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Blood, Vol. 113, Issue 22, 5650-5659, May 28, 2009
Critical role of CD11b+ macrophages and VEGF in inflammatory lymphangiogenesis, antigen clearance, and inflammation resolution
Blood Kataru et al.
113: 5650
Supplemental materials for: Koh et al
Files in this Data Supplement:
- Figure S6. Paracrine lymphangiogenic role of macrophages in LPS-and LTA-induced dermal and DLN lymphangiogenesis (JPG, 92.8 KB)
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CDL (25 mg/kg) was given intravenously to deplete macrophages at one day before and after the intradermal injection of LPS (LPS+CDL) or LTA (LTA+CDL). As a control, CL (25 mg/kg) (LPS+CL, LTA+CL) or PBS (P) only was given in the same manner. At day 3 after intradermal injection of PBS, LPS, or LTA, the ear and DLN were sampled, and CD11b+ cells from the DLN were enriched. RT-PCR comparisons of VEGF ligands in the ear skin and DLN (A), and the enriched CD11b+ macrophages from the DLN (B). Solid line, VEGF-A164; dotted line, VEGF-A120. (C and D) Densitometric analyses are presented as the relative fold to PBS after normalization with β-actin; PBS is arbitrarily regarded as 1. Bars represent the mean ± SD from 3 experiments. *, P<0.05 versus PBS; #, P<0.05 versus LPS+CL or LTA+CL.
- Figure S7. Paracrine lymphangiogenic role of macrophages in LPS-and LTA-induced dermal and DLN lymphangiogenesis (JPG, 29.7 KB)
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At day 3 after intradermal injection of PBS, LPS, or LTA, the ear and DLN were sampled, and CD11b+ cells from the DLN were enriched. Quantitative real-time RT-PCR of VEGF ligands were performed in the ear skin, DLN and the enriched CD11b+ macrophages from the DLN. Data are presented as the relative fold to PBS after normalization with GAPDH. Bars represent the mean ± SD from 3 experiments. *, P<0.05 versus PBS; #, P<0.05 versus LPS+CL or LTA+CL.
- Figure S8. Blockade of VEGF-C/D or VEGF-A delays inflammation resolution in LPS-induced inflamed skin (JPG, 97 KB)
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1 × 109 pfu Ad-sVEGFR3 (VR3) or Ad-βgal (β-gal) was injected at 12 hr before the intradermal injection of LPS into ear skin. VEGF-Trap (25 mg/kg, VT) or dimeric-Fc (Fc, 25 mg/kg) was given one day before and after the intradermal injection of LPS. As a control, PBS (P) only was given in the same manner. At day 6, the mice were anaesthetized, their ears were photographed (A), the severities of swelling (B) and erythema (C) were determined, and the ears were sectioned for HE staining (D). Pink arrows indicate delayed inflammation resolution. Scale bars, 200 µm. All bars represent mean ± SD from 5 mice. *, P<0.05 versus P; #, P<0.05 versus LPS+β-gal or LPS+Fc.
- Figure S9. Blockade of VEGF-A profoundly attenuates the LPS- and LTA-induced lymphatic vessel densities in the inflamed ear skin and DLN, and lymphatic flow and mobilization of inflammatory cells from the inflamed skin to DLN (JPG, 141 KB)
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Anti–VEGF-A antibody (Anti–VEGF-A or AV, 4 mg/kg), dimeric-Fc (Fc, 4 mg/kg) or none was treated at one day before and after the intradermal injection of PBS (P), LPS, or LTA. (A) At day 3 after the injection, the inflamed ears and DLN were sectioned for histological analysis. Tissue sections were co-immunostained for LYVE-1 and CD31. Scale bars, 50 µm. (B and C) Lymphatic (LV) and blood vessel (BV) densities in the inflamed skin (B) and DLN (C) were quantified and presented as %. (D and E) Three days later, 3 µl of FITC-conjugated dextran was intradermally injected into the inflamed skin. At 30 min later, fluorescence intensities in DLN (pink arrows) were determined with the IVIS (upper panels) and fluorescence stereomicroscope (lower panels), and quantified and presented as relative radiance (photons/sec/cm2/ steradian). (F) At day 3 after intradermal injection of LPS or LTA into the ear, the GFP+ inflammatory cells (~106 cells) were injected intradermally into the inflamed skin. At 12 hr later, the DLN were sectioned and immunostained for LYVE-1. Scale bars, 50 µm. (G) The GFP+ inflammatory cells in the section of DLN are quantified and presented as AU. All bars represent mean ± SD from 4 mice. #, P<0.05 versus LPS+Fc or LTA+Fc.
