Involvement of mitogen-activated protein kinase pathways in interleukin-8 production by human monocytes and polymorphonuclear cells stimulated with lipopolysaccharide or Mycoplasma fermentans membrane lipoproteins

Abstract

Interleukin-8 (IL-8) is a chemokine that belongs to the alpha-chemokine or CXC subfamily and is produced by a wide variety of human cells, including monocytes and polymorphonuclear cells (PMN). IL-8 is secreted in response to inflammatory stimuli, notably bacterial products such as lipopolysaccharide (LPS), but little is known about the mechanisms by which these agents mediate IL-8 induction. In this report, we show that Mycoplasma fermentans lipid-associated membrane proteins (LAMPf) induce the production of high levels of IL-8 by THP-1 (human monocyte) cells and PMN at the same extent as LPS. It was previously demonstrated that stimulation of monocytic cells with either LPS or LAMPf led to a series of common downstream signaling events, including the activation of protein tyrosine kinase and of mitogen-activated protein kinase cascades. By using PD-98059 and SB203580, two potent and selective inhibitors of MEK1 (a kinase upstream of ERK1/2) and p38, respectively, we have demonstrated that both ERK1/2 and p38 cascades play a key role in the production of IL-8 by monocytes and PMN stimulated with bacterial fractions.

FIG. 1

IL-8 production by human THP-1 cells in response to LAMPf and LPS. (A) THP-1 cells (10⁶/ml) were not stimulated (control [CTRL]) or stimulated with either LPS (1 μg/ml) or LAMPf (1 μg/ml). IL-8 production was measured by ELISA 18 h after stimulation. Data presented are means ± SEM of three distinct assays. (B) Multi-NPA using oligonucleotide probes for IL-8 and 28S rRNA (for standardization). RNAs were prepared from untreated THP-1 cells (○) and THP-1 cells induced with LAMPf (1 μg/ml) for the indicated time and subjected to multi-NPA analysis. Multi-NPA gels were exposed to a PhosphorImager screen for 2 to 4 h; the gel shown is representative of three experiments with similar results. IL-8 and 28S rRNA oligonucleotide probes protect 24 and 35 bases of the IL-8 and 28S rRNA transcripts, respectively. The IL-8 signal in each lane was quantitatively assessed and normalized to the 28S rRNA signal. (C) Effect of anti-CD14 antibody treatments on LAMPf-induced IL-8 secretion. Human THP-1 cells were incubated with anti-human CD14 monoclonal antibody MY4 at 5 μg/ml for 1 h prior to stimulation with either LAMPf (1 μg/ml) or LPS (100 ng/ml). An irrelevant antibody (NS) was used as a control. IL-8 secretion was determined after 18 h of culture and expressed as percentage of secretion normalized to stimulated cells that received no antibody treatment. Mean values of two different experiments are shown.

FIG. 2

IL-8 production by human monocytes and PMN stimulated with LAMPf or LPS. PBMC (6 × 10⁶/ml) and PMN (2 × 10⁶/ml) were unstimulated (control [CTRL]) or stimulated with either LPS (1 μg/ml) or LAMPf (1 μg/ml). IL-8 production was measured by ELISA 18 h after stimulation. Data presented are means ± SEM of four and five distinct assays for monocytes and PMN, respectively.

FIG. 3

Effect of PTK blockade on IL-8 production. Monocytes and PMN were incubated with herbimycin A (10 μM) for 1 h prior to LPS (1 μg/ml) or LAMPf (1 μg/ml) stimulation. DMSO (1%) was used as the solvent control. The IL-8 level was measured by ELISA 18 h after stimulation and normalized to cells that received no treatment prior to stimulation. Data presented are means ± SEM of four and three distinct experiments for monocytes and PMN, respectively.

FIG. 4

Effect of ERK1/2 and p38 pathway-specific inhibitors on IL-8 production by human monocytes and PMN. Cells were treated for 1 h with either PD-98059 (MEK1 inhibitor) or SB203580 (p38 inhibitor) at different concentrations and then stimulated with either LAMPf (1 μg/ml) (A) or LPS (1 μg/ml) (B). DMSO (1%) was used as the solvent control. The IL-8 level was measured by ELISA 18 h after stimulation and normalized to cells that received no treatment prior to stimulation. Results are means ± SEM of four and three separate experiments for monocytes and PMN, respectively.

FIG. 5

Effect of CAPE on NF-κB transactivation mediated by LAMPf. THP-1 cells were transiently transfected with an NF-κB- or AP-1-driven luciferase (luci) reporter plasmid. Transfected cells were stimulated with LAMPf (1 μg/ml) for 6 h prior to cell lysis. Luciferase activity was assessed in stimulated and unstimulated (control [CTRL]) cells and normalized to protein content. To assess the effect of CAPE, transfected cells were preincubated with CAPE at the indicated concentration for 1 h and then stimulated with LAMPf and analyzed for luciferase activity as indicated above. Assays were performed in duplicate, and data presented are means ± SEM of three independent experiments.

FIG. 6

Effect of an NF-κB-specific inhibitor on IL-8 production by human monocytes and PMN. Cells were incubated with CAPE (100 μM) for 1 h prior to LPS (1 μg/ml) or LAMPf (1 μg/ml) stimulation. DMSO (1%) was used as the solvent control. IL-8 production was measured by ELISA 18 h after stimulation and normalized to cells that received no treatment prior to stimulation. Data presented are means ± SEM of four and three distinct experiments for monocytes and PMN, respectively.