Journal: Cell Death & Disease

Article Title: Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma

doi: 10.1038/s41419-019-1387-6

Figure Lengend Snippet: a PDX GBM43 cells were treated with TMZ (50 μM), and RNA collected after 4 or 8 days. Microarray analysis using Affymetrix platform was performed. Gene set enrichment analysis (GSEA) for genes known to support and maintain the GIC phenotype and demonstrated significance increase after 8 days [day 4 FDR q 0.82 and day 8 FDR 0.08 and FWER p-value 0.046, TMZ-treated cells as compared with DMSO controls]. b Whole-genome ChIP-seq analysis of H3K27 trimethylation (H3K27me) and H3k27 acetylation mark (H2K27ac) on PDX GBM43 following 4 days treatment with TMZ or vehicle control. Top two tracks represent the BET file showing significant enrichment of histone mark relative to DMSO control. Bottom two lanes represent WIG file showing significant enrichment peak. Analysis of three established astrocyte enhancer tracks for the IL-8 gene showed no changes in H3K27me3; however, they did show significant change in #3 enhancer region [Chr. 4 :74783222–74783418, fold enrichment 3.2 relative to IgG input. p < 0.0001. FDR = 0.004]. Right bottom inset image shows zoomed in view of the #3 enhancer region. Left bottom inset image represents overlayed peaks for TMZ and DMSO control. c Expression of IL-8 mRNA after exposure to 50 µM TMZ was determined by quantitative real-time polymerase chain reaction (qPCR) after treatment with TMZ across 8 days. All IL-8 values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Bars represent means from three independent experiments and error bars represent the standard deviation. Multiple Student's t tests were performed. ** p < 0.01, *** p < 0.001. d Immunohistochemistry was performed on mouse brains with intracranial xenografts of GBM43. 1.5 × 10 5 GBM43 PDX cells were implanted to establish orthotropic xenograft tumors. Animals received 2.5 mg/kg of either DMSO or TMZ for five consecutive days, beginning 7 days after tumor implantation. Animals were killed 5 days after the cessation of treatment, and whole brains were extracted, flash frozen, then sectioned (8 µm) and analyzed by immunofluorescence. 4′-6-diamidino-2-phenylindole (DAPI) stained DNA (blue) in the nuclei and allophycocyanin-conjugated secondary (orange) antibody was used against primary antibody for IL-8. Dotted lines represent the edge of the tumor based on cell density and morphology. e–f PDX GBM xenografts were harvested and immediately plated in either mild differentiation media (DMEM containing 1% FBS) or GIC maintenance media (neurobasal supplemented with FGF and EGF). Cells were then treated with either DMSO or TMZ (50 μM). After 4 days, IL-8 levels were determined by ELISA. g GBM cells were treated with physiologically relevant dose of TMZ (50 μM), carmustine (BCNU, 100 μM), or equimolar DMSO. After 24 h, conditioned media was collected and IL-8 levels were quantified by ELISA. Bars represent means from three independent experiments and error bars represent the standard deviation. Multiple Student's t tests were performed. ** p < 0.01, *** p < 0.001

Article Snippet: The following antibodies were employed: mouse anti-IL-8 (R&D), rabbit anti-c-Myc (Cell Signaling, Danvers, MA, USA), rabbit anti-Sox2 (Cell Signaling), rabbit anti-Nanog (Cell Signaling), rabbit anti-LIN28A (Cell Signaling), rabbit anti-KLF4 (Cell Signaling), mouse anti-β-actin (Abgent, San Diego, CA, USA), rabbit anti-EZH2 (Cell Signaling), mouse anti-phosphoEZH2 (AbCam, Cambridge, UK), rabbit anti-Bmi1 (Cell Signaling), rabbit anti-ring1A (Cell Signaling), rabbit anti-SUZ12 (Cell Signaling), rabbit anti-H3 (Cell Signaling), rabbit anti-H3K4methyl3 (Cell Signaling), rabbit anti-H3K27acetyl (Cell Signaling), rabbit anti-H3K27methyl3 (Cell Signaling), rabbit anti-H3K36methyl3 (Cell Signaling), and rabbit anti-H2A-Ubiquitin (Cell Signaling).

