1. by Jesmond Dalli, and Charles N. Serhan
Specific lipid mediator signatures of human phagocytes: microparticles stimulate macrophage efferocytosis and pro-resolving mediators
by Jesmond Dalli, and Charles N. Serhan
Blood
Volume 120(15):e60-e72
October 11, 2012
©2012 by American Society of Hematology

2. PMN MPs enhance macrophage efferocytosis of apoptotic PMN
PMN MPs enhance macrophage efferocytosis of apoptotic PMN. PMN MPs were obtained after PMN stimulation with formyl-methionyl-leucyl-phenylalanine (1μM).
PMN MPs enhance macrophage efferocytosis of apoptotic PMN. PMN MPs were obtained after PMN stimulation with formyl-methionyl-leucyl-phenylalanine (1μM). (A) The MP population (left panel) was monitored by flow cytometry using fluorescently conjugated AnxAV (right top panel) and anti-CD66b (right bottom panel). (B) Macrophages were prepared by differentiating peripheral blood monocytes in the presence of GM-CSF for 7 days. These were incubated with MP for 5 minutes before the addition of apoptotic (Apo) PMN. Uptake of the CFDA-labeled Apo PMN (3 × 105 cells/well) was monitored after 1-hour incubation (37°C) by assessing the levels of fluorescence (see “Phagocytosis”). (B) Results are expressed as mean ± SEM (n = 4 distinct cell preparations). *P < .05 vs macrophage plus PMN group. **P < .01 vs macrophage plus Apo PMN group.
Jesmond Dalli, and Charles N. Serhan Blood 2012;120:e60-e72
©2012 by American Society of Hematology

3. SPM biosynthesis during macrophage efferocytosis of apoptotic PMNs is enhanced by PMN MPs. Macrophages were incubated for 5 minutes with PMN MPs before the addition of apoptotic PMNs (7.5 × 105 cells/well).
SPM biosynthesis during macrophage efferocytosis of apoptotic PMNs is enhanced by PMN MPs. Macrophages were incubated for 5 minutes with PMN MPs before the addition of apoptotic PMNs (7.5 × 105 cells/well). Cells were incubated for 1 hour at 37°C. At the end of the incubation, ice-cold MeOH was added and the products extracted. (A) Representative MRM traces for the identified LMs. (B) Accompanying MS/MS spectra used for identification. (C) Specific bioactive lipid mediator and precursor/pathway markers where: Q1, M-H (parent ion); and Q3, diagnostic ion in the MS-MS (daughter ion) along with mean ± SEM values for each of the mediators identified. Quantification and values obtained after PMN (3 × 105 PMN) and MP (2 × 105 MP) incubations. The detection limit was ∼ 1 pg. *Below limits. (D) D-series resolvins, protectins, and maresins. (E) Lipoxins. (F) Prostaglandins and thromboxanes. (C-F) Results are expressed as mean ± SEM; n = 4 distinct cell preparations. *P < .05 vs macrophage group. **P < .01 vs macrophage group. #P < .05 vs macrophage plus Apo PMN group.
Jesmond Dalli, and Charles N. Serhan Blood 2012;120:e60-e72
©2012 by American Society of Hematology

4. Elevated SPM biosynthesis in apoptotic PMNs
Elevated SPM biosynthesis in apoptotic PMNs. PMNs were isolated from peripheral blood of healthy volunteers and placed directly in MeOH (PMN), induced to apoptose (Apo PMN), or stimulated with zymosan (PMN + Zy).
Elevated SPM biosynthesis in apoptotic PMNs. PMNs were isolated from peripheral blood of healthy volunteers and placed directly in MeOH (PMN), induced to apoptose (Apo PMN), or stimulated with zymosan (PMN + Zy). After extraction, LM profiles were obtained using LC-MS/MS (see “Sample extraction and lipid mediator metabololipidomics”). (A) Representative MRM chromatograms of the LM identified in the apoptotic or Zy-activated neutrophils. Peak heights represent the relative levels of each LM. Cumulative levels for individual LM families are depicted as a function of color intensity, where color scales (eg, white to blue [Apo PMN = ] or white to tan [Zy PMN = ]) are set from zero to 500 pg per 5 × 106 cells. (B) Accompanying MS/MS spectra used for identification. (C) Lipid mediator and precursor/pathway marker transition where: Q1, M-H (parent ion); and Q3, diagnostic ion in the MS-MS (daughter ion), along with mean ± SEM values for each of the mediators identified. Values are for 5 × 106 PMNs. The detection limit was ∼ 1 pg. *Below limits. Cumulative values: (D) Leukotriene B4 (and 20-OH LTB4 metabolite). (E) D-series resolvins, protectins, and maresins. (F) Lipoxins. (G) E-series resolvins and lipoxins. (H) Prostaglandins and thromboxanes. (C-H) Results are expressed as mean ± SEM; n = 5 distinct cell preparations. *P < .05 vs PMNs. **P < .01 vs PMNs. ***P < .01 vs PMNs. #P < .05 vs Apo PMNs.
Jesmond Dalli, and Charles N. Serhan Blood 2012;120:e60-e72
©2012 by American Society of Hematology

