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Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration  Julia Esser, BS, Ulf.

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Presentation on theme: "Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration  Julia Esser, BS, Ulf."— Presentation transcript:

1 Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration  Julia Esser, BS, Ulf Gehrmann, MS, Fabio Luiz D'Alexandri, PhD, Alicia M. Hidalgo-Estévez, PhD, Craig E. Wheelock, PhD, Annika Scheynius, PhD, Susanne Gabrielsson, PhD, Olof Rådmark, PhD  Journal of Allergy and Clinical Immunology  Volume 126, Issue 5, Pages e4 (November 2010) DOI: /j.jaci Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Cell surface proteins and exosome formation of 4 APC subtypes. Surface marker proteins on APCs (A and B) and exosomes (B) assessed by flow cytometry. Results are for the same healthy blood donors (n = 4). C, Exosome secretion is decreased in TGF-β1–MDMs versus MDMs. Amounts of exosomal protein per 106 cells are shown (n = 9). All data are means ± SEMs. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 1 Cell surface proteins and exosome formation of 4 APC subtypes. Surface marker proteins on APCs (A and B) and exosomes (B) assessed by flow cytometry. Results are for the same healthy blood donors (n = 4). C, Exosome secretion is decreased in TGF-β1–MDMs versus MDMs. Amounts of exosomal protein per 106 cells are shown (n = 9). All data are means ± SEMs. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 2 Enzymes of the LT pathway are present in APC derived exosomes. WBs for LTC4 synthase, 50 μg total protein in exosome and cell samples; lane 1, purified recombinant LTC4 synthase (100 ng) (A). LTA4H, FLAP and HLA-DR, 50 μg total protein in all samples (B). 5-LO, HLA-DR, FLAP, and LTA4H, 25 μg total protein in all samples (C). For all proteins, similar results were obtained in at least three independent experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 3 APC exosome markers and LT pathway proteins in exosome-enriched sucrose fractions; immunoelectron microscopy for LTC4S in exosomes. Flow cytometry for HLA-DR and CD81 (A) and WBs for LTA4H, 5-LO, and FLAP (B) and LTC4S (C) in fractionated MDM vesicles. Results are representative of 3 separate experiments. Immunoelectron microscopy for LTC4S in exosomes (D); arrows, gold-labeled antibodies (black dots); bars, 200 nm. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 4 APCs and their exosomes convert LTA4 to LTC4 and LTB4; TGFβ1 up-regulates LTB4 production. LTA4 conversions in APCs (A) and their exosomes (B); Mean ± SEM, n=5 donors of cells and n=3 pooled exosomes (each pool from 3 individuals); LT formation in co-incubations of MDM exosomes (50 μg) and PMNL (1×106 cells) stimulated with AA and A23187 (C); Mean ± SEM (n=7). ND = non detectable. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 5 Migration of PMNLs to MDM and MDDC–derived exosomes. Suspensions of 1 × 105 PMNLs were added on top of the chemotaxis plate filter and allowed to migrate for 1.5 hour to PGC buffer controls or exosome suspensions (in the wells), without or with AA (40 μmol/L) and A23187 (2.5 μmol/L). Numbers of migrated cells presented as means ± SEMs (n = 6). Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 6 Plasma exosomes contain active LTA4H and LTC4S. WB analyses of LTA4H, LTC4S, FLAP, 5-LO, and β-actin (100 μg total protein; A) and LTA4 conversions in plasma exosomes (500 μg protein used for incubations) from healthy individuals (B); mean ± SEM (n = 3 [LTB4], n = 2 [LTC4]). Inserted small picture (in B): representative chromatogram for the LTC4 analysis. PGB2, prostaglandin B2. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Expression of LTA4H and 5-LO in APCs
Expression of LTA4H and 5-LO in APCs. LTA4H mRNA levels (relative to β-actin) in MDMs and MDDCs (± TGF-β1; A; mean ± SEM (n=4). WB analysis of LTA4H and 5-LO in cell lysates from APCs (± TGF-β1; 25 μg protein; B; lower band in 5-LO WB corresponds to 5-LO. Representative results of 4 independent experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Biosynthesis of 5-LO and 15-LO products in APCs depends on the blood donor and is regulated by TGF-β1. Data from incubations of APCs with AA and A Samples were analyzed by HPLC for 5-HETE (A), LTB4(B), 15-HETE (C), and 12-HETE (D); data points for MDMs and MDDCs (±TGF-β1) derived in parallel from 7 donors. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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