Triggering receptor expressed on myeloid cells (TREM-1) is regulated post-transcriptionally and its ligand is present in the sera of some septic patients

Abstract

Inflammation is necessary for survival, but it is also an important cause of human morbidity and mortality, as exemplified by sepsis. During inflammation, cells of the innate immune system are recruited and activated in response to infection, trauma or injury. These cells are activated through receptors, such as Toll-like receptors (TLRs), which recognize microbial ligands such as lipopolysaccharide (LPS). Triggering receptor expressed on myeloid cells (TREM)-1 amplifies the inflammatory response initiated by TLRs, and its expression on the surface of monocytes increases in the presence of TLR ligands. Here we have shown that in monocytes TREM-1 mRNA levels, measured by reverse transcription-polymerase chain reaction (RT-PCR), remained unchanged and TREM-1 protein levels, measured by flow cytometry, increased, indicating that LPS increases TREM-1 expression by a post-transcriptional mechanism. We also showed that TREM-1/Fc fusion protein decreased the ability of the sera of some patients with sepsis to activate monocytes, indicating that the TREM-1 ligand, whose identity is unknown, may be present in the sera of some of these patients. We describe a mechanism for the regulation of TREM-1 expression on monocytes and the possible presence of its ligand in serum; these findings help to explain the contribution of TREM-1 during systemic inflammation.

Fig 1

Effect of lipopolysaccharide (LPS) on surface triggering receptor expressed on myeloid cells (TREM)-1 expression by peripheral blood monocytes. Monocytes were stimulated with 10 or 100 ng/ml LPS, and TREM-1 expression was analysed by flow cytometry. The results are shown as individual data (cells from different donors) and mean. Significant (P < 0·05) differences between groups are shown (*Mann– Whitney U-test). MFI: mean fluorescence intensity.

Fig 2

Effect of lipopolysaccharide (LPS) on triggering receptor expressed on myeloid cells (TREM)-1 mRNA level I human peripheral blood monocytes. (a) Monocytes were stimulated with 10 ng/ml LPS for the indicated times, total mRNA was extracted with TRIZOL reagent, cDNA was synthesized and TREM-1 and DNAX protein of 12 kilodaltons (DAP12) were amplified with Taq polymerase, using the indicated primers. For TREM-1, two sequences [476 base pairs (bp) and 286 bp)] were detected by gel electrophoresis. β-actin was amplified simultaneously to verify RNA integrity and to ensure that equivalent amounts of template were used. (b, d) Monocytes were stimulated with 10 ng/ml LPS for 0, 5, 10 and 40 min, and 2 and 24 h, total RNA was extracted with TRIZOL reagent, cDNA was synthesized and the target genes were amplified by real-time polymerase chain reaction (PCR). The graphs represent the specific fluorescence (F2 or F3) divided by the background fluorescence (F1), plotted against the cycle number of the reaction. (c) Monocytes were treated as in (b), and the expression indices (E.I.) of membrane form of TREM-1 (mTREM-1) and the alternatively spliced form of TREM-1 mRNA (svTREM-1) relative to hypoxanthine phosphoribosyltransferase (HPRT) were calculated. The graphs represent the change in mTREM-1 and svTREM-1 E.I. relative to their E.I. in untreated monocytes. These results are representative of five independent experiments with cells from different donors.