N-formyl-methionyl-leucyl-phenylalanine inhibits both gamma interferon- and interleukin-10-induced expression of FcgammaRI on human monocytes

Abstract

Three different classes of receptors for the Fc portion of immunoglobulin G (FcgammaRs), FcgammaRI, FcgammaRII, and FcgammaRIII, have been identified on human leukocytes. One of them, FcgammaRI, is a high-affinity receptor capable of induction of functions that include phagocytosis, respiratory burst, antibody-dependent cell-mediated cytotoxicity (ADCC), and secretion of cytokines. This receptor is expressed on mononuclear phagocytes, and this expression is regulated by cytokines and hormones such as gamma interferon (IFN-gamma), IFN-beta, interleukin-10 (IL-10), and glucocorticoids. We have recently demonstrated that the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is capable of inducing a time-dependent downregulation of both FcgammaRIIIB and FcgammaRII in human neutrophils, altering FcgammaR-dependent functions. Considering the biological relevance of the regulation of FcgammaRI, we investigated the effect of FMLP on the overexpression of FcgammaRI induced by both IFN-gamma and IL-10 on human monocytes. We demonstrate that FMLP significantly abrogated IFN-gamma- and IL-10-induced FcgammaRI expression, although its basal level of expression was not altered. However, other IFN-gamma-mediated effects such as the overexpression of the major histocompatibility complex class II antigens and the enhancement of lipopolysaccharide-induced secretion of tumor necrosis factor alpha were not affected by FMLP treatment. The formyl peptide completely inhibited the IFN-gamma- and IL-10-induced enhancement of ADCC and phagocytosis carried out by adherent cells. The inhibitory effect of FMLP on FcgammaRI upregulation could exert an important regulatory effect during the evolution of bacterial infections.

FIG. 1

Effect of FMLP on IFN-γ-induced FcγRI expression. PBMCs (10⁶ cells/ml) were incubated with medium or 1 μM FMLP for 1 h. Then, the cells were washed and incubated for 24 h with medium (a) or IFN-γ (240 U/ml) (b). After this period, the PBMCs were stained with anti-FcγRI and anti-CD14 antibodies. The histograms represent the CD14⁺ population of PBMCs and correspond to a representative experiment of 12 experiments conducted. The background fluorescence intensity (filled peak) was obtained with control isotype antibodies. The x axis represents fluorescence intensity (arbitrary units); the y axis represents the cell number.

FIG. 2

Effect of FMLP on IL-10-induced FcγRI expression. PBMCs (10⁶ cells/ml) were incubated with medium or 1 μM FMLP for 1 h. Then, the cells were washed and incubated with IL-10 (100 U/ml) for 24 h. After this period, the PBMCs were stained with anti-FcγRI and anti-CD14 antibodies. The histograms represent the CD14⁺ population of PBMCs and correspond to a representative experiment of eight experiments conducted. The background fluorescence intensity (filled peak) was obtained with control isotype antibodies. The x axis represents fluorescence intensity (arbitrary units); the y axis represents the cell number.

FIG. 3

Effect of FMLP on ADCC enhancement induced by IFN-γ in total and adherent PBMCs. PBMCs (4 × 10⁶ cells/ml) were incubated in polypropylene tubes with medium or 1 μM FMLP for 1 h. Then, the cells were washed and incubated with medium or IFN-γ (240 U/ml) for 24 h. After this period, ADCC assay was performed (see Materials and Methods). The adherent PBMC population was obtained as indicated in Materials and Methods and was treated with 1 μM FMLP and IFN-γ as indicated for PBMCs. After 24 h of incubation, ADCC assay was performed on the 96-well round-bottom plates in the presence or absence of catalase (5,650 U/ml). The figure shows the results of a representative experiment of six experiments conducted. Data are expressed as percent ADCC of that for the control ± standard deviation ∗, significance (P < 0.05) of difference for results of six experiments compared to the results for IFN-γ-treated cells by the Wilcoxon test (two tailed).

FIG. 4

Effect of FMLP on ADCC enhancement induced by IL-10 in adherent PBMCs. Adherent PBMCs were obtained as indicated in Materials and Methods and were incubated with medium or 1 μM FMLP for 1 h. Then, the cells were washed and incubated with medium or IL-10 (100 U/ml). After 24 h of incubation, ADCC assay was performed on the 96-well round-bottom plates in the presence or absence of catalase (5,650 U/ml). The figure shows the results of a representative experiment of six experiments conducted. Data are expressed as percent ADCC of that for the control ± standard deviation. ∗, significance (P < 0.05) of difference for results of six experiments compared to the results for IL-10-treated cells by the Wilcoxon test (two tailed).

FIG. 5

Effect of FMLP on IFN-γ-induced enhancement of phagocytosis. PBMCs (2 × 10⁶ cells/ml) were incubated with medium or 1 μM FMLP for 1 h. Then, the cells were washed and incubated with medium or IFN-γ (240 U/ml) for 24 h. After this period, the level of phagocytosis was determined (see Materials and Methods) (n = 5), ∗, significantly different (P < 0.008) from the control by the Mann-Whitney test (two tailed).