Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Schematic of the proposed mechanism by which interleukin (IL)-18 amplifies macrophage (Mφ) M2 polarization and its morphological alteration, leading to excessive angiogenesis. IL-18 amplifies IL-10-induced increases in the production of osteopontin (OPN) and thrombin as soluble mediators derived from Mφ, yielding the generation of thrombin-cleaved form of OPN (Thr-OPN). Subsequently, Thr-OPN binds to integrins α4/α9 receptors on Mφ, which in turn augments M2 polarization of Mφ with higher expression of CD163 and its morphological alteration. Furthermore, CD163 may be responsible for mediating the direct cell–cell interaction between these Mφs and endothelial cells, ultimately resulting in the excessive angiogenesis.

Article Snippet: After that, they were incubated with primary Abs directed against OPN (1:50 dilution; R&D Systems, AF808), thrombin (1:50 dilution; Abcam, ab92621), integrin α4 (1:50 dilution; R&D Systems, BBA37), integrin α9 (1:50 dilution; R&D Systems, FAB3827), or CD163 (1:20 dilution; Bioss, bs-2527R) in blocking buffer at 4°C overnight.

Techniques: Derivative Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Interleukin (IL)-18 amplifies macrophage (Mφ) M2 polarization and angiogenic capacity. (A) Representative FACS density plots for the expression of CD86 and CD163 in each Mφ subset. Upper, Mφ (–); middle; Mφ [tumor necrosis factor (TNF)-α]; lower, Mφ (IL-10). The numbers in each quartile of the plots are percentages of each cell population. (B) Relative mean fluorescence intensities (MFIs) of CD54, CD86, CD163, and CD206 in each Mφ subset were measured by FACS analysis, n = 3 (*** p < 0.001, * p < 0.05 vs. untreated, ## p < 0.01 vs. IL-10 alone). (C) Relative MFI of IL-18Rβ in each Mφ subset was determined by FACS analysis, n = 4 (*** p < 0.001 vs. untreated, ### p < 0.001 vs. IL-10 alone). (D,E) The total areas and lengths of tube-like structures measured by the Matrigel tube formation assay where b.End5 was cocultured with each Mφ subset, n = 6 (*** p < 0.001, ** p < 0.01, * p < 0.05 vs. untreated, ## p < 0.01, # p < 0.05 vs. IL-10 alone). (F) Representative pictures of tube-like structures visualized by calcein acetoxymethylester staining. Scale bar represents 100 µm. All data are presented as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.

Article Snippet: After that, they were incubated with primary Abs directed against OPN (1:50 dilution; R&D Systems, AF808), thrombin (1:50 dilution; Abcam, ab92621), integrin α4 (1:50 dilution; R&D Systems, BBA37), integrin α9 (1:50 dilution; R&D Systems, FAB3827), or CD163 (1:20 dilution; Bioss, bs-2527R) in blocking buffer at 4°C overnight.

Techniques: Expressing, Fluorescence, Tube Formation Assay, Staining

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Osteopontin (OPN) drives enhancement in macrophage (Mφ) M2 polarization and angiogenic capacity. (A) Representative images of protein expression profiles obtained by comprehensive protein array in each Mφ subset. Red arrowheads indicate OPN. (B) The mRNA expression level of Spp1 relative to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) was analyzed by real-time reverse transcription polymerase chain reaction in each Mφ subset and was normalized to Mφ (–), n = 6 [*** p < 0.001 vs. untreated, # p < 0.05 vs. interleukin (IL)-10 alone]. (C) The protein expression level of OPN relative to GAPDH was measured by western blotting and was normalized to Mφ (–), n = 10. Lower panels are typical images of each protein (*** p < 0.001 vs. untreated, # p < 0.05 vs. IL-10 alone). (D) Representative confocal laser scanning immunofluorescence overlay images of OPN (red) and DAPI (blue) in each Mφ subset. Scale bar represents 20 µm. Images in the right row are magnified regions from white or yellow rectangles in the panels of corresponding groups. Scale bar represents 10 µm. (E) Relative mean fluorescence intensity (MFI) of CD163 was measured by FACS analysis in each Mφ subset. An anti-OPN antibody (Ab) and its isotype-matched control Ab were used at 3 µg/mL, n = 4 (*** p < 0.001 vs. untreated, ## p < 0.01, # p < 0.05 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). (F) The total areas and lengths of tube-like structures were determined by the Matrigel tube formation assay where b.End5 was cocultured with each Mφ subset, n = 12 (*** p < 0.001, ** p < 0.01, * p < 0.05 vs. untreated, # p < 0.05 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). All data are expressed as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.

