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: Cells were then stained with anti-mouse Abs against phycoerythrin (PE)-conjugated CD 54 (60 ng; BioLegend, 116108), allophycocyanin (APC)-conjugated CD86 (6.0 ng; Miltenyi Biotec, 130-102-558), fluorescein isothiocyanate (FITC)-conjugated CD68 (25 ng; Bio-Rad Laboratories, MCA1957F), FITC-conjugated CD163 (200 ng; Bioss, bs-2527R-FITC), FITC-conjugated CD206 (200 ng; Bio-Rad Laboratories, MCA2235F), APC-conjugated M-CSF1 receptor (M-CSF1R) (60 ng; BioLegend, 135510), Alexa Fluor 647-conjugated IL-18Rβ (80 ng; Bioss, bs-2616R-A647), FITC-conjugated integrin α4 (130 ng; Thermo Fisher Scientific, 11-0492-82), or PE-conjugated integrin α9 (0.50 µL; Thermo Fisher Scientific, PA5-46896) for 30 min at 4°C.

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: Cells were then stained with anti-mouse Abs against phycoerythrin (PE)-conjugated CD 54 (60 ng; BioLegend, 116108), allophycocyanin (APC)-conjugated CD86 (6.0 ng; Miltenyi Biotec, 130-102-558), fluorescein isothiocyanate (FITC)-conjugated CD68 (25 ng; Bio-Rad Laboratories, MCA1957F), FITC-conjugated CD163 (200 ng; Bioss, bs-2527R-FITC), FITC-conjugated CD206 (200 ng; Bio-Rad Laboratories, MCA2235F), APC-conjugated M-CSF1 receptor (M-CSF1R) (60 ng; BioLegend, 135510), Alexa Fluor 647-conjugated IL-18Rβ (80 ng; Bioss, bs-2616R-A647), FITC-conjugated integrin α4 (130 ng; Thermo Fisher Scientific, 11-0492-82), or PE-conjugated integrin α9 (0.50 µL; Thermo Fisher Scientific, PA5-46896) for 30 min at 4°C.

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: Cells were then stained with anti-mouse Abs against phycoerythrin (PE)-conjugated CD 54 (60 ng; BioLegend, 116108), allophycocyanin (APC)-conjugated CD86 (6.0 ng; Miltenyi Biotec, 130-102-558), fluorescein isothiocyanate (FITC)-conjugated CD68 (25 ng; Bio-Rad Laboratories, MCA1957F), FITC-conjugated CD163 (200 ng; Bioss, bs-2527R-FITC), FITC-conjugated CD206 (200 ng; Bio-Rad Laboratories, MCA2235F), APC-conjugated M-CSF1 receptor (M-CSF1R) (60 ng; BioLegend, 135510), Alexa Fluor 647-conjugated IL-18Rβ (80 ng; Bioss, bs-2616R-A647), FITC-conjugated integrin α4 (130 ng; Thermo Fisher Scientific, 11-0492-82), or PE-conjugated integrin α9 (0.50 µL; Thermo Fisher Scientific, PA5-46896) for 30 min at 4°C.

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: Cells were then stained with anti-mouse Abs against phycoerythrin (PE)-conjugated CD 54 (60 ng; BioLegend, 116108), allophycocyanin (APC)-conjugated CD86 (6.0 ng; Miltenyi Biotec, 130-102-558), fluorescein isothiocyanate (FITC)-conjugated CD68 (25 ng; Bio-Rad Laboratories, MCA1957F), FITC-conjugated CD163 (200 ng; Bioss, bs-2527R-FITC), FITC-conjugated CD206 (200 ng; Bio-Rad Laboratories, MCA2235F), APC-conjugated M-CSF1 receptor (M-CSF1R) (60 ng; BioLegend, 135510), Alexa Fluor 647-conjugated IL-18Rβ (80 ng; Bioss, bs-2616R-A647), FITC-conjugated integrin α4 (130 ng; Thermo Fisher Scientific, 11-0492-82), or PE-conjugated integrin α9 (0.50 µL; Thermo Fisher Scientific, PA5-46896) for 30 min at 4°C.

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: Cells were then stained with anti-mouse Abs against phycoerythrin (PE)-conjugated CD 54 (60 ng; BioLegend, 116108), allophycocyanin (APC)-conjugated CD86 (6.0 ng; Miltenyi Biotec, 130-102-558), fluorescein isothiocyanate (FITC)-conjugated CD68 (25 ng; Bio-Rad Laboratories, MCA1957F), FITC-conjugated CD163 (200 ng; Bioss, bs-2527R-FITC), FITC-conjugated CD206 (200 ng; Bio-Rad Laboratories, MCA2235F), APC-conjugated M-CSF1 receptor (M-CSF1R) (60 ng; BioLegend, 135510), Alexa Fluor 647-conjugated IL-18Rβ (80 ng; Bioss, bs-2616R-A647), FITC-conjugated integrin α4 (130 ng; Thermo Fisher Scientific, 11-0492-82), or PE-conjugated integrin α9 (0.50 µL; Thermo Fisher Scientific, PA5-46896) for 30 min at 4°C.

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: Cells were then stained with anti-mouse Abs against phycoerythrin (PE)-conjugated CD 54 (60 ng; BioLegend, 116108), allophycocyanin (APC)-conjugated CD86 (6.0 ng; Miltenyi Biotec, 130-102-558), fluorescein isothiocyanate (FITC)-conjugated CD68 (25 ng; Bio-Rad Laboratories, MCA1957F), FITC-conjugated CD163 (200 ng; Bioss, bs-2527R-FITC), FITC-conjugated CD206 (200 ng; Bio-Rad Laboratories, MCA2235F), APC-conjugated M-CSF1 receptor (M-CSF1R) (60 ng; BioLegend, 135510), Alexa Fluor 647-conjugated IL-18Rβ (80 ng; Bioss, bs-2616R-A647), FITC-conjugated integrin α4 (130 ng; Thermo Fisher Scientific, 11-0492-82), or PE-conjugated integrin α9 (0.50 µL; Thermo Fisher Scientific, PA5-46896) for 30 min at 4°C.

Techniques: Immunofluorescence, Tube Formation Assay

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Deletion of JNK2 prevents vitamin-D-deficiency-induced hypertension and atherosclerosis in mice

doi: 10.1016/j.jsbmb.2017.09.014

Figure Lengend Snippet: JNK2−/− LDLR−/− and LDLR−/− mice were fed vitamin D-sufficient or deficient high fat diet for 10 weeks (n=4M/4F mice per group). Peritoneal macrophages harvested after HFD were assessed for (A) flow cytometry of membrane expression of M1 macrophage markers (CCR7 and CD86) and M2 markers (MR and CD163) and (B–D) mRNA expression of macrophage cytokines. Data are expressed as mean ± SEM, and horizontal lines demonstrate comparison between individual groups where *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Macrophage cell surface marker analysis was performed using a FACStar Plus with PE-conjugated anti-CCR7 and anti-CD86 (E-Bioscience) for M1 macrophage membrane protein expression and FITC-conjugated anti-CD163 (Bioss USA) and anti-MR (R&D Systems) for M2 macrophage membrane protein expression in unstimulated peritoneal macrophages collected from HFD-fed mice [ 28 , 39 ].

Techniques: Flow Cytometry, Expressing

Journal: American Journal of Physiology - Cell Physiology

Article Title: Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease

doi: 10.1152/ajpcell.00386.2023

Figure Lengend Snippet: Hemoglobin scavenger receptor CD163 is associated with hepatobiliary injury in sickle cell disease (SCD). A : representative images of immunofluorescence (IF) analysis of SCD patient biopsied liver samples show abundant CD163-positive cells. B , left : representative IF images (merged and single channels) of colocalization assay show higher percentage of CD163 colocalized with positive F4/80 staining of Kupffer cells in SCD mouse liver. Right : bar graph depicts the Pearson’s coefficient of colocalization in control (CON) and SCD mouse liver. C , left : representative IF images (merged and single channels) of colocalization assay show higher percentage of CD163 colocalized with positive CD11b staining of monocytes in SCD mouse liver. Right : bar graph depicts the Pearson’s coefficient of colocalization in control and SCD mouse liver. The error bars represent SD. Arrowheads indicate liver sinusoidal endothelial cells. * P < 0.05, ** P < 0.01. Scale bars, 50 µm.

Article Snippet: CD163 activity was blocked with Antigenic Blocking Peptide CD163 N-epitope (FabGennix), which was administered subcutaneously at the dose of 1 mg/kg mouse body weight or as mentioned in the text.

