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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
Mice. Specific pathogen-free C57BL/6J mice were purchased from The Jackson Laboratory. GFP+ mice (C57BL/6J genetic background) were a gift from Dr. Masaru Okabe (Osaka University, Japan) (1). K14-VEGF-C transgenic mice (FVB/N genetic background) were generated and maintained as previously described (2), and transferred to KAIST. Mice were bred in our pathogen-free animal facility, and male mice aged 7–8 weeks were used unless otherwise indicated. Animal care and experimental procedures were performed under approval from the Animal Care Committee of KAIST. All animals were provided water and a standard diet (PMI LabDiet; Purina Mills Inc.) ad libitum. Ear skin inflammation model. LPS (from Escherichia coli 0111:B4) and MDP were purchased from Sigma-Aldrich. Highly purified and structurally intact LTA was prepared from Staphylococcus aureus (American Type Culture Collection) as previous described (3). Mice were anesthetized by intramuscular injection of a combination of anesthetics (80 mg/kg ketamine and 12 mg/kg xylazine), then a single intradermal injection of LPS (10 µg in 10 µl PBS) or LTA+MDP (10:1 ratio, 10 µg LTA+1 µg MDP in 10 µl PBS) (4) was performed into the dorsal side of the ear using a 31-gauge syringe. For control mice, the same amount of PBS was injected in the same manner. Depletion of macrophages and blockades of VEGF ligands. For systemic depletion of macrophages, mice were given an intravenous injection of clodronate liposome (CDL, 25 mg/kg) (5, 6) through the tail vein at one day before and after the intradermal injection of LPS or LTA. As a control, the same amount of control liposome (CL) was injected in the same manner. To block VEGF-C/D, mice were treated with a single intravenous injection of 1 × 109 pfu Ad-sVEGFR3 (7) at 12 hr before the intradermal injection of LPS or LTA. As a control, the same amount of Ad-βgal was injected in the same manner. To block VEGF-A, mice were given a subcutaneous injection of VEGF-Trap (25 mg/kg) (8,9) or anti–VEGF-A blocking antibody (4 mg/kg, R&D Systems) under the back skin at 1 day before and after the intradermal injection of LPS or LTA. As a control, the same amount of dimeric human Fc portion of IgG was injected in the same manner. Histological and morphometric analyses. Mice were anesthetized by intramuscular injection of anesthetics, as described above, at the indicated times after the intradermal injection of LPS or LTA. The ears were excised from the base, fixed in 4% paraformaldehyde for 2 hr, incubated in 20% sucrose solution for 6 hr at 4°C, embedded in optimum cutting temperature compound, and frozen. Twenty-µm thick sections of the ear were cut around the inflamed region. Cervical DLN from the inflamed ear were visualized by intradermal injection of 3 µl of Evans blue dye (Sigma-Aldrich, 1.5% in PBS), collected, cleared of surrounding adipose tissue, and fixed and embedded using a modified AMeX method (10). Briefly, the DLN were fixed in acetone overnight at ¬20°C, then incubated in methyl benzoate (99%) for 30 min, xylene for 30 min at room temperature (RT), liquid paraffin for 2 hr at 60°C, and embedded in paraffin at RT. Whole DLN were cut serially at 10-µm thickness. The central mid-sections of each DLN were chosen for immunostaining. Sections were incubated for 1 hr at RT with blocking solution containing 5% goat serum (Jackson ImmunoResearch) in PBST (0.3% Triton X-100 in PBS). After blocking, the sections were incubated overnight at 4°C with one or more of the following primary antibodies: (a) for lymphatic vessels, rabbit anti LYVE-1 antibody, 1:1,000 (Chemicon International) or rat anti-mouse LYVE-1 antibody, clone Han-1, 1:1,000 (Aprogen, Daejeon, Korea); (b) for blood vessels, hamster anti¬PECAM-1 antibody, clone 2H8, 1:1,000 (Chemicon International); (c) for monocytes/macrophages, rat