Techniques: Microarray, ChIP-sequencing, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Immunohistochemistry, Tumor Implantation, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma

doi: 10.1038/s41419-019-1387-6

Figure Lengend Snippet: a IL-8 mRNA expression levels were analyzed using the Affymetrix U133a platform on the Cancer Genome Atlas (TCGA) for different WHO grades of glioma. GBM (Grade IV glioma) patients had higher IL-8 expression level than low-grade (Grade II, Grade III) glioma patients. b All GBM patients were stratified into IL-8-upregulated and IL-8-downregulated groups based on IL-8 gene expression using quartile (Q1, Q3) as split points. High expression of IL-8 correlated with reduced median survival. Survival curves were generated via the Kaplan–Meier method and compared by log-rank test. ** p < 0.01. Multivariate stepwise Cox proportional hazards model with stepwise variable selection was conducted to examine whether IL-8 could be an independent factor for predicting survival with major clinical variables adjusted. This analysis confirmed that IL-8 was an independent prognostic factor for survival in all GBM patients (HR [95% CI]: all GBM 1.07 [1.01, 1.14], p = 0.0467). c Index (95% CI) or C statistics are provided. c Within grade IV glioma subtypes, proneural GBM patients had the lowest level of IL-8 expression. Boxplots represent means and interquartile range. One-way ANOVAs with Bonferroni correction for the multiple comparisons were performed. * p < 0.05, *** p < 0.001. d Patients with proneural GBM were stratified into IL-8-upregulated and IL-8-downregulated groups based on IL-8 gene expression using quartile (Q1, Q3) as split points. Kaplan–Meier survival curves and multivariate stepwise Cox proportional hazards models were generated as in B. e In TCGA database (U133a) GBM patients with IL-8-downregulated also have a longer time to recurrence, compared with IL-8-upregulated patients. f The Ivy Glioblastoma Atlas Project (IVY GAP) was employed to determine the location of IL-8 in glioblastoma samples. Each column represents the data for one biopsy from a tumor. Microdissection for the noted anatomically portions of the tumor and subsequent mRNA extraction and expression analysis demonstrated that IL-8 is upregulated in the perinecrotic zone and pseudopalisading cells. Heatmap illustrates most significantly and differential expressed genes with a false discovery rate < 0.01. mRNA expression in each anatomical compartment were compared. IL-8 was significantly upregulated in the perinecrotic zone. Bars represent means from three independent experiments and error bars represent the standard deviation. Multiple Student's t tests were performed. *** p < 0.001. g Brain tumor samples from primary biopsies or surgical resections were stained for IL-8 at the Northwestern Brain Tumor Tissue Bank. Histological and morphological analysis confirm that IL-8 is present in the perinecrotic zone and pseduopalisading cells. Scale bar 50 microns

Article Snippet: The following antibodies were employed: mouse anti-IL-8 (R&D), rabbit anti-c-Myc (Cell Signaling, Danvers, MA, USA), rabbit anti-Sox2 (Cell Signaling), rabbit anti-Nanog (Cell Signaling), rabbit anti-LIN28A (Cell Signaling), rabbit anti-KLF4 (Cell Signaling), mouse anti-β-actin (Abgent, San Diego, CA, USA), rabbit anti-EZH2 (Cell Signaling), mouse anti-phosphoEZH2 (AbCam, Cambridge, UK), rabbit anti-Bmi1 (Cell Signaling), rabbit anti-ring1A (Cell Signaling), rabbit anti-SUZ12 (Cell Signaling), rabbit anti-H3 (Cell Signaling), rabbit anti-H3K4methyl3 (Cell Signaling), rabbit anti-H3K27acetyl (Cell Signaling), rabbit anti-H3K27methyl3 (Cell Signaling), rabbit anti-H3K36methyl3 (Cell Signaling), and rabbit anti-H2A-Ubiquitin (Cell Signaling).