5. Macrophage phagocytosis of apoptotic PMNs
Macrophage phagocytosis of apoptotic PMNs. A nidus for transcellular biosynthesis.
Macrophage phagocytosis of apoptotic PMNs. A nidus for transcellular biosynthesis. MPs or PMNs were preincubated with deuterium-labeled substrate (AA, EPA, and DHA, 100 ng each) before coincubation with macrophages for 1 hour at 37°C. Incubations were stopped with ice-cold MeOH and LM extracted. The levels of deuterium-labeled LM biosynthesized during the phagocytosis of apoptotic neutrophils were assessed by LC-MS/MS. (A) Representative MRM chromatograms of the identified LM. (B) Accompanying MS/MS spectra used for identification. (C) LM and precursor/pathway marker transition Q1-Q3 transitions along with mean ± SEM values for each of the mediators identified. The detection limit was ∼ 1 pg. *Below limits of detection. Cumulative values: (D) D-series resolvins, protectins, and maresins. (E) E-series resolvins and lipoxins. (F) Lipoxins. (G) Prostaglandins and thromboxanes. (C-G) Results are ± SEM; n = 5 distinct cell preparations.
Jesmond Dalli, and Charles N. Serhan Blood 2012;120:e60-e72
©2012 by American Society of Hematology

6. Distinct macrophage subtypes biosynthesize specific lipid mediator profiles.
Distinct macrophage subtypes biosynthesize specific lipid mediator profiles. M1 and M2 macrophages were prepared from primary human monocytes after incubation with GM-CSF (20 ng/mL), IFN-γ (20 ng/mL), and LPS (100 ng/mL) to produce M1 or M-CSF (20 ng/mL) and IL-4 (20 ng/mL) to obtain M2. Incubations were stopped with ice-cold MeOH and taken for LC-MS/MS analysis (see “Sample extraction and lipid mediator metabololipidomics”). (A) Representative MRM chromatograms for the identified LM. Peak heights represent the relative levels of each mediator in the different macrophage subtypes. Cumulative levels for each lipid mediator category are represented as a function of color intensity, where color scales (eg, white to green for M1 macrophages and white to purple for M2 macrophages) are set from zero to pg per 2.5 × 106 cells. (B) Accompanying MS/MS spectra used for identification. (C) Lipid mediator and precursor/pathway marker transition along with mean ± SEM values for each of the mediators identified. The detection limit was ∼ 1 pg. *Below limits. Cumulative values: (D) D-series resolvins, protectins, and maresins. (E) Lipoxins. (F) E-series resolvins and lipoxins. (G) Prostaglandins and thromboxanes. (H) EPA-derived prostaglandins and thromboxanes. (I) Leukotriene B4 (and 20-OH LTB4 metabolite). (J) Leukotriene B5. (D-J) Results are ± SEM; n = 8 distinct cell preparations. *P < .05 vs M1 group. **P < .01 vs M1 group.
Jesmond Dalli, and Charles N. Serhan Blood 2012;120:e60-e72
©2012 by American Society of Hematology

7. PMN and macrophage characteristic lipid mediator constitution.
PMN and macrophage characteristic lipid mediator constitution. Lipid mediator levels were assessed for (A) ionophore-activated, zymosan-stimulated, apoptotic PMN and intact PMN (B) M1 and M2 macrophages. Results are representative of 4-8 separate cell preparations. For quantitative values, see Figures 3 and 5.
Jesmond Dalli, and Charles N. Serhan Blood 2012;120:e60-e72
©2012 by American Society of Hematology