anti-mouse CD11b antibody, clone M1/70, 1:1,000 (BD Pharmingen); (d) for dividing cells, rabbit polyclonal anti-PH3 antibody, 1:500 (Upstate Biotechnology Inc.). After several washes in PBST, sections were incubated for 2 hr at RT with one or more secondary antibodies: (a) FITC-conjugated anti-rabbit or anti-rat antibody (diluted 1:500; Jackson ImmunoResearch); (b) Cy3-conjugated anti-hamster or anti-rat IgG antibody (diluted 1:500; Jackson ImmunoResearch). DAPI (diluted 1:1,000, 1.0%; Sigma-Aldrich) was used for nuclear staining. Fluorescent signals were visualized and digital images were obtained using a Zeiss LSM 510 confocal microscope equipped with argon and helium–neon lasers (Carl Zeiss, Germany) or using a Zeiss ApoTome microscope coupled to a monochrome charge-coupled device camera (AxioVision, Carl Zeiss). Morphometric measurements of lymphatic vessels in ear skin and DLN were made from immunostained tissue sections by photographic analysis using ImageJ software (http://rsb.info.nih.gov/ij) after converting the images into 8-bit gray scale. Measurements of the densities of the lymphatic and blood vessels in ear skin were made at a screen magnification of 200×, each 1.0 mm2 in area, whereas those in DLN were made in total sectioned area (0.3–1.5 mm2), and 4 to 5 mice were used per group. A lymphatic sprout is defined as a longitudinal process that is greater than 5 µm in length and emanates from the main lymphatic vessels. The number of sprouts in the lymphatic vessels of skin and DLN was counted, each 1.0 mm2 in area, and 4 to 5 mice were used per group. To exclude background fluorescence, only pixels over a certain level (>50 intensity value) were taken. In some instances, the sectioned tissues were stained with H&E using standard methods. Flow cytometric analysis of CD11b+ macrophages from ear skin and DLN. After anesthesia, the ears and DLN were harvested and dissected into small pieces by a micro-scissor, and the pieces were incubated with 2 ml of Hank’s balanced salt solution (Sigma-Aldrich) containing 0.2% collagenase type-II (Worthington) for 1 hr at 37°C. After inactivation of collagenase activity with bovine serum, the cell suspension was filtered through a 70-µm nylon filter (BD Bioscience), and centrifuged at 400g for 5 min. The filtered cells were washed with a FACS buffer (0.5% BSA and 0.01M EDTA in PBS), and the washed cells were stained with one or more of the following fluorescent conjugated antibodies for 30 min at 4°C: perCP-cy5.5-conjugated rat anti-mouse CD11b, FITC-conjugated rat anti-mouse Gr-1, and PE-conjugated rat anti-mouse F4/80 (BD Biosciences). Then the cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences) using Cell Quest software. A total of ~100,000 cells were counted per sample. Data was analyzed by using FlowJo software (BD Biosciences). Enrichment of CD11b+ cells from DLN by MACS. At 3 days after the intradermal injection of LPS or LTA, single cell suspensions of DLN were made by incubation with collagenase type-II, and the suspended cells were washed with MACS buffer (Miltenyi Biotec). CD11b+ macrophages in the cells were enriched by using anti-mouse CD11b antibody-coupled MicroBeads (Miltenyi Biotec) and a Magnetic Cell Sorter (MACS, Miltenyi Biotec) according to the manufacturer’s instructions. The purity of subpopulations of the enriched CD11b+ macrophages (~5,000 cells) was ~95% according to the FACS analysis. Assessment of inflammation in the ear skin. Ear thickness was measured by a Flat Anvil type caliper (Cat# 7301, Mitutoyo Corporation, Kawasaki, Japan). Swelling and erythema were graded by severities according to Table S1 and S2. RT-PCR. Total RNA from ear skin, whole DLN, and MACS-enriched CD11b+ cells from the DLN were extracted by using Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer’s instructions. Each cDNA was made with reverse transcription system (Promega), and semi-quantitative RT-PCR was