N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibits tumour necrosis factor-alpha (TNF-alpha) production on lipopolysaccharide (LPS)-stimulated human neutrophils

Abstract

During gram-negative infections bacterial components, such as LPS and formylated peptides, exert profound physiological effects on polymorphonuclear neutrophils (PMN) resulting in increased neutrophil effector activities, including the generation of oxidative metabolites, degranulation, phagocytosis and cytokine release. There is not enough evidence about the relationships between LPS and formylated bacterial peptides in the triggering and regulation of the immune inflammatory response. In this study, we present evidence indicating that pretreatment of human PMN with a prototype formylated peptide such as fMLP results in the inhibition of TNF-alpha secretion, a key molecule that plays a central role in the pathogenesis of septic shock. This inhibitory effect of fMLP does not appear to alter the expression of LPS receptors or the transcriptional pathway of the TNF-alpha mRNA, but instead, fMLP reduces the expression of the membrane form of TNF-alpha on the PMN surface. These findings indicate that fMLP, a typical proinflammatory agent, could play, at least in determined conditions, an anti-inflammatory role.

Fig. 1

Inhibitory effect of fMLP on TNF-α secretion. Polymorphonuclear neutrophils (PMN; 3 × 10⁶/ml) were stimulated with LPS (5 μg/ml), fMLP (10⁻⁶m), or simultaneously with LPS + fMLP. In addition, PMN were treated with fMLP for 1 h, before LPS addition (fMLP/LPS), or were treated with LPS for 15 min before fMLP addition (LPS/fMLP). After 18 h of incubation at 37°C, all supernatants were collected and assayed for TNF-α activity on L-929 cells. *Statistical difference (P < 0.001) compared with LPS-stimulated PMN.

Fig. 2

Polymorphonuclear neutrophils (PMN; 3 × 10⁶/ml) were stimulated or not with 100 U/ml of recombinant human IFN-γ for 3 h at 37°C. Then LPS (5 μg/ml) was added (IFN/LPS). After 18 h of incubation at 37°C, all supernatants were collected and assayed for TNF-α activity on L-929 cells. *Statistical difference (P < 0.001) compared with LPS-stimulated PMN.

Fig. 3

(a) CD14 expression and (b) FITC-LPS binding of fMLP-treated polymorphonuclear neutrophils (PMN). PMN were treated (––) or not (·····) with fMLP (10⁻⁶m) for 1 h at 37°C. Then the cells were incubated with FITC-LPS (1 μg/10⁶ PMN) during 1 h at 37°C or with MoAb anti-CD14 (0.4 μg/10⁶ PMN) for 30 min at 4°C followed by goat anti-mouse IgG-FITC. Fluorescence intensity of 10 000 cells was analysed for each sample. The solid thin graphs represent the background fluorescence of cells incubated with (a) an isotype-matched IgG2b control MoAb and (b) buffer. Abscissa, Fluorescence intensity; ordinate, number of cells.

Fig. 4

CD11/CD18 expression of fMLP-treated polymorphonuclear neutrophils (PMN). PMN were treated (––) or not (·····) with fMLP (10⁻⁶m) for 1 h at 37°C. Then the cells (10⁶) were incubated for 30 min at 4°C with 3 μl of MoAbs FITC–anti-CD18, FITC–anti-CD11a/CD18, PE–anti-CD11b/CD18, or PE–anti-CD11c/CD18. Fluorescence intensity of 10 000 cells was analysed for each sample. Abscissa, Fluorescence intensity: FL-1, FITC; FL-2, PE; ordinate, number of cells.

Fig. 5

FACS analysis of IFN-γ-treated polymorphonuclear neutrophils (PMN). (a) PMN were treated (·····) or not (^––) with IFN-γ (100 U/ml) for 1 h at 37°C. Then the cells were incubated with FITC-LPS (1 μg/10⁶ PMN) during 1 h at 37°C (b) or with MoAb anti-CD14 (0.4 μg/10⁶ PMN) for 30 min at 4°C followed by goat anti-mouse IgG-FITC (a). Fluorescence intensity of 10 000 cells was analysed for each sample. The solid thin graphs represent the background fluorescence of cells incubated with (a) an isotype-matched IgG2b control MoAb and (b) buffer.

Fig. 6

Effect of fMLP on the expression of membrane TNF-α. Polymorphonuclear neutrophils (PMN; 3 × 10⁶/ml) were treated with fMLP (10⁻⁶ m) during 15 min and then stimulated with LPS (5 μg/ml) for 1 h at 37°C. After washing, the cells (10⁶ PMN) were incubated during 30 min at 4°C with 25 μg/ml of an anti-TNF-α polyclonal antibody, followed by goat anti-rabbit FITC-IgG. The solid thin graph represent the background fluorescence of non-stimulated cells (control). The solid histogram was obtained using, as a control, a normal rabbit IgG. Fluorescence intensity of 10 000 cells was analysed for each sample.

Fig. 7

RNA extraction was performed after 3 h of LPS (5 μg/ml) stimulation. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification and Southern blot analysis were carried out as described in Materials and Methods. The values for TNF-α transcripts from each sample were normalized individually to the corresponding level of the housekeeping gene β-actin. Lane 1, control non-stimulated polymorphonuclear neutrophils (PMN); lane 2, LPS-stimulated PMN; lane 3, fMLP (10⁻⁶m)-stimulated PMN; lane 4, PMN treated with fMLP (10⁻⁶m) for 1 h followed by LPS stimulation; lane 5, TNF-α (355 bp) and β-actin (661 bp) controls; lane 6, negative controls. The figure shows a representative experiment of three performed with similar results.