Techniques: Expressing, Generated, Selection, Laser Capture Microdissection, Standard Deviation, Staining

Journal: Cell Death & Disease

Article Title: Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma

doi: 10.1038/s41419-019-1387-6

Figure Lengend Snippet: a Representative immunohistochemical staining for IL-8 in the matched primary (P) and recurrent (R) clinical GBM samples. Top row low-powered and bottom row high-powered magnification of IL-8 staining. Scale bar for all top row images 1 mm, all middle row images 5 mm, and for the bottom row images 250 µm. These sets of patient samples show IL-8 upregulation in the matched recurrent tissues. b Same as previous, but this set of patient samples shows a decrease in IL-8 staining for recurrent GBM as compared with their matched primary GBM. c Quantitative analysis of the percent of IL-8 positive cells in the matched primary and recurrent GBM tissue. Representative immunohistochemical analysis of IL-8 expressing ( d ) tumor cells (left, black arrow) and ( e ) infiltrative macrophage (right, red arrow)

Article Snippet: The following antibodies were employed: mouse anti-IL-8 (R&D), rabbit anti-c-Myc (Cell Signaling, Danvers, MA, USA), rabbit anti-Sox2 (Cell Signaling), rabbit anti-Nanog (Cell Signaling), rabbit anti-LIN28A (Cell Signaling), rabbit anti-KLF4 (Cell Signaling), mouse anti-β-actin (Abgent, San Diego, CA, USA), rabbit anti-EZH2 (Cell Signaling), mouse anti-phosphoEZH2 (AbCam, Cambridge, UK), rabbit anti-Bmi1 (Cell Signaling), rabbit anti-ring1A (Cell Signaling), rabbit anti-SUZ12 (Cell Signaling), rabbit anti-H3 (Cell Signaling), rabbit anti-H3K4methyl3 (Cell Signaling), rabbit anti-H3K27acetyl (Cell Signaling), rabbit anti-H3K27methyl3 (Cell Signaling), rabbit anti-H3K36methyl3 (Cell Signaling), and rabbit anti-H2A-Ubiquitin (Cell Signaling).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cell Death & Disease