performed with appropriate primers (Table S3) and cycles. Quantitative real-time RT¬PCR was performed with the SYBR Premix Ex Taq™ (Takara, Japan) using the iCycler iQ5 Real-time PCR system (Bio-Rad, Hercules, CA). PCR reactions were performed with the appropriate primers (Supplemental Table 3) for 40 cycles. Monitoring lymph flow. After anesthesia, the skin over the the DLN region was carefully dissected for clear imaging of the DLN. Two intravital imaging methods were applied to monitor lymph flow from ear skin to cervical DLN. Firstly, 3 µl of FITC-conjugated dextran (MW 2,000,000, Sigma-Aldrich) was intradermally injected at the inflammatory site at day 3 after the LPS or LTA injection. The flow of the fluorescent FITC-dextran from the ear to DLN was monitored at 2, 5, 10, 20, and 30 min after the injection using the IVIS imaging system (Xenogen). Fluorescence intensity was quantified in the DLN at each time point using Living Image software (Xenogen) and expressed as radiance (photon/sec/cm2/steradian). Secondly, at 30 min after the FITC-dextran injection, the DLN were photographed and quantified by a stereomicroscope connected to a fluorescent lamp using a FITC filter (Stemi SV-6, Carl Zeiss). Assay for inflammatory cell migration from the inflammation site to DLN. GFP+ mice were intraperitoneally injected with 1 ml of 3.0% thioglycolate (Sigma-Aldrich) in saline, and inflammatory cells were collected day 3 later by peritoneal washing with ice cold DMEM culture medium. The cells were washed in ice cold DPBS and counted. Approximately, 106 of GFP+ inflammatory cells in 10 µl of PBS were adaptively transferred by injection at the inflamed site of the ear. At 12 hr later, DLN were collected, sectioned for immunohistology, and a cell suspension was made by collagenase digestion for the flow cytometric analysis. Monitoring antigen clearance. FITC-labeling of LPS was performed according to the manufacturer’s protocol (Molecular Probe). After anesthesia, a single intradermal injection of FITC-LPS (10 µg/ear in 10 µl PBS) was made into the ear in the mice treated with the Ad-sVEGFR3, Ad-βgal, VEGF-Trap, or dimeric-Fc. The fluorescent intensity of FITC-LPS in the ear was measured using the IVIS imaging system (Xenogen) and quantified using Living Image software (Xenogen) at days 0, 1, and 3 after the injection. At day 3 after the injection, the ears were harvested, and histological analyses were performed to detect the FITC-LPS remaining at the injection sites. Statistics. Values presented are means ± standard deviation (SD). Significant differences between means were determined by Student’s t-test or analysis of variance
followed by the Student-Newman-Keuls test. Statistical significance was set at P<0.05. REFERENCES 1. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. 'Green mice' as a
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3. Kim HJ, Yang JS, Woo SS, et al. Lipoteichoic acid and muramyl dipeptide synergistically induce maturation of human dendritic cells and concurrent expression of proinflammatory cytokines. J Leukoc Biol. 2007;81:983–989.
4. Han SH, Kim JH, Martin M, Michalek SM, Naam MH. Pneumoccal Lipoteichoic acid (LTA) is not as potent as Staphylococcal LTA in stimulating Tool like receptor-2. Infec Immun. 2003;71:5541–5548.
5. Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA. Clodronate-liposome–mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach. Br J Cancer. 2006;95:272–281.
6. Cho CH, Koh YJ, Han J, et al. Angiogenic role of LYVE-1–positive macrophages in adipose tissue. Circ Res.2007;100:e47–57.
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8. Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A. 2002;99:11393–11398.
9. Jeon BH, Jang C, Han J, et al. Profound but dysfunctional lymphangiogenesis via vascular endothelial growth factor ligands from CD11b+ macrophages in advanced ovarian cancer. Cancer Res. 2008;68:1100–1109.
