Specificity of eicosanoid production depends on the TLR-4-stimulated macrophage phenotype - PubMed (original) (raw)

Specificity of eicosanoid production depends on the TLR-4-stimulated macrophage phenotype

Paul C Norris et al. J Leukoc Biol. 2011 Sep.

Abstract

Eicosanoid metabolism differs in profile and quantity between macrophages of different tissue origin and method of elicitation, as well as between primary and immortalized macrophages after activation with inflammatory stimuli. Using a lipidomic approach, we comprehensively analyzed the eicosanoids made by murine RPMs, TGEMs, BMDM, and the macrophage-like cell line RAW after stimulation with the TLR-4-specific agonist KLA. Direct correlation among total COX metabolites, COX side-products (11-HETE, 15-HETE), COX-2 mRNA, and protein at 8 h was found when comparing each cell type. Comprehensive qPCR analysis was used to compare relative transcript levels between the terminal prostanoid synthases themselves as well as between each cell type. Levels of PGE(2), PGD(2), and TxB(2) generally correlated with enzyme transcript expression of PGES, PGDS, and TBXS, providing evidence of comparable enzyme activities. PGIS transcript was expressed only in RPM and TGEM macrophages and at an exceptionally low level, despite high metabolite production compared with other synthases. Presence of PGIS in RPM and TGEM also lowered the production of PGE(2) versus PGD(2) by approximately tenfold relative to BMDM and RAW cells, which lacked this enzyme. Our results demonstrate that delayed PG production depends on the maximal level of COX-2 expression in different macrophages after TLR-4 stimulation. Also, the same enzymes in each cell largely dictate the profile of eicosanoids produced depending on the ratios of expression between them, with the exception of PGIS, which appears to have much greater synthetic capacity and competes selectively with mPGES-1.

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Figures

Figure 1.

Figure 1.. Lipidomic analysis of RPM, TGEM, BMDM, and RAW macrophages.

Heat map representing fold-change in the extracellular medium levels of 140 eicosanoid species after stimulation with the TLR-4-specific receptor agonist KLA (100 ng/ml) relative to unstimulated PBS control at 0, 8, and 24 h time-points. Increases in metabolite levels are indicated by red, decreases by green, and detectable but unchanged levels by gray. Metabolites below the limit of detection are indicated by black; n = 3 individual biological replicates/time-point/group. PGDH, PG dehydrogenase; β-ox, β-oxidation; LTAH, LTA4 hydrolase; LTCS, LTC4 synthase; HEDH, HETE dehydrogenase; sEH, soluble epoxide hydrolase.

Figure 2.

Figure 2.. Quantitative eicosanoid production profiles of different TLR-4 agonist-stimulated macrophages.

Different macrophage types incubated in the absence (open bars) and presence (shaded bars) of KLA (100 ng/ml) from the same experiment as Fig. 1. Extracellular medium was removed at 8 h (upper row) and 24 h (lower row) and was analyzed for eicosanoid levels by MS. The data are expressed as mean values ±

sem

of three biological replicates.

Figure 3.

Figure 3.. COX-2 activity and expression.

Comparison of 8 h (A) total arachidonate COX-derived eicosanoids, (B) 11-HETE, and (C) 15-HETE from the same experiment as in Fig. 1; relative expression level of (D) COX-2 mRNA and (E) COX-1 mRNA at 0, 8, and 24 h; and (F) 8 h COX-2 protein in RPM, TGEM, BMDM, and RAW macrophages after KLA (100 ng/ml) stimulation. The data are expressed as mean values ±

sem

of three biological replicates. WB, Western blot.

Figure 4.

Figure 4.. Expression of terminal prostanoid synthases.

Comparison of transcript expression of (A) PGIS, (B) TBXS (TBXAS), (C) mPGES-1, and (D) H-PGDS synthases after 0, 8, and 24 h of KLA (100 ng/ml) stimulation in RPM (blue), TGEM (green), BMDM (purple), and RAW (red) macrophages. The data are expressed as mean values ±

sem

of three biological replicates.

Figure 5.

Figure 5.. Correlation between PGE2:PGD2 ratio and respective enzyme transcript expression ratio.

(A) Eicosanoid ratios of PGE2:PGD2 after 8 h and 24 h KLA stimulation, where PGD2, PGJ2, 15d PGD2, and 15d PGJ2 were summed. (B) Transcript expression ratios of mPGES-1:H-PGDS using average expression of 0 h and 8 h time-points (0–8 h) and 0, 8, and 24 h time-points (0–24) in RPM (blue), TGEM (green), BMDM (purple), and RAW (red) macrophages stimulated with KLA (100 ng/ml). The data are expressed as mean values ±

sem

of three biological replicates.

Figure 6.

Figure 6.. Overview of prostanoid synthase expression and relative activity in different macrophage cell types after long-term TLR-4 activation.

Metabolism of AA in (A) RPM, (B) TGEM, (C) BMDM, and (D) RAW through induced COX-2 leads to a pool of PGH2 that is metabolized by TBXS, PGIS, PGDS (H-PGDS), and induced PGES (mPGES-1). Percentages of metabolites (circles) and enzyme transcripts (squares) in each cell type are represented by area after 8 h KLA stimulation. Metabolites and expressed levels of PGES, PGDS, and TBXS are roughly proportional, although the presence of PGIS appears to selectively draw substrate away from PGES but not PGDS. Low constitutive expression level of PGIS in RPM and TGEM macrophages with disproportionately high levels of PGI2 suggests a significantly higher synthetic rate compared with other prostanoid synthases.

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