Article Snippet: After that, they were incubated with primary Abs directed against OPN (1:50 dilution; R&D Systems, AF808), thrombin (1:50 dilution; Abcam, ab92621), integrin α4 (1:50 dilution; R&D Systems, BBA37), integrin α9 (1:50 dilution; R&D Systems, FAB3827), or CD163 (1:20 dilution; Bioss, bs-2527R) in blocking buffer at 4°C overnight.

Techniques: Expressing, Protein Array, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Fluorescence, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Thrombin contributes to macrophage (Mφ) M2 polarization and angiogenic capacity through proteolytic modification for osteopontin (OPN). (A) The mRNA expression level of Prothrombin relative to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) was analyzed by reverse transcription polymerase chain reaction in each Mφ subset and were normalized to Mφ (–), n = 7 [*** p < 0.001, ** p < 0.01 vs. untreated, ## p < 0.01 vs. interleukin (IL)-10 alone]. (B,C) The protein expression levels of (B) thrombin or (C) OPN N-Half relative to GAPDH were measured by western blotting in each Mφ subset and were normalized to Mφ (–). Lower panels are typical images of each protein. (B) n = 8 (*** p < 0.001, * p < 0.05 vs. untreated, # p < 0.05 vs. IL-10 alone). (C) n = 16 (*** p < 0.001, * p < 0.05 vs. untreated, ## p < 0.01, # p < 0.05 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). (D) Representative confocal laser scanning immunofluorescence images of OPN (red), thrombin (green), and their merge with DAPI (blue) in each Mφ subset. Scale bar represents 20 µm. Higher magnification images are from the white rectangle region in merged panel of Mφ (IL-10 + IL-18). Scale bar represents 10 µm. (E) Relative mean fluorescence intensity (MFI) of CD163 was measured by FACS analysis in each Mφ subset. Hirudin, a specific thrombin inhibitor, was used at 1 µg/mL, n = 3 (*** p < 0.001 vs. untreated, ### p < 0.001 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). (F) The total areas and lengths of tube-like structures were determined by the Matrigel tube formation assay where b.End5 were cocultured with each Mφ subset. Hirudin was used at 1 µg/mL, n = 6 (*** p < 0.001, ** p < 0.01 vs. untreated, ## p < 0.01, # p < 0.05 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). All data are expressed as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.

Article Snippet: After that, they were incubated with primary Abs directed against OPN (1:50 dilution; R&D Systems, AF808), thrombin (1:50 dilution; Abcam, ab92621), integrin α4 (1:50 dilution; R&D Systems, BBA37), integrin α9 (1:50 dilution; R&D Systems, FAB3827), or CD163 (1:20 dilution; Bioss, bs-2527R) in blocking buffer at 4°C overnight.

Techniques: Modification, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Fluorescence, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Intergins α4/α9 are responsible for the action of osteopontin (OPN) in the augmentation of macrophage (Mφ) M2 polarization and angiogenic capacity. (A) Relative mean fluorescence intensities (MFIs) of integrins α4/α9 were measured by FACS analysis in each Mφ subset. Anti-integrin α4 or α9 antibodies (Abs) and each isotype-matched control Ab were used at 10 µg/mL, n = 4 [*** p < 0.001 vs. untreated, ### p < 0.001 vs. interleukin (IL)-10 alone]. (B) Representative confocal laser scanning immunofluorescence overlay images of integrin α4 (green) and DAPI (blue), as well as those of integrin α9 (red) and DAPI (blue) in Mφ (–) and Mφ (IL-10 + IL-18). Scale bar represents 20 µm. (C) Relative MFI of CD163 was measured by FACS analysis in each Mφ subset. Anti-integrin α4 or α9 Abs and each isotype-matched control Ab were used at 10 µg/mL, n = 4 (*** p < 0.001 vs. untreated, ### p < 0.001 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). (D) The total areas and lengths of tube-like structures were determined by the Matrigel tube formation assay where b.End5 was cocultured with each Mφ subset. Anti-integrin α4 or α9 Abs and each isotype-matched control Ab were used at 10 µg/mL, n = 6 (*** p < 0.001, ** p < 0.01, * p < 0.05 vs. untreated, ### p < 0.001, ## p < 0.01, # p < 0.05 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). Integrin α4 = ITGA4; Integrin α9 = ITGA9. All data are expressed as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.

