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Blood, Vol. 113, Issue 11, 2605-2613, March 12, 2009
Toll-like receptor 4 in lymphatic endothelial cells contributes to LPS-induced lymphangiogenesis by chemotactic recruitment of macrophages
Blood Kang et al.
113: 2605
Supplemental materials for: Kang et al
Isolation and culture of primary mouse LEC isolation from the peritoneal lymphangioma model
The peritoneal lymphangioma model was generated by injection of 200 µl of a 1:1 emulsion of PBS and incomplete Freund’s adjuvant into the peritoneal cavity of both HeN and HeJ mice at day 1 and 15. One month after the initial injection, white lymphangioma plaques were harvested from the peritoneal side of the diaphragm and liver surface, digested with collagenase-A for 1 hr in 32°C in a humidified 5% CO2 incubator, and then cultured and maintained in EBM2-MV medium up to passage 3 according to previous report (Methods Enzymol. 26:415–438, 2007). Assays for proliferation/migration and tube formation of LEC
For the proliferation/migration assay, ~3 × 105 of LEC were seeded in each well of a 6 well plate and cultured to ~90% confluence. A p1000 pipet blue tip (Greiner Bio-One, Frickenhausen, Germany) was used to make scrapes in each well. The cells were incubated with the indicated dose of LPS in EBM supplemented with 2% FBS for 24 hr. EBM containing 2% FBS was used as a negative control, and EBM containing 10% serum with growth supplements was used as a positive control. Photographs were taken using DpxViewPro software (Kane Computing, Northwich, Cheshire, UK) at 0 and 24 hrs after the scratch was made. The distance from the initial scrape line to the final position of the cells after migration was measured at three points of each image, and the mean value of the three points was used for quantification. For the tube formation assay, 50 µl of Growth Factor Reduced Matrigel™ (BD-Pharmingen) was loaded into each well of a 96 well plate on ice and allowed to solidify for 60 min at 37°C. LEC (2 × 104) were resuspended in 150 µl of EBM containing 2% FBS with the indicated dose of LPS, and seeded into the matrigel coated well. After 6 hr of incubation in a 37°C humidified CO2 incubator, cells were photographed using the DpxViewPro software. The total lengths of tube-like structures exceeding 25 µm were measured and quantified in each image. Immunostaining and morphometric analysis
Cultured cells were fixed with 95% methanol at −20°C for 3 min. Mice were anesthetized by intramuscular injection of a combination of anesthetics (80 mg/kg ketamine and 12 mg/kg xylazine) and the tissues were fixed with 1% paraformaldehyde in PBS at room temperature (RT) for 1 hr or with 100% acetone at −20°C for 6 hr. The samples were blocked by incubation in 5% goat serum (Jackson ImmunoResearch, West Grove, PA) in PBST (PBS containing Triton). Primary antibodies include mouse anti-human CD31 (Dako, Carpinteria, CA), rabbit anti-human Prox-1 (Reliatech, Braunschweig, Germany), mouse anti-human CD45 (BD-Pharmingen), and rabbit anti-human LYVE-1 (Upstate Biotechnology Inc., Lake Placid, NY), mouse anti-human podoplanin (Signet, Dedham, MA), rabbit anti-human TLR4 (Santa Cruz), rabbit anti-human p65 (Santa Cruz), rat anti-mouse LYVE-1 (Aprogen, Daejeon, Korea), rabbit anti-mouse PH3 (Upstate), hamster anti-mouse CD31 (Chemicon International, Temecula, CA), and rat anti-mouse CD11b antibodies (BD-Pharmingen), rabbit anti-mouse CCL2 (Santa Cruz). FITC-conjugated anti-rat, FITC-conjugated anti-rabbit, Cy3-conjugated anti-rat, Cy3-conjugated anti-rabbit and Cy3-conjugated anti-mouse antibody (all from Jackson ImmunoResearch) was used as secondary antibodies. DAPI (Molecular Probes, Eugene, OR) was used to stain nuclei. For 3,3′-diaminobenzidine (DAB) immunostaining, samples were incubated with HRP-conjugated anti-rat antibody (Jackson ImmunoResearch) or HRP-conjugated anti-rabbit antibody (Amersham, Piscataway, NJ) and developed with a DAB substrate kit (Vector, Burlingham, CA) according to the manufacturer’s instructions. The stained samples were visualized and digital images were obtained either using a Zeiss inverted microscope or a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY). Morphometric measurements and analysis were performed by ImageJ software (http://rsb.info.nih.gov/ij).
