”

“ Immunostaining of the GBM2 monolayer showed cytosolic IL-1β immunoreactivity in a subset of untreated cells (Figure 2C) . Together, these results show the ability of human malignant glioma cells to produce IL-1 protein spontaneously and following stimulation with IL-1. Non-neoplastic (or low grade) human astrocytes do not produce IL-1 protein even when they express IL-1 mRNA. The ability to produce IL-1 protein appears to be common to all GBM cells tested. ”

“ In order to test whether IL-1 promotes angiogenesis we performed a tube formation assay using HUVEC as the source of endothelial cells and IL-1-stimulated U87 cell conditioned medium (CM) as the incubation medium. The BD BioCoat Angiogenesis System-Endothelial Cells Tube Formation Matrigel Matrix 96-well plate was used to measure tube formation. U87 glioma CM was prepared by treating cultures with medium alone, IL-1, or IL-1 plus IL-1ra, followed by extensive washing of cells and further incubation with fresh medium. ”

“ We used a combination of cell culture, real-time PCR, ELISA, western blot, immunocytochemistry, siRNA and plasmid transfection, micro-RNA analysis, angiogenesis (tube formation) assay, and neurotoxicity assay.
Results
Glioblastoma cells produced large quantities of IL-1 when activated, resembling macrophages/microglia. The activation signal was provided by IL-1 but not the pathogenic components LPS or poly IC. Glioblastoma cells were highly sensitive to IL-1 stimulation, suggesting its relevance in vivo. ”

“ The most studied NLR is NLRP3 as it senses a diverse type of stimuli and plays a significant role in many inflammatory diseases [51] , [52] – [53] . Very little is known about the mechanism of inflammasome activation in non-myeloid cells. In keratinocytes, NLRP3 inflammasome is activated by UV irradiation [54] , [55] . In human astrocytes (study limited to cells derived from a single donor) IL-1β production/secretion was induced by ATP (alone) via NLRP2 inflammasome [56] . The latter finding is highly unusual as ATP does not provide signal 1 in most cells. ”

“ Fluorescence microscopy at 24 h after transfection reveals that control GFP vector but not IL-1/GFP vector-transfected astrocytes show green fluorescence. In contrast, HEK293 cells showed fluorescence in both conditions. (C) Western blot analysis of HEK293 cells (left) and human astrocytes (right) transfected with none (control) , GFP plasmid (GFP) , or IL-1 plasmid (IL-1/GFP) . Incubation with anti-IL-1β antibody shows positive bands only in IL-1/GFP-transfected HEK cells and not in human astrocytes. ”

“ Figure S2 U251 secretome neurotoxicity assay. U251 cells were stimulated with IL-1+IFNγ and the neurotoxicity assay was performed as described in Figure 9. Mixed primary human fetal neuronal glial cultures were stimulated with medium alone (Ctr = 1) , IL-1β + IFNγ ( = 2) , conditioned medium from IL-1β/IFNγ-stimulated U251 cells prepared as described ( = 3) or conditioned medium from U251 cells stimulated with IL-1β/IFNγ + IL-1ra as described ( = 4) . ”

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“ Data are mean ± SD ***p<0.001.Glioma releases neurotoxic substances. (A, B) GBM2 conditioned media (CM) were prepared by incubating cells with medium alone (Ctr) , IL-1ra (1 μg/ml) , IL-1α (10 ng/ml) or both, as indicated (right panel, B) . Control cultures were exposed to fresh medium (Ctr medium) or recombinant IL-1α (left panel, A) . Human fetal mixed neuronal cultures were exposed to CM and neurotoxicity was measured by trypan blue exclusion at 72 h post incubation. Arrows indicate the examples of dead (blue) cells. ”

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