IL-1 expression in patient-derived glioblastoma cells.

<p>Three patient-derived glioma cell lines (one low grade astrocytoma and two GBM cells) were tested for IL-1 protein production spontaneously (Ctr) and following IL-1/IFNγ stimulation. ELISA was performed with cell lysates (intracellular = i.c.) and culture supernatants (secreted = s). (A) IL-1 protein production was seen in GBM cells only. In one of them (GBM2), IL-1 production occurred spontaneously (Ctr) and was potentiated by IL-1 (± IFNγ). A portion of IL-1 was secreted. (B) Dose-dependent induction of IL-1: production of IL-1β was examined in GBM2 cells after stimulation with different concentrations (0.1 pg/ml to 100 ng/ml) of IL-1α. (C) IL-1β immunostain of unstimulated GBM2 cells reveals scattered positive cells, while preimmune IgG (negative control) showed no positive cells. Data shown are mean ± SD (n = 3), ***p<0.001, **p<0.01, *p<0.05 vs. control (Ctr).</p

Aberrant Expression of Interleukin-1β and Inflammasome Activation in Human Malignant Gliomas

<div><p>Objective</p><p>Glioblastoma is the most frequent and malignant form of primary brain tumor with grave prognosis. Mounting evidence supports that chronic inflammation (such as chronic overactivation of IL-1 system) is a crucial event in carcinogenesis and tumor progression. IL-1 also is an important cytokine with species-dependent regulations and roles in CNS cell activation. While much attention is paid to specific anti-tumor immunity, little is known about the role of chronic inflammation/innate immunity in glioma pathogenesis. In this study, we examined whether human astrocytic cells (including malignant gliomas) can produce IL-1 and its role in glioma progression.</p><p>Methods</p><p>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.</p><p>Results</p><p>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 <i>in vivo</i>. 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. IL-1 also activated Stat3, a transcription factor crucial in glioma progression. IL-1 activated glioblastoma-conditioned media enhanced angiogenesis and neurotoxicity.</p><p>Conclusions</p><p>Our results demonstrate unique, species-dependent immune activation mechanisms involving human astrocytes and astrogliomas. Specifically, the ability to produce IL-1 by glioblastoma cells may confer them a mesenchymal phenotype including increased migratory capacity, unique gene signature and proinflammatory signaling.</p></div

miR-132, miR-212 or the proteasome inhibitor lactacystin do not affect the expression of IL-1 in human astrocytes.

<p>Human astrocytes were transfected with specific or control anti-miR inhibitors (10 nM) for 48 h, and then stimulated with IL-1α for 24 h. (A) The expression of miR-132 was quantified by TaqMan real-time RT-PCR. Specific anti-miRs but not control anti-miR suppress miR-132 expression. (B) The culture supernatants were examined for the presence of IL-1β protein by sensitive ELISA with a lower detection limit of 3.9 pg/ml. There was no detectable IL-1β protein production in any of the human astrocyte cultures examined. (C) The effect of the proteasome inhibitor lactacystin on astrocyte IL-1β was examined. Astrocytes were treated with lactacystin at indicated concentrations with or without IL-1α, then cell lysates were subjected to ELISA after 24 h. IL-1β protein was undetectable under any conditions. Mean ± SD from triplicate cultures.</p

IL-1-activated U87 secretome promotes angiogenesis <i>in vitro</i>.

<p>Tube formation assay was performed using the BD BioCoat Angiogenesis System-Endothelial Cells Tube Formation Matrigel Matrix 96-well plate as described in the Materials and Methods. U87 conditioned media (CM) were prepared by incubating cultures with medium alone (Ctr), IL-1β (10 ng/ml), or IL-1β plus IL-1ra (1 µg/ml). HUVEC at 2.5×10⁴ cells were suspended in U87 CM and then added to the plates for 18 h at 37°C. Cells were then fixed in 4% PFA and viewed by phase-contrast microscopy. (A) Representative photography. (B) The number of closed network of vessel-like tubes was counted from three experiments. Data are mean ± SD ***p<0.001.</p

Annexin A2 promotes glioma IL-1 synthesis and release.