- Figure S10. Blockade of VEGF-A with anti–VEGF-A antibody delays inflammation resolution in LPS-induced inflamed skin (JPG, 63.7 KB)
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Anti–VEGF-A antibody (AV, 4 mg/kg) or dimeric-Fc (Fc, 4 mg/kg) was given one day before and after the intradermal injection of LPS. As a control, PBS (P) only was given in the same manner. At day 6, the mice were anaesthetized, their ears were photographed (A), the severities of swelling (B) and erythema (C) were determined, and the ears were sectioned for HE staining (D). Pink arrows indicate delayed inflammation resolution. Scale bars, 200 µm. All bars represent mean ± SD from 4 mice. *, P<0.05 versus P; #, P<0.05 versus LPS+Fc.
- Figure S11. Blockade of VEGF-C/D or VEGF-A profoundly attenuates the LPS- and LTA-induced mobilization of inflammatory cells from the inflammation site of ear skin to DLN (JPG, 129 KB)
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1 × 109 pfu AdsVEGFR-3, Adβ-gal or none was injected at 12 hr before the intradermal injection of PBS, LPS, or LTA. VEGF-Trap (25 mg/kg), dimeric-Fc (Fc, 25 mg/kg) or none was given one day before and after the intradermal injection of PBS, LPS, or LTA. At day 3 after injection of LPS or LTA into the ear, the DLN were sampled for flow cytometry. Flow cytometric analyses of CD11b+/Gr-1+ macrophages in the DLN are shown. Three independent experiments show similar results.
- Figure S12. Blockade of VEGF-A with anti–VEGF-A antibody delays FITC-LPS from the inflammation site to DLN in the inflamed skin (JPG, 84.9 KB)
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Anti–VEGF-A antibody (anti–VEGF-A, 4 mg/kg) or dimeric-Fc (Fc, 4 mg/kg) was given one day before and after FITC-LPS into ear skin. (A and B) At days 0, 1, and 3 after the injection of FITC-LPS, the remaining FITC-LPS was quantified with IVIS and presented as relative radiance (photons/sec/cm2/steradian). All dots represent mean ± SD from 4 mice. *, P<0.05 versus Fc. (C) At day 3 after the intradermal injection of FITC-LPS, the ears were sectioned and immunostained for LYVE-1, and merged. Scale bars, 50 µm. Four independent experiments show similar results.
- Figure S13. K14-VEGF-C transgenic mice display greater inflammatory cell migration and faster resolution of inflammation regardless macrophage depletion (JPG, 109 KB)
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(A–D) PBS (P), CDL (25 mg/kg, CD) or CL (25 mg/kg, C) was given intravenously to K14-VEGF-C mice twice at day 1 and 3. At day 4, 3 µl of FITC-conjugated dextran was intradermally injected into the inflamed skin. Fluorescence intensities were captured in DLN at 30 min after the injection using IVIS (A), and quantified and presented as radiance (photon/sec/cm2/steradian) (B). The GFP+ inflammatory cells (~106 cells) were injected intradermally into the ear skin. At 12 hr later, the DLN were sectioned and immunostained for LYVE-1 (C). Scale bars, 50 µm. The GFP+ inflammatory cells in the DLN were quantified and presented as AU (D). (E and F) PBS (P), CDL (25 mg/kg, CD) or CL (25 mg/kg, C) was given intravenously at one day before and after the intradermal injection of LPS. At 6 days after the intradermal injection of LPS or LTA, the ears were photographed (E), and the severities of erythema and swelling were scored. All bars represent mean ± SD from 4–5 mice.
- Figure S14. Role of macrophages in lymphangiogenesis and resolution of inflammation during bacterial pathogen-induced acute inflammation (JPG, 98.2 KB)
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When bacterial pathogens invade the dermis of the skin, residential and circulating monocyte-driven macrophages are activated, and cytokines and lymphangiogenic growth factors are secreted. These macrophages and lymphangiogenic growth factors in the inflamed tissue and DLN actively participate in lymphatic vessel expansion, antigen clearance, and resolution of inflammation by enhancing lymphangiogenesis, lymph flow, and mobilization of inflammatory cells from the inflamed tissue to DLN. Of these, a subset of CD11b+/Gr-1+/F4/80+ macrophages, and the lymphangiogenic growth factors VEGF-C, -D and -A, could be key components in promoting antigen clearance and resolution of inflammation during bacterial pathogen-induced acute inflammation.
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