(a) Induction kinetics of the mRNAs of IL-4, IL-4Rα, CCR2, and CD11b. The induction of Ccl2 peaked at 1 day after the exposure followed by the peak induction of Il4 and Il4ra. (n = 4–14 eyes/group). …
Induction kinetics of the mRNAs of IL-4, IL-4Rα, CCR2, and CD11b.
Iba1 (cyan), CCL2 (red) and CD11b (green) expressing cells accumulated in the laser treated area at 1 to 3 days. The CCL2 and CD11b expression peaked at 1 to 3 days and decreased gradually …
Bone marrow-derived cells (green) are located in the subretinal layer around the laser irradiation lesion 3 days after laser irradiation. Bone marrow-derived cells (green) expressing CD11b (cyan) …
(a) Effect of systemic administration of recombinant murine IL-4 (rIL-4) or recombinant IL-13 (rIL-13) in the inductive phase in a CNV model. (n = 7–19 eyes/group). (b) Inhibitory effect of systemic …
Requirements of IL-4/IL-4Rα in the inductive phase of CNV.
The distribution of GFP transgenic bone marrow cells (green) shows dynamic changes after laser irradiation. GFP transgenic bone marrow cells remain around the choroidal scar at 1 day after the laser …
CCR2 (cyan) expressing bone marrow-derived cells (green) are incorporated into CD31 (red) expressing vascular endothelial morphology (arrows) and were spatially distinct from monocytes lineage …
(a) Induction of Ccl2 and Flt1 in bone marrow-derived endothelial progenitor cells by murine IL-4. IL-4 stimulated bone marrow-derived EPCs induced Ccl2 and Flt1 in a dose dependent manner. This …
Induction of Ccl2 and Flt1 in bone marrow-derived EPC and retinal vascular endothelial cells by IL-4.
IL-4 exposure did not affect the induction of angiogenesis-related mRNAs of EPCs including Vegf, Kdr, Flt4, Tek, Angpt1, Selp, Ednra, Cdh5, F2r, F2rl1 after 24 hr. (RT-PCR, n = 5–8/group).
Profile of angiogenic mRNAs of bone marrow-derived EPCs after IL-4 exposure.
IL-4 exposure significantly stimulated CCL2 and VEGFR-1 protein secretion from EPC after 24 hr. (ELISA, n = 5/group). *p<0.05, **p<0.0005, ***p<0.0001. ANOVA with post hoc test.
The CCL2 and VEGFR-1 protein levels in bone marrow-derived EPCs after IL-4 exposure.
(a) IL-4-induced tube formation of retinal vascular endothelial cells. Human and murine IL-4 exposure (10 ng/ml) significantly stimulated tube formation of human and murine retinal microvascular …
IL-4-induced tube formation inEPCs and retinal vascular endothelial cells.
(a) IL-4Rα-mediated transcriptional networks associated with angiogenesis in bone marrow-derived EPCs after IL-4 treatment. Z score = 2.781, p=1.8 × 10−4. (b) IL-4Rα-mediated transcriptional …
IL-4Rα-mediated transcriptional networks of bone marrow-derived EPCs.
(a) Requirement of IL-4 in bone marrow for choroidal neovascularization (CNV). Bone marrow chimeric mice were constructed on backgrounds of wild type, Il4-/-, or Il4ra-/- mice by transfer of wild …
Requirement of IL-4 in bone marrow for CNV.
Bone marrow-derived cells (green) and endothelial cells (red) were localized in the retinal tissue of wild type and Il4ra-/- bone marrow chimeric mice on Il4ra-/- background and wild type, Il4-/- …
Cytokines (pg/ml) | Control (n = 104) | age-related macular degeneration (n = 234) | P value |
---|---|---|---|
IL-4 | 0.3 ± 0.1 | 0.9 ± 0.1 | p=0.0000 |
IL-13 | 3.8 ± 0.7 | 5.2 ± 0.7 | NS |
Two-tailed t test; Mean ± standard error of the means (SEMs).
Relative risk ratio | Relative risk ratio | ||||
---|---|---|---|---|---|
N | IL-4 (quintile) | P value | IL-13 (quintile) | P value | |
Control | 104 | - | - | - | - |
Typical AMD | 33 | 2.11 ± 0.33 | 0.000 | 2.09 ± 0.39 | 0.000 |
Polypoidal choroidal vasculopathy (PCV) | 78 | 1.70 ± 0.17 | 0.000 | 1.39 ± 0.15 | 0.002 |
Retinal angiomatous proliferation (RAP) | 11 | 2.46 ± 0.69 | 0.001 | 1.48 ± 0.37 | 0.11 |
Multinomial logistic regression analysis after age adjustment; Mean ± standard error of the means (SEMs).
