Journal: Journal of Biological Chemistry

Article Title: 5-Oxo-6,8,11,14-eicosatetraenoic Acid Stimulates the Release of the Eosinophil Survival Factor Granulocyte/Macrophage Colony-stimulating Factor from Monocytes

doi: 10.1074/jbc.m401537200

Figure Lengend Snippet: FIG. 5. Mediation of 5-oxo-ETE-induced eosinophil survival by monocyte-derived GM-CSF. Left, in control experiments, monocyte- depleted eosinophils were incubated for 48 h with vehicle (control (Con); open bar), 1 nM IL-5 (hatched bars), or 1 nM GM-CSF (GM; cross- hatched bars) in the presence or absence of monoclonal antibody against IL-5 (aIL5) or GM-CSF (aGM). Right, monocytes from four different donors were incubated for 24 h with either vehicle (open bar) or 1 M 5-oxo-ETE (5o; closed bars). The conditioned media (MCM) were then incubated for 60 min at 37 °C in the presence or absence of monoclonal antibody (20 g/ml) against IL-5 or GM-CSF. Aliquots (10 l) of the conditioned media were then incubated for 48 h with monocyte-depleted eosinophils from two different donors, and cell survival was determined by flow cytometry. The average percent survival for the two eosinophil preparations was calculated for each of the monocyte preparations, and n was taken to be equal to the number of monocyte donors (i.e. n 4). ***, p 0.001.

Article Snippet: The neutralizing monoclonal antibodies against recombinant human IL-5 and recombinant human GM-CSF were purchased from R&D Systems (Minneapolis, MN).

Techniques: Derivative Assay, Control, Incubation, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: 5-Oxo-6,8,11,14-eicosatetraenoic Acid Stimulates the Release of the Eosinophil Survival Factor Granulocyte/Macrophage Colony-stimulating Factor from Monocytes

doi: 10.1074/jbc.m401537200

Figure Lengend Snippet: FIG. 6. 5-Oxo-ETE induces GM-CSF release from monocytes. A, monocytes were incubated for different times at 37 °C with either vehicle (control; E) or 1 M 5-oxo-ETE (G), and the amounts of GM-CSF in the media were determined by enzyme-linked immunosorbent assay as described under “Experimental Procedures.” B, monocytes were in- cubated for 24 h at 37 °C with either vehicle or different concentrations (1 pM to 100 nM) of either 5-oxo-ETE (G) or LTB4 (E), and the amounts of GM-CSF in the media were measured. The inset shows the responses to higher concentrations (10 nM to 10 M) of 5-oxo-ETE (G) and LTB4 (E). Because of limitations in the numbers of monocytes, only a limited number of concentrations of LTB4 were tested. C, purified eosinophils from three different donors were incubated for 48 h at 37 °C with either vehicle or different concentrations (10 fM to 100 pM) of recombinant human GM-CSF, and survival was assessed following staining with FITC-labeled annexin V and propidium iodide. *, p 0.05; **, p 0.01; ***, p 0.001.

Article Snippet: The neutralizing monoclonal antibodies against recombinant human IL-5 and recombinant human GM-CSF were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Staining, Labeling