<p>The role of Annexin A2 in IL-1 production was examined by ELISA following transfection with A2-specific or control siRNA. (A) Western blot showing robust amounts of A2 protein expression in U87 and U251 compared to normal human astrocytes. (B) Western blot showing decreased A2 expression in U251 cells transfected with A2-specific siRNA vs. control siRNA. (C, D) The amount of IL-1β protein (intracellular and secreted) was determined by ELISA in U251 and U87 cultures following A2 knockdown. Data are mean ± SD (n = 3) *** p<0.001, ** p<0.01 (si Ctr vs. si A2), t-test.</p

Glioma releases neurotoxic substances.

<p>(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. (C) Number of trypan blue+ cells are counted in each conditions and data are pooled from three independent experiments. *** p<0.001, **p<0.01, * p<0.005 Neurotoxic activity was induced by glioma secretome (activated or non-activated), as well as recombinant IL-1α. IL-1ra completed reversed neurotoxicity in glioma CM.</p

IL-1 (± IFNγ) activates Stat3 in gliomas.

<p>U87 and U251 cells were stimulated with IL-1 (A) or IL-1±IFNγ (B) and cells were harvested at indicated time points (min) for western blot. Blots were incubated with antibody to pStat3, total Stat3, as well as β-actin. Both cultures showed Stat3 phosphorylation reaching maximal at 60 min post IL-1 stimulation.</p

Human astrocyte IL-1β expression is actively suppressed.

<p>Human astrocytes or HEK293 cells were transfected with IL-1β/GFP or GFP expression plasmids as described in the Materials and Methods. (A, B) 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. The last lane (Std.) is a positive control (17 kDa recombinant IL-1β).</p

Human glioma cells produce IL-1 mRNA and proteins in response to IL-1 stimulation.

<p>The production of IL-1β protein (intracellular A and secreted B) was examined in U251, U87, human fetal astrocytes and microglia (from left to right) in response to IL-1α, IL-1α/IFNγ, poly IC (PIC) and LPS. The GBM cell lines (U87 and U251) produced IL-1β in response to IL-1α with IFNγ acting as a primer. Little or no IL-1 was induced by poly IC and none by LPS. Microglial IL-1 protein was induced by LPS >poly IC >> IL-1 (± IFNγ). Human astrocytes did not produce IL-1β protein. (C) By real-time PCR, all four cultures including human astrocytes expressed IL-1β mRNA in response to IL-1/IFNγ. Data shown are mean ± SD (n = 3). ***p<0.001, **p<0.01, *p<0.05, n.s. = not significant compared to control (Ctr). Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103432#pone.0103432.s003" target="_blank">Table S1</a>.</p

Inflammasome activation in GBM cells.

<p>(A) Western blot showing induction of intracellular 32 kDa pro-IL-1β and secreted 17 kDa IL-1β following IL-1 stimulation. Addition of ATP and nigericin (Nig.) increased IL-1β processing (decrease of proIL-1 and increase of secreted IL-1) in GBM2 and U87 cells. NLRP3 protein was detected in unstimulated (Ctr) cultures and showed a marginal increase after IL-1 stimulation. ATP and Nig substantially increased NLRP3 protein expression. NLRP3 protein was complexed with ASC (immunoprecipitation with anti-ASC antibody) in all conditions and the complex formation was increased by ATP and Nig. Numbers are densitometric ratios to β-actin for intracellular proteins and densitometry of IL-1β measured in concentrated culture supernatants (see Materials and Methods). Average densitometry data (NLRP3, pro-IL-1β and secreted IL-1β) from two experiments for both GBM2 and U87 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103432#pone.0103432.s001" target="_blank">Figure S1</a>. (B) ELISA of GBM2 cells confirm enhanced IL-1β processing by ATP and nigericin. (C) Suppression of NLRP3 expression by siRNA in U87 cells. (D) NLRP3 siRNA-transfected U87 cells show significant reduction in the amount of secreted IL-1β in all three conditions (IL-1, + ATP, + nigericin) compared to control siRNA-transfected cells. Data shown are mean ± SD (n = 3) ***p<0.001, ** p<0.01, *p<0.05 by t-test.</p