Neutrophil elastase enhances IL-12p40 production by lipopolysaccharide-stimulated macrophages via transactivation of the PAR-2/EGFR/TLR4 signaling pathway.
Proteinase-activated receptor 2 (PAR-2) and toll-like receptor 4 (TLR4) are involved in innate immune responses and signaling cross-talk between these receptor molecules has the potential to augment an ongoing inflammatory response. The aim of this study was to evaluate the possible cooperative influence of PAR-2 and TLR4 on IL-12p40 production by macrophages after stimulation with lipopolysaccharide (LPS). During culture, GM-CSF upregulated PAR-2 expression by macrophages in a time-dependent manner. Stimulation with LPS enhanced IL-12p40 production by macrophages in a concentration-dependent manner. While human neutrophil elastase (HNE) did not induce IL-12p40 production, pretreatment of macrophages with HNE synergistically increased the IL-12p40 protein level after LPS exposure. Silencing of TLR4 with small interfering RNA blunted the synergistic enhancement of IL-12p40 by HNE combined with LPS. Silencing of β-arrestin 2, p22phox, or ERK1/2 also inhibited an increase of IL-12p40. Interestingly, transfection of macrophages with small interfering RNA duplexes for DUOX-2, EGFR, TLR4, or TRAF6 significantly blunted the increase of IL-12p40 in response to treatment with HNE plus LPS. U73122 and Rottlerin also inhibited the increased production of IL-12p40. In conclusion, HNE is involved in transactivation of TLR4 through activation of DUOX-2/EGFR and synergistically enhances IL-12p40 production by macrophages stimulated with LPS.
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan; Graduate School of Medical Science, Kumamoto University Medical School, Chuo-ku Honjo 1-1-1, Kumamoto 860-8556, Japan.
Rui
R
Yamaguchi
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan.
Takatoshi
T
Yamamoto
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan.
Arisa
A
Sakamoto
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan.
Shinji
S
Narahara
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan.
Hiroyuki
H
Sugiuchi
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan.
Yasuo
Y
Yamaguchi
D002478:Cells, Cultured; D000074623:Dual Oxidases; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D053762:Interleukin-12 Subunit p40 / Q000096:biosynthesis*; D019272:Leukocyte Elastase / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D020239:Receptor Cross-Talk; D044464:Receptor, PAR-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D053762:Interleukin-12 Subunit p40; D008070:Lipopolysaccharides; D044464:Receptor, PAR-2; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D000074623:Dual Oxidases; C431515:DUOX2 protein, human; C512478:EGFR protein, human; D066246:ErbB Receptors; D019272:Leukocyte Elastase
Epidermal growth factor receptor; Granulocyte–macrophage colony-stimulating factor; Human neutrophil elastase; Interleukin-12; Toll-like receptor 4