Article Title: Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma

doi: 10.1038/s41419-019-1387-6

Figure Lengend Snippet: a Limiting dilution neurosphere assays were performed on two cell lines—GBM43 and GBM6—after treatment with 50 ng/ml of IL-8. Stem cell frequency for GBM43 with IL-8 35.7, lower limit 50.7 and upper limit 25.1 as compared with no IL-8 118.6, lower limit 169.2 and upper limit 83.1, p = 0.0156; for GBM6 with IL-8 27.1, lower limit 39.2 and upper limit 18.8 as compared with no IL-8 60.7, lower limit 85 and upper limit 18.8, p = 0.001. b To determine the ability of IL-8 to influence cellular plasticity, we employed a reporter cell line in which RFP expression is controlled the OCT4 promoter. Cells were treated with 50 ng/ml of IL-8, and RFP expression was monitored by FACS over 6 days. Treatment increased both Oct4 and Sox2. Bars represent means from three independent experiments and error bars represent the standard deviation. Multiple Student's t tests were performed. ** p < 0.01, *** p < 0.001. c The network of GIC-promoting genes in patient tumors, we selected the top GIC-associated genes activated during TMZ therapy (Fig. and Supplementary Table ) and correlated with their levels with IL-8 mRNA using the GlioVis data portal for visualization and analysis of brain tumor expression database (gliovis.bioinfo.cnio.es; dataset LeeY) 16–18. IL-8 expression was significantly correlated with critical GIC-associated genes including KLF4, CD44, HIF1A, HIF2A, Myc, and Twist expression (Fig. 5c). The IL-8 expression in different anatomical location and potential colocalization of these GIC-specific genes with areas of high IL-8 transcript level (Fig. 5c, heatmap). d Immunoblot analysis of endogenous glioma-initiating cell-associated transcription factors expression upon stimulation with escalation dose of IL-8 (0–100 ng/ml) for 24 h. Protein extracts of IL-8-treated PDX lines GBM43 and GBM6 were immunoblotted with antibody against several GIC markers, including c-myc, Sox2, Nanog, KLF4, OCT4, or an antibody against β-actin as a control for equal loading. e GBM43 PDX cells were treated with neutralizing antibody against IL-8 or control IgG antibody (100 ng/ml) prior to treatment with DMSO or 50 µM TMZ. Neutralizing antibody was added every day for 8 days,and protein extracts from this experiment were immunoblotted with antibody against c-myc, Sox2, OCT4, or an antibody against β-actin as a control for equal loading. f Schematic diagram of experiment design for in vivo testing. Top graph, U251 cells were infect with lentivirus (Sigma Mission shRNA) shRNA against IL-8 or scrambled shRNA (control) with 10 infectious unit/cell. In total, 2 × 10 5 transduced cells were stereotactically injected into the right hemisphere of the brain of athymic nude mice ( n = 8 per group, four males and four females). Two weeks after implantation, two groups of mice, control, and knock down, were treated with vehicle treated (DMSO, top curve) or TMZ (2.5 mg/kg) intraperitoneally. Survival curves were obtained by the Kaplan–Meier method, and overall survival time was compared between groups using log-rank test. All statistical tests were two-sided. Bottom graph, to examine the role of IL-8 in GBM progression in a more clinically relevant manner, next the same method was used to knockdown the IL-8 expression in GBM43 PDX line. In total, 1.5 × 10 5 cells were injected stereotactically into the right hemisphere of the brain of athymic nude mice ( n = 8 per group, four males and four females). Survival curves were obtained by the Kaplan–Meier method, and overall survival time was compared between groups using log-rank test

Article Snippet: The following antibodies were employed: mouse anti-IL-8 (R&D), rabbit anti-c-Myc (Cell Signaling, Danvers, MA, USA), rabbit anti-Sox2 (Cell Signaling), rabbit anti-Nanog (Cell Signaling), rabbit anti-LIN28A (Cell Signaling), rabbit anti-KLF4 (Cell Signaling), mouse anti-β-actin (Abgent, San Diego, CA, USA), rabbit anti-EZH2 (Cell Signaling), mouse anti-phosphoEZH2 (AbCam, Cambridge, UK), rabbit anti-Bmi1 (Cell Signaling), rabbit anti-ring1A (Cell Signaling), rabbit anti-SUZ12 (Cell Signaling), rabbit anti-H3 (Cell Signaling), rabbit anti-H3K4methyl3 (Cell Signaling), rabbit anti-H3K27acetyl (Cell Signaling), rabbit anti-H3K27methyl3 (Cell Signaling), rabbit anti-H3K36methyl3 (Cell Signaling), and rabbit anti-H2A-Ubiquitin (Cell Signaling).

Techniques: Expressing, Standard Deviation, Western Blot, In Vivo, shRNA, Injection