Article Snippet: After that, they were incubated with primary Abs directed against OPN (1:50 dilution; R&D Systems, AF808), thrombin (1:50 dilution; Abcam, ab92621), integrin α4 (1:50 dilution; R&D Systems, BBA37), integrin α9 (1:50 dilution; R&D Systems, FAB3827), or CD163 (1:20 dilution; Bioss, bs-2527R) in blocking buffer at 4°C overnight.

Techniques: Fluorescence, Immunofluorescence, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: CD163 is a critical factor for determining the angiogenic capacity of macrophage (Mφ). (A) Upper; representative confocal laser scanning immunofluorescence overlay images of CD163 (green) and DAPI (blue) in Mφ (–) and Mφ [interleukin (IL)-10 + IL-18]. Scale bar represents 20 µm. Lower; Three-dimensional images of each upper panel. Higher magnification image in the panel of Mφ (IL-10 + IL-18) is from white rectangle region. Scale bar represents 10 µm. White arrowheads indicate CD163 highly expressed and localized at pseudopodia. (B) The total areas and lengths of tube-like structures were determined by the Matrigel tube formation assay where b.End5 were cocultured with each Mφ subset. An anti-CD163 antibody (Ab) and its isotype-matched control Ab were used at 4 µg/mL, n = 3 (*** p < 0.001, * p < 0.05 vs. untreated, ### p < 0.001, ## p < 0.01, # p < 0.05 vs. IL-10 alone, ††† p < 0.001 vs. IL-10 + IL-18). (C) Representative images (upper) and corresponding three-dimensional images (lower) of tube-like structures as well as (D) total areas and lengths of tubular structure where b.End5 (green) and Mφs (IL-10 + IL-18) (red) were cocultured on Matrigel for 16 h with an anti-CD163 Ab or its isotype-matched control Ab. Scale bar represents 100 µm, n = 8 (*** p < 0.001 vs. untreated, ### p < 0.001 vs. IL-10 + IL-18). All data are expressed as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.

Article Snippet: After that, they were incubated with primary Abs directed against OPN (1:50 dilution; R&D Systems, AF808), thrombin (1:50 dilution; Abcam, ab92621), integrin α4 (1:50 dilution; R&D Systems, BBA37), integrin α9 (1:50 dilution; R&D Systems, FAB3827), or CD163 (1:20 dilution; Bioss, bs-2527R) in blocking buffer at 4°C overnight.

Techniques: Immunofluorescence, Tube Formation Assay

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: Expression of CD24 was cell type‐dependent in colorectal cancer cell lines. (A) Flow cytometric analysis of different colorectal cancer cell lines at the protein level. The cells were stained with anti‐CD24 monoclonal antibodies and FITC‐conjugated secondary antibodies. Abscissa, fluorescence intensity (log scale); ordinate, cell number (linear scale). Filled trace, background staining; open trace, staining with CD24. (B) Differentiated levels of CD24 mRNA were detected in colorectal cancer cell lines by RT‐PCR.

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Bioprocessing, Fluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: Effect of CD24 on the growth of SW480 cells. (A) CD24 mRNA was detected in cell clones of SW480 using RT‐PCR. (B) Flow cytometry confirmed the expression of CD24 at the protein level. Abscissa, fluorescence intensity (log scale); ordinate, cell number (linear scale). Filled trace, background staining; open trace, staining with CD24. (C) Increased growth ability was detected in SW480CD24+1 cells compared with SW480vec cells and parental SW480 cells using the WST‐8 assay (***P < 0.001). Ordinate, absorbance at 450 nm. (D) CD24 promoted tumor growth in nude mice (***P < 0.001). BALB/c nude mice were injected subcutaneously with 4×106 cells and tumor volume was measured once every 5 days. (E) Histological analysis of implanted tumors with H&E staining (×200).