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (M. musculus) | C57BL/6J (wt) | PMID:15729571 | RRID:IMSR_JAX:000664 | |
Genetic reagent (M. musculus) | C57BL/6-Tg(CAG-EGFP) | PMID:9175875 | RRID:IMSR_JAX:003291 | |
Genetic reagent (M. musculus) | C57BL/6-Il4tm1Nnt/J | PMID:8906833 | RRID:IMSR_JAX:002518 | |
Genetic reagent (M. musculus) | BALB/c-Il4ratm1Sz/J | PMID:9380721 | RRID:IMSR_JAX:003514 | |
Genetic reagent (M. musculus) | Il13ra1tm1Twy | PMID:18066066 | RRID:MGI:3772446 | Dr. Marc E Rothenberg, Cincinnati Children's Hospital Medical Center University of Cincinnati College of Medicine |
Cell line (M. musculus) | C57BL/6 Mouse Primary Retinal Microvascular Endothelial Cells | Cell Biologics | C57-6065 | |
Cell line (H. sapiens) | Primary Human Retinal Microvascular Endothelial Cells | Cell Systems | ACBRI 181 | |
Antibody | anti IL-4 (rat monoclonal) | Biolegend | Cat. #: 504108, RRID:AB_315322 | IHC(1:200) |
Antibody | anti IL-4 (rabbit polyclonal) | abcam | Cat. #: ab9622, RRID:AB_308736 | IHC(1:200) |
Antibody | anti Phospho-Tyr497 IL-4R/CD124 (rabbit polyclonal) | Assay Biotechnology Company | Cat. #: A1064, RRID:AB_10683571 | IHC(1:200) |
anti CD124 (rat monoclonal) | BD Pharmingen | Cat. #: 552288, RRID:AB_394356 | 0.1–10 ng/ mL TVI | |
Antibody | anti IL-13 receptor alpha 1 (rabbit polyclonal) | abcam | Cat. #: ab-79277, RRID:AB_1640587 | IHC(1:200) |
anti IL13 antibody (rabbit polyclonal) | GeneTex, Inc | Cat. #: GTX59763, | 0.1–10 ng/ mL TVI | |
Antibody | anti CD11b (rat monoclonal) | eBioscience | Cat. #: 14-0112-82, RRID:AB_467108 | IHC(1:200) |
Antibody | anti CD11b (rat monoclonal) Alexa Fluor 594 | Biolegend | Cat. #: 101254, RRID:AB_2563231 | IHC(1:100) |
Antibody | CD11b (M1/70) (rat monoclonal) FITC | eBioscience | Cat. #: 11-0112-41, RRID:AB_11042156 | IHC(1:100) |
Antibody | anti CCR2 (rabbit polyclonal) DyLight 550 | Novus Biologicals | Cat. #: NBP1-48338R | IHC(1:100) |
Antibody | anti MCP-1 (hamster monoclonal) | Biolegend | Cat. #: 505906, RRID:AB_2071552 | IHC(1:200) |
Antibody | anti Iba1 (rabbit polyclonal) | FUJIFILM Wako Pure Chemical Corporation | Cat. # 019–19741, RRID:AB_839504 | IHC(1:200) |
Antibody | anti CD31 (rabbit polyclomal) | abcam | Cat. #: ab28364, RRID:AB_726362 | IHC(1:200) |
Antibody | anti-mouse CD31(rat monoclonal) Alexa Fluor 647 | Biolegend | Cat. #: 102516, RRID:AB_2161029 | IHC(1:100) |
Antibody | anti rat IgG (goat polyclonal) Brilliant Violet 421 | Biolegend | Cat. #: 405414, RRID:AB_10900808 | IHC(1:100) |
Antibody | anti rabbit IgG (goat polyclonal) DyLight 488 | Vector Laboratories | Cat. #: DI-1488, RRID:AB_2336402 | IHC(1:100) |
Antibody | anti rabbit IgG (donkey polyclonal) Alexa Fluor 555 | Biolegend | Cat. #: 406412, RRID:AB_2563181 | IHC(1:100) |
Antibody | anti rabbit IgG (goat polyclonal) HiLyte Fluor 555 | AnaSpec | Cat. #: AS-61056–05 H555 | IHC(1:100) |
Antibody | anti hamster IgG (goat polyclonal) DyLight 594 | Biolegend | Cat. #: 405504, RRID:AB_1575119 | |
Antibody | anti rabbit IgG (goat polyclonal) PE | Santa Cruz Biotechnology | Cat. #: sc-3739, RRID:AB_649004 | IHC(1:100) |
Antibody | anti rabbit IgG (donkey polyclonal) Alexa Fluor 647 | abcam | Cat. #: ab150075, RRID:AB_2752244 | IHC(1:100) |
Antibody | anti rabbit IgG (donkey polyclonal) DyLight 649 | Biolegend | Cat. #: 406406, RRID:AB_1575135 | IHC(1:100) |
Antibody | anti mouse IgG2A (rat monoclonal) | R and D | Cat. #: mab006, RRID:AB_357349 | 0.1–10 ng/ mL TVI |