Files in this Data Supplement:
- Table S1. Primers for quantitative RT-PCR (PDF, 52.7 KB)
- Figure S1. The increased density of lymphatic vessels by LPS inejction in HeN mice results from proliferation of LEC (JPG, 49.3 KB)
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After daily intraperitoneal injections of PBS or LPS (0.5 mg/kg/day) for 7 days, diaphragms were harvested, sectioned, and immunostained with LYVE-1 (green), PH3 (red), and PECAM (blue). (A) White arrowheads indicate LYVE-1+/PH3+ LEC. Scale bars, 100 µm. (B) Numbers of LYVE-1+/PH3+ cells were counted in a given area (0.21 mm2) of the muscular region of the diaphragm. Bars represent mean ± SD (n=4–5). *, P<0.05 versus PBS.
- Figure S2. Comparison of VEGF-A–induced lymphangiogenesis in the peritoneal side of diaphragm between HeN and HeJ mice (JPG, 73.1 KB)
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(A) HeN and HeJ mice were treated intraperitoneally with PBS (P) or human VEGF-A (10 µg/day, V) for 7 days and diaphragms were immunostained for LYVE-1 (red). Representative images of LYVE-1+ lymphatic vessels and lymphatic branching (white arrows) in the peritoneal side of diaphragm muscle. Scale bars, 300 µm. (B) Densities of LYVE-1+ diaphragmatic lymphatic vessels were measured in each given area (3.64-SA–less mm2) and values were presented as a percentage per each area (n=4). Numbers of LYVE-1+ lymphatic branching exceeding 50 µm in a given area (1 mm2) were counted and presented as actual number per field (n=4). Bars represent mean ± SD. *, P<0.05 versus PBS. NS, not statistically significant between V in HeN versus V in HeJ.
- Figure S3. Morphology and expression of selective markers of primary cultured BEC and LEC (JPG, 84.4 KB)
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(A) Phase contrast images of primary cultured human BEC and LEC. Scale bars, 100 µm. (B) Double immunostaining reveals expression of LYVE-1, podoplanin, Prox-1 and CD31 in primary cultured LEC. Scale bars, 100 µm.
- Figure S4. Visualization of lymphatic vessels in κB-lacZ mice (JPG, 41.6 KB)
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After daily intraperitoneal injections of PBS or LPS (0.5 mg/kg/day) for 7 days in the κB-lacZ mice, diaphragms were harvested, whole mounted, and stained for β-gal (blue) and LYVE-1 (brown). LYVE-1+ lymphatic vessels in the peritoneal side of diaphragm show constitutive NF-κB expression in both the PBS (upper panel) and LPS-treated (lower panel) mice.
- Figure S5. LPS does not directly promote cell proliferation/migration, tube formation or expression of lymphangiogenic molecules in LEC (JPG, 104 KB)
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(A and B) Images of proliferating/migrating LEC in response to scratches were taken 0 and 24 hr after the indicated doses of LPS stimulation. Serum (S), 10% serum with growth supplements as a positive control. Scale bars, 100 µm. Migration distances from the initial scrape points were measured and quantified. Bars represent mean ± SD (n=4). *, P<0.05 versus 0 ng/ml of LPS. (C and D) Images of LEC tube formations were taken 6 hr after indicated doses of LPS stimulation. Serum (S), 10% serum with growth supplements as positive control. Scale bars, 100 µm. Number of tube-like structures exceeding 25 µm in length were quantified. Bars represent mean ± SD (n=5). *, P<0.05 versus 0 ng/ml of LPS. (E) LECs were stimulated with indicated doses of LPS for 4 hr and the mRNA levels of VEGF-C, VEGF-D, VEGFR-2, and VEGFR-3 were analyzed by quantitative RT-PCR. mRNA levels for each gene were normalized to GAPDH and presented as fold increase compared to 0 ng/ml of LPS. Bars represent mean ± SD (n=4). *, P<0.05 versus 0 ng/ml of LPS.
- Figure S6. Comparison of LPS-induced chemokine expressions in the LEC between HeN and HeJ mice (JPG, 102 KB)
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(A) Primary cultured LEC from HeN and HeJ mice were double- immunostained for Prox-1 and CD31. Scale bars, 100 µm. (B) Primary cultured LEC from HeN and HeJ mice were stimulated with LPS (500 µg/ml) for 4 hr, and the CCL2, CCL5 and CX3CL1 mRNA levels were analyzed by quantitative RT-PCR. The mRNA level of each gene were normalized to GAPDH and presented as a fold increase compared to the control. Bars represent mean ± SD (n=4). *, P<0.05 versus LPS−. (C) Representative images for CCL2 expression in the peritoneal side of diaphragm of HeN and HeJ mice. After daily intraperitoneal injections of LPS (0.5 mg/kg/day) for 7 days in the HeN and HeJ mice, diaphragms were harvested, sectioned, and immunostained for LYVE-1 (green) and CCL2 (pink), and stained nuclei with DAPI (blue). Note that CCL2 strongly expresses in the LYVE-1+ lymphatic vessels (white arrows) in the peritoneal side of diaphragm of HeN mouse, whereas almost no expression of CCL2 in the lymphatic vessels of HeJ mouse. Three independent experiments show similar findings.
- Figure S7. Schematic diagram of adoptive cell transfer experiment (JPG, 65.5 KB)
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Thioglycollate (0.3%) was injected intraperitoneally into the HeN mouse and the peritoneal macrophages were harvested 3 days later. (A) The collected macrophages were tagged with CFSE green dye and then adoptively transferred by intraperitoneal injection just after LPS (0.5 mg/kg) injection into HeN or HeJ mouse. The migratory fate of the injected macrophages was analyzed after the indicated time. (B) The collected macrophages were analyzed by flow cytometery using antibodies for F4/80, CD11b and CD11c. Numbers represent mean ± SD. *, P<0.05 versus HeN mice.
- Figure S8. Z-stack imaging analysis of transferred cells and diaphragmatic lymphatic vessels after the adoptive cell transfer (JPG, 49.5 KB)
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The diaphragms were analyzed 4 h after the intraperitoneal injection of LPS (0.5 mg/kg) and transfer of ~2 × 107 of CFSE-tagged peritoneal macrophages (green) into the peritoneal cavity. Whole diaphragms were harvested, immunostained with LYVE-1 (red), and peritoneal lymphatic vessels and injected cells (green) were imaged by Z-stack. Each cell (a, b, c, d) in the left panel was further visualized by Z-stack analysis. Each horizontal and vertical section of the image is presented as each upper and right small square.
- Figure S9. Macrophage depletion abolishes LPS-induced lymphangiogenesis in the diaphragmatic lymphatic vessels (JPG, 67.3 KB)
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Endogenous macrophages were depleted by intraperitoneal injection with CDL (50 mg/kg), or CL as control, at every 3 days from the same day of starting the PBS or LPS treatment. After 7 days of PBS or LPS (0.5 mg/kg/day) treatments, from the four groups (CL+PBS, CL+LPS, CDL+PBS, and CDL+LPS), diaphragms were harvested, whole mounted, immnostained with LYVE-1+, and the peritoneal side of lymphatic vessels were visualized by DAB. Scale bar, 300 µm. Four independent experiments show similar finding.
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