Eicosanoids in the innate immune response: TLR and non-TLR routes

Abstract

The variable array of pattern receptor expression in different cells of the innate immune system explains the induction of distinct patterns of arachidonic acid (AA) metabolism. Peptidoglycan and mannan were strong stimuli in neutrophils, whereas the fungal extract zymosan was the most potent stimulus in monocyte-derived dendritic cells since it induced the production of PGE(2), PGD(2), and several cytokines including a robust IL-10 response. Zymosan activated kappaB-binding activity, but inhibition of NF-kappaB was associated with enhanced IL-10 production. In contrast, treatments acting on CREB (CRE binding protein), including PGE(2), showed a direct correlation between CREB activation and IL-10 production. Therefore, in dendritic cells zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE(2), thus unraveling a functional cooperation between eicosanoid production and cytokine production.

Figure 1

Distribution of [³H]AA label in the different lipid fractions in PMN and supernatants. PMN at a concentration of 10⁷ cells/ml were labelled with 0.2 μCi of [³H]AA and stimulated for 1 hour in the presence of 10 μg/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 N₂ 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 [³H]AA released into the cell supernatants assayed. Data represent mean ± 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 PGE₂/PGD₂ and 20-OH-LTB₄. 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 LTB₄, m/z 351–195 for 20-OH-LTB₄, m/z 303–205 for arachidonic acid, m/z 351–189 for PGE₂/PGD₂ are shown in (d).

Figure 2

Sequence of the 5′-UTR of COX-2. The sequence and the predicted secondary structure of the 5′-UTR of COX-2 calculated using RNAfold software are shown. Polypyrimidine tracts are underlined in the sequence (a). Agarose gel electrophoresis of PCR reactions in RNA obtained from resting PMN using different combinations of primers (b). Stick diagrams show the location of primers used for PCR reactions, and the lanes are marked according to the combination of primers selected to carry out the reactions. FP indicates forward primer, RP indicates reverse primer. Reproduced with permission [19].

Figure 3

Cells and receptors involved in AA metabolism in the innate immune system. PMN respond to cooperative binding of dectin-1 and CR3, to the cross-linking of FcγR, to mannose-based PAMP, and to G-protein-coupled receptors (GPCR) in the presence of priming agents. The receptor involved in the response to PGN has not been characterized as yet. Macrophages express a similar array of receptors showing productive binding. In DC the most efficient response is obtained by triggering dectin-1 and DC-SIGN by PAMP containing β-glucan and mannose polymers, respectively.

Figure 4

Coimmunoprecipitation of dectin-1 and DC-SIGN. DC were incubated in the presence and absence of 1 mg/ml zymosan for 10 minutes and then the cell lysates were used for immunoprecipitation with either 5 μg anti-DC-SIGN or 5 μg anti-glutathione S-transferase (GST) control mAb and blotting with anti-DC-SIGN, anti-dectin-1, or anti-GST Ab (a). HEK293 cells were transfected with the indicated expression vectors and the cell lysates immunoprecipitated with either 2 μg anti-DC-SIGN or 0.5 μg anti-Myc mAb (b). Upper panels show the expression of the Myc tag and DC-SIGN in the immunoprecipitates. The lower panels show the expression of the proteins in the cell lysates. Cells transfected with pEF4 empty vector were used where no expression vector is indicated. Immunoprecipitations have been conducted with 2 mg protein per condition. The lanes corresponding to cell lysates were loaded with 40 μg protein. Reproduced with permission [53].

Figure 5

DC-SIGN and dectin-1 cluster around zymosan particles. DC at a concentration of 5 · 10⁵ cells/ml were layered over glass coverslips and incubated with Alexa-Fluor 488 (green) labeled zymosan particles (5 particles per cell) for different times at 37°C. Anti-DC-SIGN mAb and goat anti-mouse IgG Ab labelled with Alexa-Fluor 594 (red) were used for staining DC-SIGN. Note that upon zymosan stimulation, DC-SIGN clusters around zymosan, but not around engulfed particles inside the cell (arrowheads) (a). DC were treated with anti-dectin-1 mAb and goat anti-mouse IgG Ab labelled with Alexa-Fluor 594 (b). Cells incubated in the absence of zymosan particles were stained for DC-SIGN and dectin-1 (c, d). Cells incubated in the presence of zymosan particles were assayed for CD45 staining at the times indicated (e). Reproduced with permission [53].

Figure 6

Diagram of AA metabolism in DC stimulated with zymosan particles. The mannan and β-glucan components of zymosan are recognized by at least DC-SIGN, TLR2, and dectin-1. This gives rise to a series of signaling events implicating activation of Syk and Src families of tyrosine kinases. Both routes converge to activate phospholipase Cγ and through the generation of diacylglycerol activate protein kinase C and mitogen-activated protein kinase (MAPK) cascades. Phosphorylation by MAPK and Ca²⁺-driven translocation of cPLA₂ explain AA release from cell phospholipids. Activation of IκB kinases via MyD88 is a major factor explaining COX-2 induction. The CARD9/MALT1/BCL10/TRAF6 complex is involved in the activation of IκB kinases. DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PLCγ, phospholipase Cγ; P-Y, phosphotyrosine.