Antibody | anti mouse Fc gamma RII/RIII (CD32/CD16)(goat polyclonal) | R and D | Cat. #: AF1460-SP, Accession # P08101 | IHC (0.2 μg/mL) |
Peptide, recombinant protein | recombinant murine IL-4 | R and D | Cat. #: 404 ML | |
Peptide, recombinant protein | recombinant human IL-4 | Peprotec | Cat. #: AF-200–04 | |
Peptide, recombinant protein | recombinant murine IL-13 | Peprotec | Cat. #: 210–13 | |
Chemical compound, drug | ZM306416 hydrochloride | abcam | Cat. #: ab144576 | |
Chemical compound, drug | ZM323881 hydrochloride | R and D | Cat. #: 2475/1 | |
Chemical compound, drug | bovine serum albumin | Sigma-Aldrich | Cat. #: A2153 | |
Chemical compound, drug | fetal bovine serum | Sigma-Aldrich | Cat. #: 12103C | |
Chemical compound, drug | Medetomidine | Chemscene LLC | Cat. #: 86347-14-0 | |
Chemical compound, drug | Butorphanol tartrate | FUJIFILM Wako Pure Chemical Corporation | Cat. #: 58786-99-5 | |
Chemical compound, drug | Midazolam | FUJIFILM Wako Pure Chemical Corporation | Cat. #: 59467-70-8 | |
Chemical compound, drug | Tropicamide, Phenylephrine Hydrochloride | Santen Pharmaceuitical Co., Ltd. | Cat. #: 1319810Q1053 | |
Chemical compound, drug | Hydroxy methyl cellulose | SENJU Pharmaceutical Co.,Ltd | Cat.#: 131980AQ1038 | |
Chemical compound, drug | RNAlater solution | Ambion | Cat. #: AM7021 | |
Chemical compound, drug | Triton X-100 | Sigma-Aldrich | Cat. #: X100 100ml | |
Chemical compound, drug | Tween 20 | Sigma-Aldrich | Cat. #: P1379-100ml | |
Chemical compound, drug | Paraformadehyde | Electron Microscopy Science | Cat. #: 15710 | |
Commercial assay or kit | ISOLECTIN B4 Fluorescein | Vector Laboratories | Cat. #: FL-1201, RRID:AB_2314663 | IHC(1:100) |
Commercial assay or kit | ISOLECTIN B4 DyLight 594 | Vector Laboratories | Cat. #: FL-1207 | IHC(1:100) |
Commercial assay or kit | DAPI | Roche Diagnostics | Cat. #: 10 236 276 001 | 1 μg/ml |
Commercial assay or kit | TO-PRO-3 iodide | Molecular Probes, Inc | Cat. #: T-3605 | IHC(1:100) |
Commercial assay or kit | VECTASHIELDAntifade Mounting Medium | Vector Laboratories | Cat. #: H-1000, RRID:AB_2336789 | |
Commercial assay or kit | Fluorescence Mounting Medium | DAKO | Cat. #: 15710 | |
Commercial assay or kit | SurePrint G3 Mouse GE 8 × 60K Microarray | Agilent Technologies | Cat. #: AGLMO002 | |
Commercial assay or kit | QuantiTect Reverse Transcription Kit | Qiagen | Cat. #: 205311 | |
Commercial assay or kit | QuantiTect SYBR Green PCR kit | Qiagen | Cat. #: 204143 | |
Commercial assay or kit | ELISA kits | ThermoFisher Scientific | Cat. #: BMS6005, EMFLT1 | |
Commercial assay or kit | PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling | Sigma-Aldrich | Cat. #: PKH26GL-1KT | |
Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat. #: 74104 | |
Commercial assay or kit | DMEM:F12 | ThermoFisher Scientific | Cat. #: 11330057 | |
Commercial assay or kit | recombinant human GMCSF | Peprotec | Cat. #: AF-300–03 | |
Commercial assay or kit | recombinant murine GMCSF | Peprotec | Cat. #: 315–03 | |
Software, algorithm | Ingenuity Pathway Analysis, March 2020 | Qiagen | RRID:SCR_008653 | |
Software, algorithm | Adobe Photoshop CS5, Version 12.0.5 | Adobe | RRID:SCR_014199 | |
Software, algorithm | STATA 16.1 | StataCorp LLC | RRID:SCR_012763 | |
Software, algorithm | GeneSpring Software | Agilent Technologies | RRID:SCR_009196 |
Sequences of primer pairs used in quantitative reverse-transcription polymerase chain reaction.