Journal: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: A , Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin ( B ) and macrophages ( C ), CD19 ( E ), and myeloperoxidase (MPO) of the AAA wall. B , Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C , Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D , CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E , CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F , MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P , Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P , Macrophage: green, CD11b ( G )/LYVE-1 ( H )/VEGF-C ( I )/MMP-9 ( J )/TGF-β1 ( K )/IL-4 ( L )/IL-8 ( M )/MIP-1α ( N )/IFN-γ ( O )/MCP-1 ( P ): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. Q , Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. R , Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. S , Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm ( A–F ) and 10 µm ( G–S ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining

Journal: The Journal of Experimental Medicine

Article Title: Regulation of inflammatory responses by IL-17F

doi: 10.1084/jem.20071978

Figure Lengend Snippet: IL-17F is produced by IL-17–expressing T cells. (A) 293T cells transfected with pcDNA–IL-17 or pcDNA–IL-17F, or vector only, were fixed and stained with the indicated antibodies. (B) CD4 + T cells from OT-II mice were differentiated into Th1, Th2, and Th17 lineages. On day 5 of culture, CD4 + T cells were restimulated with PMA and ionomycin and stained with appropriate antibodies. (C) Splenocytes were activated with PMA and Ionomycin for 5 h, and IL-17– and IL-17F–expressing cells were assessed by intracellular staining on CD4 + or TCRγδ + gates. (D) IL-17F and IL-17F–expressing cells in CNS infiltrates of mice with EAE, and in the lamina propria and intestinal intraepithelial lymphocytes were analyzed by intracellular staining with CD4 + gating. CNS infiltrates were isolated from perfused mice on day 12 after the second immunization. Data are representative of at least two independent experiments with similar results (percentages are shown).

Article Snippet: Polyclonal antibody against IL-17F was made by PRF&L by immunizing rabbits with purified IL-17F–Ig fusion proteins.

Techniques: Produced, Expressing, Transfection, Plasmid Preparation, Staining, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Regulation of inflammatory responses by IL-17F

doi: 10.1084/jem.20071978

Figure Lengend Snippet: IL-17F induces proinflammatory cytokines and chemokines in vitro via IL-17RA, TRAF6, and Act1. (A) MEFs were treated with 100 ng/ml IL-17 and IL-17F, and RNA samples were analyzed for expression of the indicated genes using real-time PCR. (B) MEFs from WT and IL-17RA KO mice were treated with 100 ng/ml IL-17 or IL-17F. After overnight incubation, cell supernatants were subjected for ELISA. After administration of recombinant IL-17 and IL-17F in WT and IL-17RA KO mice, neutrophil numbers were counted. The results shown are averaged from three animals in each group. (C) WT and TRAF6-deficient MEFs were treated with cytokines overnight. Culture supernatants were subjected for ELISA. (D) Real-time PCR was performed using cDNAs from WT and Act1-deficient MEFs treated with the indicated cytokines. p-values were calculated between cytokine treatment and medium alone (A) or between the groups indicated by a horizontal line (B–D) using an unpaired Student's t test. The experiments were repeated two to three times with consistent results. Results are mean ± SD. **, P < 0.005.

Article Snippet: Polyclonal antibody against IL-17F was made by PRF&L by immunizing rabbits with purified IL-17F–Ig fusion proteins.

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Regulation of inflammatory responses by IL-17F

doi: 10.1084/jem.20071978

Figure Lengend Snippet: CC10–IL-17F mice develop lung inflammation and mucus hyperplasia. (A) MLE12 were treated with 100 ng/ml IL-17F for 4 h and subjected to real-time PCR analysis. (B) Schematic illustration of the construct in the IL-17F transgenic mice. hGH, human growth hormone. (C) RT-PCR detection of IL-17 and IL-17F mRNA in the lung. (D) Immunohistochemistry of lung sections from IL-17F transgenic mice. Frozen lung tissues were stained with CD4, CD8, B220, and CD11C. (E and F) H&E staining of lung sections from 5–7-mo-old IL-17F transgenic mice. High power magnification shows the accumulation of mixed inflammatory cells, macrophages, and Charcot-Leyden–like crystals. (G) PAS staining of transgenic mouse lung shows mucus overproduction. (H) RT-PCR of homogenized lung from IL-17F transgenic mice and age-matched WT. Data shown were repeated at least twice with similar results. Results are mean ± SD. Bars: (D and E) 100 μm; (F and G) 300 μm.

Article Snippet: Polyclonal antibody against IL-17F was made by PRF&L by immunizing rabbits with purified IL-17F–Ig fusion proteins.

Techniques: Real-time Polymerase Chain Reaction, Construct, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining

Journal: The Journal of Experimental Medicine

Article Title: Regulation of inflammatory responses by IL-17F

doi: 10.1084/jem.20071978

Figure Lengend Snippet: Analysis of T and B cell responses in IL-17 and IL-17F KO animals. IL-17F KO, IL-17 KO, and WT control mice were immunized with KLH in CFA (three per group). 7 d later, the mice were killed and spleens and blood were collect. (A) Splenocytes from the immunized mice were restimulated with KLH for 3 d, and cytokine expression was measured by ELISA. (B) KLH-specific antibodies were measured in the sera by ELISA. The sera were subject to a threefold serial dilution, and the antibody concentrations are shown as the mean for each group. Results are mean ± SD.