Techniques: Immunofluorescence, Staining, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease

doi: 10.1152/ajpcell.00386.2023

Figure Lengend Snippet: CD163 blocker competes with hemoglobin (Hb) for binding. A : schematic showing the experimental plan to analyze the effect of CD163 blocker in sickle cell disease (SCD) mouse liver. B : bar graph depicting the results of heme ELISA assay post administration of CD163 blocker in the plasma of SCD mice. C : schematic showing the experimental plan to examine the mode of action of the CD163 blocker used in blocking CD163-Hb binding. HO-1, heme oxygenase-1. D : bar graphs depicting the results of heme ELISA assay post CD163 blocker administration in the plasma of SCD mice. oxHb, oxyhemoglobin; Veh, vehicle. The error bars represent SD. * P < 0.05. Figure created with BioRender.com.

Article Snippet: CD163 activity was blocked with Antigenic Blocking Peptide CD163 N-epitope (FabGennix), which was administered subcutaneously at the dose of 1 mg/kg mouse body weight or as mentioned in the text.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Blocking Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease

doi: 10.1152/ajpcell.00386.2023

Figure Lengend Snippet: Heme oxygenase-1 (HO-1) positively regulates CD163 level in sickle cell disease (SCD) mouse liver. A and B : representative immunohistochemistry image ( A ) and colocalization analysis ( B ) show increased colocalization of CD163 and HO-1 in hepatic Kupffer cells of SCD mouse liver. CON, control. C : qRT-PCR analysis of hepatocytes and nonhepatocytes isolated from control mouse liver exhibits increased expression of both CD163 and HO-1 in the nonhepatocyte cell population. D : the representative images acquired during proximity ligation assay (PLA) show increased interactions between CD163 and HO-1 in SCD mice compared to littermate control mice. Scale bars, 50 µm. E : fluorescence intensity quantification of PLA demonstrating significantly higher intensity in the SCD group compared to control mice. FOV, field of view. F and G : series of Western blot micrographs (for the treatment pattern see ) showing the levels of HO-1 in liver lysates of control mice, untreated SCD mice, and SCD mice after treatment with iron dextran ( F ) and treatments with clodronate and hemoglobin as well as undergoing hypoxic conditions ( G ). H , left : representative Western blot micrographs showing decreased levels of CD163 protein expression after HO-1 knockdown in Hep3B human hepatoma cell line compared with untreated cells. Right : densitometric analysis of micrographs shows significantly reduced levels of CD163 protein expression after HO-1 knockdown and confirms the efficiency of performed knockdown in control and SCD mouse liver tissue. * P < 0.05; # P < 0.06–0.08.

Article Snippet: CD163 activity was blocked with Antigenic Blocking Peptide CD163 N-epitope (FabGennix), which was administered subcutaneously at the dose of 1 mg/kg mouse body weight or as mentioned in the text.

Techniques: Immunohistochemistry, Control, Quantitative RT-PCR, Isolation, Expressing, Proximity Ligation Assay, Fluorescence, Western Blot, Knockdown

Journal: American Journal of Physiology - Cell Physiology

Article Title: Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease

doi: 10.1152/ajpcell.00386.2023

Figure Lengend Snippet: The CD163 and heme oxygenase-1 (HO-1) signaling pathway in sickle cell disease (SCD)-related liver complications. Schematic diagram depicting the HO-1-CD163 interaction in SCD mouse liver. Cell-free hemoglobin (Hb) released after intravascular hemolysis is scavenged by plasma haptoglobin (HP), which chaperones it to the liver for hemoglobin scavenger receptor CD163-dependent clearance by macrophages. Cell-free Hb on its own can also bind to CD163, albeit less efficiently. In both cases, it leads to endocytosis and degradation of Hb and the release of heme. HO-1 metabolizes heme to produce carbon monoxide, iron, and biliverdin. Along with its role in heme degradation, HO-1 also regulates CD163 expression in the hepatic Kupffer cells. Loss of HO-1 in the liver reduces CD163 expression, which can further impact the Hb-heme metabolism in SCD liver. RBC, red blood cell. Figure created with BioRender.com.

Article Snippet: CD163 activity was blocked with Antigenic Blocking Peptide CD163 N-epitope (FabGennix), which was administered subcutaneously at the dose of 1 mg/kg mouse body weight or as mentioned in the text.

Techniques: Clinical Proteomics, Expressing