Eicosanoids in the Innate Immune Response: TLR and Non-TLR Routes

Figure 1

Distribution of $[{}_{}{}^3\text{H}]$AA label in the different lipid fractions in PMN and supernatants. PMN at a concentration of ${10}^7$ cells/ml were labelled with 0.2 $\mu$Ci of $[{}_{}{}^3\text{H}]$AA and stimulated for 1 hour in the presence of 10 $\mu$g/ml PGN or 25 mg/ml mannan, or left untreated. PMN and supernatants were subjected separately to extraction in chloroform/methanol ($1:2$, v/v), according to the Bligh and Dyer procedure. The lipids extracted into the chloroform layer were dried under ${\text{N}}_2$ stream and developed by thin layer chromatography on silica gel plates in the system n-hexane/diethyl ether/acetic acid ($70:30:1$, v/v). The radioactivity distributed in the different lipid fractions was quantitated using K/tritium imaging screens. The migration of the standards is indicated (a). Cells were incubated with calpeptin and pyrrolidine-1 prior to the addition of the stimuli and the $[{}_{}{}^3\text{H}]$AA released into the cell supernatants assayed. Data represent $\text{mean}\pm \text{SEM}$ of three experiments in duplicate. ${}^{*}P<0.05$. Reproduced with permission [10] (b). Chromatogram of a supernatant of human PMN stimulated with mannan showing the retention times of ${\text{PGE}}_2$/${\text{PGD}}_2$ and 20-OH-LT${\text{B}}_4$. Both compounds have been identified by their mass spectra (c). Fragmentation spectra in the negative ion model using MRM (multiple reaction monitoring) of the specific transitions m/z 335–195 for ${\text{LTB}}_4\text{,}$ m/z 351–195 for 20-OH-${\text{LTB}}_4\text{,}$ m/z 303–205 for arachidonic acid, m/z 351–189 for ${\text{PGE}}_2$/${\text{PGD}}_2$ are shown in (d).