Journal: Cell Death & Disease

Article Title: Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma

doi: 10.1038/s41419-019-1387-6

Figure Lengend Snippet: a Correlation between IL-8 and 12042 genes from TCGA was determined by Pearson correlation coefficients. Sixty-eight genes with coefficients > 0.5 or < −0.5 and false discovery rate (FDR) < 0.05 were selected. Unsupervised hierarchical clustering of those genes found two clusters with the highest cophenetic coefficient at 0.95 and an average silhouette width at 0.46. Principal component analysis was used to validate these clusters. Enrichment analysis found one cluster of genes was enriched at cell chemotaxis (GO: 0060326, adjusted p -value 1.058e-11) and cytokine activity (GO: 0005125, adjusted p -value 2.495e-9), while another cluster of genes enriched at wounding (GO: 009611, adjusted p -value 0.002) and hypoxia (GO: 0001666, adjusted p -value 0.004). b Representative immunoblot of different histone marks. A panel of PDX lines from a different subtype of GBMs was exposed to IL-8 (50 ng/ml) for 24 h. Nuclei were extracted from the harvested cells and subjected to immunoblot analysis for suppressive histone marks H3K27 and H3K9 trimethylation (me3) and activating mark H3K27 acetylation(ac). Immunoblotting for total histone three was performed to confirm the equal loading. c The extracted nuclei from the U251 IL-8 knockdown cells as described in Fig. were subjected to immunoblot analysis for various histone marks as described above. d GBM43 cells were treated with IL-8 (50 ng/ml) for 2 and 24 h, cytoplasmic and nuclear extract was prepared, cells were harvested, and immunoprecipitation assays were performed with the anti-EZH2 antibody. Immunoprecipitated protein was subjected to immunoblot analysis with antibodies against phosphor-EZH2 (S21, and Thr345), and SUZ12. β-Actin and histone 3 were used as loading controls. e IL-8 (50 ng/ml)-treated GBM43 PDX lines were harvested at 6, 12, and 24 h post IL-8 exposure. mRNA was extracted and subjected to reverse-transcription polymerase chain reaction (RT-PCR) analysis of OLIG2, SERPINB2, IL6R , and BMP2K transcripts. Bars represent means from two experiments in triplicate and error bars represent the standard deviation. Multiple Student's t tests were performed. ** p < 0.01, **** p < 0.0001. f A panel of GBM PDX lines was treated with TMZ (50 µM) for 48 h. Nuclei were extracted from the harvested cells and subjected to immunoblot analysis for suppressive histone marks H3K27me3 and H3K9me2 and activating mark H3K27ac and H3K4me3. Immunoblotting for total histone three was performed to confirm equal loading. g The U251 IL-8 control and knockdown cells as described in Fig. were treated with TMZ(50 µM) for 4 days. Nuclei were extracted from the harvested cells and subjected to immunoblot analysis for suppressive histone marks H3K27me3 and H3K9me2 and activating mark H3K27ac. Left, representative densitometry analysis is expressed as percent of control shRNA (shCtrl). h The GBM43 PDX line was treated with TMZ(50 µM) in the presence of 3-deazaneplanocin A (DZNep, EZH2-I, 5 µmol/L), a histone methyltransferase EZH2 inhibitor for 8 days. Cells were harvested and the GIC population was analyzed by FACS analysis of the CD133 and CD15 positive cells. Bars represent means from two experiments in triplicate and error bars represent the standard deviation. Multiple Student's t tests were performed. *** p < 0.001

Article Snippet: The following antibodies were employed: mouse anti-IL-8 (R&D), rabbit anti-c-Myc (Cell Signaling, Danvers, MA, USA), rabbit anti-Sox2 (Cell Signaling), rabbit anti-Nanog (Cell Signaling), rabbit anti-LIN28A (Cell Signaling), rabbit anti-KLF4 (Cell Signaling), mouse anti-β-actin (Abgent, San Diego, CA, USA), rabbit anti-EZH2 (Cell Signaling), mouse anti-phosphoEZH2 (AbCam, Cambridge, UK), rabbit anti-Bmi1 (Cell Signaling), rabbit anti-ring1A (Cell Signaling), rabbit anti-SUZ12 (Cell Signaling), rabbit anti-H3 (Cell Signaling), rabbit anti-H3K4methyl3 (Cell Signaling), rabbit anti-H3K27acetyl (Cell Signaling), rabbit anti-H3K27methyl3 (Cell Signaling), rabbit anti-H3K36methyl3 (Cell Signaling), and rabbit anti-H2A-Ubiquitin (Cell Signaling).