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Clone Assay, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Expressing, Fluorescence, Staining, Injection

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: The activities of ERK1/2, Raf‐1, p38 MAPK, and JNK in SW480CD24+1 and SW480CD24+4 cells were assessed by western blotting. Overexpression of CD24 induced the activation of (A) ERK1/2, (B) p38 MAPK, and (D) the upstream molecule of ERK1/2, Raf‐1. (C) However, no activation was observed in JNK1/2. β‐Actin expression was used to normalize for equal loading. (E) Immunohistochemical analysis of implanted tumors with antibodies to CD24, p‐ERK, and p‐p38 (×400).

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Over Expression, Activation Assay, Expressing, Immunohistochemical staining

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: Activation of ERK1/2 and p38 MAPK was required for CD24‐induced proliferation. Western blotting analysis confirmed that (A) ERK1/2 and (C) p38 MAPK activation was suppressed when cells were treated with 10 μm U0126 or 20 μm SB203580. The cells were incubated with RPMI‐1640 medium containing 1% FBS for 12 h. Starved cells were treated with U0126 or SB203580 2 h before complete medium was added. The total protein and phosphorylation levels of ERK1/2 and p38 MAPK expression were determined by western blotting analysis 30 min later. Cell growth in the presence of (B) U0126 or (D) SB203580 was assessed after 24 and 72 h treatment by WST‐8 assay. All experiments were repeated twice. Ordinate, absorbance at 450 nm.

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot, Incubation, Phospho-proteomics, Expressing

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: Knockdown of CD24 inhibited cell growth and reduced the activation of ERK1/2, Raf‐1, and p38 MAPK. (A) CD24 mRNA was detected in parental SW620 and treated cells using RT‐PCR. (B) Flow cytometry confirmed downregulation of CD24 at the protein level. Abscissa, fluorescence intensity (log scale); ordinate, cell number (linear scale). Filled trace, background staining; open trace, staining with CD24. (C) WST‐8 assay showed that knockdown of CD24 inhibited cell growth in SW620 cells (***P < 0.001). (D) Western blotting showed that downregulation of CD24 reduced the activation of ERK1/2, Raf‐1, and p38 MAPK, but had no effect on JNK1/2. NC, SW620 cells transfected with negative control sequence; KD, SW620 cells treated with siRNA duplex targeting CD24.

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Fluorescence, Staining, Western Blot, Transfection, Negative Control, Sequencing

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: CD24 expression increased with colorectal cancer progression. (A) Normal mucosa showing negative expression of CD24. (B) Weak expression in a stage I tumor. (C) An invasive stage II carcinoma showing moderate staining. Note an obvious interface between normal mucosa and cancer. (D) Strong expression in a stage III colorectal cancer. The scale of scale bars: 10 μm.

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Staining

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: Association between CD24 expression and clinicopathological parameters

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Cancer Science

Article Title: CD24‐dependent MAPK pathway activation is required for colorectal cancer cell proliferation

doi: 10.1111/j.1349-7006.2009.01370.x

Figure Lengend Snippet: Activation of ERK1/2 and p38 MAPK in human colorectal cancer strongly correlated with cytoplasmic CD24 expression. (A–C) Serial sections of a stage II tumor showing weak expression for (A) CD24, (B) p‐ERK1/2, and (C) p‐p38 MAPK. (D–F) Serial sections of a stage III tumor demonstrating moderate immunostaining for (D) CD24, (E) p‐ERK1/2 and (F) p‐p38 MAPK. The scale of scale bars: 10 μm.

Article Snippet: CD24 (C‐20) polyclonal antibody, Raf‐1 (C‐12) antibody, p‐Raf‐1 (Ser338), p‐ERK (E‐4), and β‐actin (C‐4) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Expressing, Immunostaining