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Files in this Data Supplement:
- Table S1. Grades of swelling and erythema in C57BL/6J mice (PDF, 16.7 KB)
- Table S2. Grades of swelling and erythema in FVB/N mice (PDF, 16.7 KB)
- Table S3. PCR primers (PDF, 32.6 KB)
- Figure S1. Generation of ear skin acute inflammation model, and tracking and analysis of DLN (JPG, 101 KB)
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(A) LPS (10 µg in 10 µl PBS) or LTA (LTA+MDP; 10:1 ratio, 10 µg LTA+1 µg MDP in 10 µl PBS) is injected intradermally into the dorsal side of the ear. As a control, the same amount of PBS is injected in the same manner. (B) Ear skin DLN are tracked by intradermal injection of 3 µl of Evans blue dye. (C) Lymphatic (red) and blood (green) vessels are visualized in the central mid-section of DNL with immunostaining for LYVE-1 and CD31 after fixation by the AMeX method. Scale bar, 200 µm.
- Figure S2. Intradermal LPS or LTA increase lymphatic and blood vessel densities, lymphatic sprouting, thickness, and recruitment of CD11b+/Gr-1+ macrophages (JPG, 112 KB)
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At day 3 after intradermal injection of PBS, LPS, or LTA, the inflamed ears were excised and sectioned for histological analysis or digested for flow cytometry. (A) Tissue sections were co-immunostained for LYVE-1 (for lymphatic vessels, green), CD31 (for blood vessels, red), CD11b (macrophages, red), or DAPI (for nuclei, blue) and merged. Note that LPS and LTA cause greater ear skin lymphatic and blood vessel densities, lymphatic sprouts (yellow arrows), skin thickness, and recruitment of CD11b+ macrophages compared to PBS. Scale bars, 50 µm. (B) Flow cytometric analysis of CD11b+/Gr-1+ macrophages in the ear skins. (C–F) Quantification of lymphatic (LV) and blood (BV) densities (arbitrary unit, %), number of lymphatic sprouts, ear thickness, and number of CD11b+/Gr-1+ cells in the gated area (No/GA) are shown. All bars represent mean ± SD from 4–5 mice. *, P<0.05 versus PBS.
- Figure S3. Intradermal LPS or LTA increase lymphatic vessel densities and sprouts, and recruitment of CD11b+/Gr-1+/F4/80+ macrophages in the DLN (JPG, 133 KB)
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At day 3 after intradermal injection of PBS, LPS, or LTA into the ear, the DLN were sampled and sectioned for histological analysis or digested for flow cytometry. (A) Tissue sections were co-immunostained for LYVE-1, CD31, or CD11b, and merged. Note that LPS and LTA cause increased lymphatic vessel densities and sprouts (yellow arrows), and increased CD11b+ macrophage infiltration compared to PBS. Scale bars, 50 µm. (B) Flow cytometric analysis of CD11b+/Gr-1+ macrophages in the DLN. Note that ~74% of CD11b+/Gr-1+ cells are F4/80+ macrophages. HE staining shows that macrophages with typical indented nuclei are abundant in the pericapsular region of DLN treated with LPS or LTA. (C–E) Quantification analyses of lymphatic (LV) and blood vessel (BV) densities (%), number of lymphatic sprouts, and number of CD11b+/Gr-1+ cells in the gated area (No/GA) are shown. All bars represent mean ± SD from 4–5 mice. *, P<0.05 versus PBS.
- Figure S4. Intradermal LPS or LTA increase proliferation of LEC in inflamed skin and DLN (JPG, 71 KB)
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At day 3 after intradermal injection of PBS, LPS, or LTA, the ear and DLN were sampled and sectioned for histological analysis. Tissue sections were co-immunostained for LYVE-1 and PH3 and merged. Note that LPS and LTA increase the number of LYVE-1+/PH3+ cells (white arrows) compared to PBS. Scale bars, 50 µm.
- Figure S5. Depletion of macrophages delays inflammation resolution in the LPS- or LTA-induced inflamed skin (JPG, 92.9 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 control, CL (25 mg/kg) (LPS+CL, LTA+CL) or PBS (P) only was given in the same manner. At 6 days after the intradermal injection of LPS or LTA, the ears were photographed (A), the severities of swelling (B) and erythema (C) were determined, and the ears were sectioned for HE staining (D). Scale bars, 200 µm. All bars represent mean ± SD from 4–5 mice. *, P<0.05 versus P; #, P<0.05 versus LPS+CL or LTA+CL.
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