Techniques: Chemotaxis Assay, Activity Assay, Western Blot, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, shRNA

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: ( A ) Relative IL-8 mRNA expression in M2 macrophages treated with leptin with or without the inhibitors. M2 macrophages were pretreated with JAK inhibitor AG490 (50 μmol/L), PI3K inhibitor LY294002 (10 μmol/L), ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative IL-8 mRNA levels were analyzed by qRT-PCR. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01. ( B ) A representative image of Western blot for IL-8 protein expression in M2 macrophages treated in the same manner as (A). ( C ) A representative image of Western blot showing the time course of leptin-induced p38 and ERK 1/2 phosphorylation. M2 macrophages were treated with leptin (100 ng/mL) for 0–24 hours. ( D ) A representative image of Western blot showing leptin-induced ObR-dependent phosphorylation of ERK 1/2 and p38 and production of IL-8. M2 macrophages were pretreated with a polyclonal anti-human ObR antibody (4 μg/mL) for 16 hours and then treated with PBS or leptin (100 ng/mL). ( E ) Luciferase reporter assay to measure leptin-mediated IL-8 promoter activation. M2 macrophages were pretreated with ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative luciferase units (RLU) normalized to β-galactosidase activity are shown. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Luciferase, Reporter Assay, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: The mouse xenograft model and leptin injection were conducted as in Figure . In the leptin-treated group, polyclonal rabbit anti-mouse IL-8 neutralization antibody (Abbexa Ltd, Cambridge, UK) was injected intraperitoneal in the mice at an initial dose of 0.2 mL per mouse and then 0.1 mL per mouse twice per week for 2 weeks. Polyclonal rabbit IgG (Bioss Ltd, Beijing, China) was used as the control. Mice received the injection of leptin and the antibodies for 2 weeks. ( A ) Tumor volume in the PBS, leptin, leptin + anti-IgG, and leptin + anti-IL8 groups. * P < 0.05. ( B ) Photos of tumor xenografts at day 21 after transplantation of human breast cancer cells. ( C ) Tumor wet weight at day 21 after the cell transplantation. ** P < 0.01. ( D ) Mouse survival. * P < 0.05. ( E ) Representative images of H & E staining of the lung tissue and the figure showing the number of metastatic nodules in the lung. ** P < 0.01. ( F ) Representative images of H & E staining of the liver tissue. ( G ) Representative images of IHC staining of CD68, Ki-67, and IL-8 in the tumor tissue and the figure showing quantitative analysis of the staining. ** P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Injection, Neutralization, Control, Transplantation Assay, Staining, Immunohistochemistry

Journal: Immunology

Article Title: Novel role of regulatory T cells in limiting early neutrophil responses in skin

doi: 10.1111/j.1365-2567.2010.03333.x

Figure Lengend Snippet: Depletion of CD25+ cells promotes neutrophil accumulation at the site of tumour inoculation. Mice were treated with either isotype control monoclonal antibodies (GL113; a) or CD25-specific monoclonal antibodies (PC61; b) and injected 1 day later with 105 B16Fas ligand. At 24 hr post-injection, mice were killed and the skin surrounding the injection site was collected for histology. Interleukin-8 receptor (IL-8R) -stained 5-μm paraffin-mounted sections were generated to identify neutrophils. Areas of neutrophil infiltration are enclosed by the blue lines. Sections are representative of at least five mice per group at 24 hr. (c) Examples of neutrophils (indicated by arrows) which stain dark brown with multi-lobed nuclei. (d) The numbers of neutrophils, assessed as polymorphonuclear cells with IL-8R expression, are given where each symbol represents the score for one mouse. Statistical significance was evaluated by Mann–Whitney U-test (*P < 0·05). (e) The relative expression of CXCL1 and CXCL2, assessed by real-time PCR, in the skin of GL113-treated and PC61-treated B16FasL-inoculated mice.

Article Snippet: Neutrophils were detected using rabbit anti-mouse interleukin-8 receptor B (IL-8RB; K-19; Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation with biotinylated swine anti-rabbit abs (Dako, Glostrup, Denmark).

Techniques: Injection, Staining, Generated, Expressing, MANN-WHITNEY, Real-time Polymerase Chain Reaction