Article Snippet: Polyclonal antibody against IL-17F was made by PRF&L by immunizing rabbits with purified IL-17F–Ig fusion proteins.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Serial Dilution

Journal: The Journal of Experimental Medicine

Article Title: Regulation of inflammatory responses by IL-17F

doi: 10.1084/jem.20071978

Figure Lengend Snippet: IL-17 but not IL-17F is required for the initiation of EAE. (A) EAE was induced in IL-17F KO, IL-17 KO, and WT control mice. Data shown are a combination of two independent experiments ( n = 10 for each group). (B) Infiltrates in the CNS from the EAE mice were isolated after perfusion on day 12 after the second immunization, and CD4 + and CD11b + cells were assessed by FACS. Horizontal bars indicate mean values. Data shown represent two independent experiments with similar results. (C) Chemokine expression in the CNS from the EAE mice was measured by real-time RT-PCR. (D) Splenocytes from the EAE mice were stimulated with MOG peptide, and cytokine expression levels were measured by ELISA. Data shown represent two independent experiments with similar results. (E) WT, IL-17 KO, and IL-17F KO mice were immunized with MOG/CFA and administered with pertussis toxin on the next day. 7 d after immunization, the mice were killed and IL-17–, IL-17F–, and IFN-γ–producing cells in the spleen and draining lymph nodes were analyzed by intracellular staining. Data shown are gated on CD4 + cells and are averaged from three to four mice in each group. Results are mean ± SD. *, P < 0.05; and **, P < 0.005 using the Student's t test. n.s., not significant.

Article Snippet: Polyclonal antibody against IL-17F was made by PRF&L by immunizing rabbits with purified IL-17F–Ig fusion proteins.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Regulation of inflammatory responses by IL-17F

doi: 10.1084/jem.20071978

Figure Lengend Snippet: IL-17 KO and IL-17F KO mice exhibited differential acute and chronic allergic responses. (A) Recruitment of neutrophils by FAP. WT, IL-17 KO, IL-17F KO, and IL-17RA KO mice were challenged once intranasally with either PBS or OVA with FAP. 18 h later, BALF cells were collected and stained with Gr.1 by FACS. Data shown are a combination of two independent experiments with similar results ( n = 6–8 mice per group). (B) Expression of chemokines. Whole-lung mRNA was collected and subjected to quantitative real-time RT-PCR. Data are expressed as the fold induction relative to PBS-challenged animals. (C–E). WT, IL-17, and IL-17F KO mice were subjected to an asthma model. (C) Cellular profiles in BAL fluid upon OVA challenge. Cells were harvested from BALF, stained by May-Grünwald-Giemsa, and counted under a microscope. Eos, eosinophil; Lymp, lymphocyte; Mac, macrophage; Neu, neutrophil. Data shown represent two independent experiments with consistent results ( n = 5). (D) Major basic protein 1 (MBP-1) and eosinophil peroxidase (EPO) expression in BAL cells. Real-time RT-PCR was performed using cDNA derived from BAL cells. CD11b expression in BAL cells was evaluated by FACS. Statistical analysis was performed between the indicated group and WT. (E) Expression of type 2 cytokines in lung lymph nodes and spleen. 3 d after culture in the presence of OVA, the levels of IL-4, IL-5, and IL-13 were measured by ELISA. Data shown in B, D, and E were repeated twice with similar results. Results are mean ± SD. *, P < 0.05; and **, P < 0.01 using the Student's t test. n.s., not significant.

Article Snippet: Polyclonal antibody against IL-17F was made by PRF&L by immunizing rabbits with purified IL-17F–Ig fusion proteins.

Techniques: Staining, Expressing, Quantitative RT-PCR, Microscopy, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Regulation of inflammatory responses by IL-17F

doi: 10.1084/jem.20071978

Figure Lengend Snippet: IL-17 and IL-17F differentially regulate DSS-induced colitis. (A) Acute colitis was induced in WT, IL-17 KO, and IL-17F KO mice by oral feeding of DSS ( n = 5–8 per group). Weight loss during colitis progression is shown. (B) Mice with colitis were killed on day 8, and the middle segment of the colon was fixed, sectioned, and stained with H&E. Bar, 1 mm. (C) Clinical scores are indicated based on microscopic examination of an epithelial lesion in the colon. (D) Chemokine mRNA expression was assessed in colon tissues by real-time RT-PCR. Data were expressed as the relative abundance of Actb. The experiments were repeated at least two to three times with consistent results. Results are mean values. *, P < 0.05; and **, P < 0.005 using the Student's t test.

Article Snippet: Polyclonal antibody against IL-17F was made by PRF&L by immunizing rabbits with purified IL-17F–Ig fusion proteins.

Techniques: Staining, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: A , Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin ( B ) and macrophages ( C ), CD19 ( E ), and myeloperoxidase (MPO) of the AAA wall. B , Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C , Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D , CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E , CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F , MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P , Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P , Macrophage: green, CD11b ( G )/LYVE-1 ( H )/VEGF-C ( I )/MMP-9 ( J )/TGF-β1 ( K )/IL-4 ( L )/IL-8 ( M )/MIP-1α ( N )/IFN-γ ( O )/MCP-1 ( P ): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. Q , Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. R , Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. S , Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm ( A–F ) and 10 µm ( G–S ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining