Takatoshi Yamamoto

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2

BACKGROUND Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. AIM This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. METHODS Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. DISCUSSION Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway.

Granulocyte–macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation.

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.

Surfactant Protein D Inhibits Interleukin 12p40 Production by Macrophages Through the SIRPα/ROCK/ERK Signaling Pathway

Objective Interleukin (IL)‐12 has a pivotal profibrotic role in the development of idiopathic pulmonary fibrosis (IPF). Medical research trials based on IPF registry databases have actively recruited patients. Surfactant protein D (SP‐D) is a useful biomarker in patients with IPF. SP‐D binds to signal regulatory protein &agr; (SIRP&agr;), which acts as an inhibitory receptor, and this SP‐D/SIRP&agr; interaction may have an anti‐inflammatory effect. Accordingly, the inhibitory effect of SP‐D on IL‐12p40 production by lipopolysaccharide (LPS)‐stimulated macrophages was investigated. Materials and Methods Human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐stimulated macrophages (day 9 of culture) was used to investigate IL‐12p40 production after stimulation with SP‐D. Results GM‐CSF was found to upregulate SIRP&agr; expression by macrophages. PD98059 (an extracellular signal‐regulated kinase [ERK] inhibitor) blunted induction of SIRP&agr; expression by GM‐CSF. SP‐D significantly attenuated IL‐12p40 production by macrophages after stimulation with LPS. Silencing of SIRP&agr;/&bgr;/&ggr; significantly reversed this inhibitory effect of SP‐D. In contrast, neither SB023580 (a p38&agr;/&bgr; MAPK inhibitor) nor BIRB796 (a p38&ggr;/&dgr; MAPK inhibitor) attenuated the inhibitory effect of SP‐D on LPS‐stimulated production of IL‐12p40. Silencing of SHP also had no influence on this effect of SP‐D. Interestingly, a Rho‐associated protein kinase (ROCK) inhibitor (Y‐27632) abolished the inhibition of LPS‐stimulated IL‐12p40 production by SP‐D, whereas silencing of ERK 2 significantly blunted this effect of Y‐27632. Conclusions These findings suggest that SP‐D inhibits LPS‐stimulated production of IL‐12p40 via the SIRP&agr;/ROCK/ERK signaling pathway.

Roles of myeloperoxidase and GAPDH in interferon-gamma production of GM-CSF-dependent macrophages

Interferon (IFN)-gamma is highly expressed in atherosclerotic lesions and may have an important role in atherogenesis. Myeloperoxidase (MPO), the most abundant protein in neutrophils, is a marker of plaque vulnerability and a possible bridge between inflammation and cardiovascular disease. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has also been implicated in the pathogenesis of atherosclerosis. The present study investigated the role of neutrophil activation in atherosclerosis. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IFN-gamma protein by GM-CSF-dependent-macrophages was investigated by enzyme-linked immunosorbent assay after stimulation with MPO. GM-CSF enhanced macrophage expression of the mannose receptor (CD206), which is involved in MPO uptake. MPO increased IFN-gamma production by GM-CSF-dependent macrophages in a concentration-dependent manner. Pretreatment of macrophages with small interfering RNA (siRNA) for CD206 or extracellular signal-regulated kinase (ERK)-2 attenuated IFN-gamma production, while siRNA for ERK-1 did not. GAPDH is known to bind to adenylate/uridylate (AU)-rich elements of RNA and may influence IFN-gamma protein expression by binding to the AU-rich element of IFN-gamma mRNA. Interestingly, pretreatment with siRNA for GAPDH significantly reduced IFN-gamma production by macrophages, while it did not affect TF protein expression. In conclusion, MPO upregulates IFN-gamma production by GM-CSF-dependent-macrophages via the CD206/ERK-2 signaling pathway, while silencing GAPDH reduces IFN-gamma production.

Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, β-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

Differential regulation of IL-23 production in M1 macrophages by TIR8/SIGIRR through TLR4- or TLR7/8-mediated signaling

HighlightsSIGIRR is a negative regulator of TLR4 signaling.SIGIRR is a positive regulator of TLR7/8 signaling.SIGIRR deficiency promotes IRF4 expression.SIGIRR deficiency modulates IL‐23 expression via IRF4. Abstract Cross‐talks between toll‐like receptors (TLRs) including various negative regulatory mechanisms are many unknown. We investigated the differential mechanism of IL‐23 production in M1 macrophages by single immunoglobulin interleukin‐1 receptor‐related (SIGIRR) molecule through TLR4 or TLR7/8. IL‐12p40 production by M1 macrophages pretreated with human neutrophil elastase (HNE) was synergistically enhanced IL‐12p40, but not IL‐23 production, after exposure to lipopolysaccharide (LPS). LPS (a TLR4 agonist) induced a slight increase of IL‐23 production, while Resiquimod (a TLR7/8 agonist) significantly enhanced IL‐23 production. Expression of SIGIRR protein, a negative regulator of TLR4, was higher in M1 macrophages than in monocytes. Interestingly, SIGIRR siRNA induced a slight increment of IL‐23 production after exposure of macrophages to LPS, while IL‐23 production in response to Resiquimod was significantly upregulated by SIGIRR siRNA. Silencing SIGIRR enhanced IRF4 protein level determined by western blotting or ELISA. IRF4 siRNA dramatically restored IL‐23 production after exposure to Resiquimod in macrophages transfected with SIGIRR siRNA. In conclusion, production of IL‐23 is differentially regulated in M1 macrophages by SIGIRR through TLR4‐ or TLR7/8‐mediated signaling. SIGIRR is both a negative regulator of TLR4 and a positive regulator of TLR7/8.

A protease‐activated receptor 2 agonist (AC‐264613) suppresses interferon regulatory factor 5 and decreases interleukin‐12p40 production by lipopolysaccharide‐stimulated macrophages: Role of p53

The transcription factor interferon regulatory factor 5 (IRF5) has a key role in the production of interleukin (IL)‐12 by macrophages. IRF5 is also a central mediator of toll‐like receptor signaling and is a direct target of p53. Activation of protease‐activated receptor 2 (PAR‐2) upregulates p53 and suppresses apoptosis. This study investigated the influence of human neutrophil elastase (HNE) and PAR‐2 agonists on expression of IRF5 and IL‐12p40 by macrophages stimulated with lipopolysaccharide. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐dependent macrophages showed upregulation of IRF5 expression, while HNE reduced expression of p53 and IRF5 in a concentration‐dependent manner. HNE also caused a concentration‐dependent decrease of IRF5 in macrophages transfected with small interfering RNA to silence p53, while silencing of β‐arrestin 2 blunted the reduction of p53 or IRF5 by HNE. Incubation of macrophages with a PAR‐2 agonist, AC‐264613, caused a decrease of IRF5 expression and also significantly reduced p53 protein expression. HNE upregulated the expression of tumor necrosis factor receptor‐associated factor 6 (TRAF6) and caused transactivation of TLR4, while AC‐264613 did not promote TLR4 transactivation. In conclusion, the PAR‐2 agonist AC‐264613 attenuated IRF5‐associated IL‐12p40 production by macrophages.

Chemokine profiles of human visceral adipocytes from cryopreserved preadipocytes: Neutrophil activation and induction of nuclear factor-kappa B repressing factor.

AIMS In obesity, infiltration of adipose tissue by proinflammatory immune cells causes chronic low-grade inflammation. We investigated the chemokine profiles of human visceral adipocytes by the reverse transcription polymerase chain reaction and the effect of human neutrophil elastase (HNE) on monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels. MAIN METHODS Human adipocytes were obtained from cryopreserved omental preadipocytes of subjects with a body mass index (BMI) <30kg/m(2) or >30kg/m(2) and were cultured to assess chemokine production. KEY FINDINGS Chemokine responses associated with obesity-related inflammation were well preserved in cultured human adipocytes derived from cryopreserved preadipocytes. Visceral adipocytes from subjects with a BMI >30kg/m(2) expressed mRNA for MCP-1, regulated on activation, normal T cell expressed and secreted (RANTES), epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), interleukin-8 (IL-8), lymphotactin-β, and fractalkine. Although visceral adipocytes from subjects with a BMI <30kg/m(2) also expressed MCP-1, RANTES, ENA-78, and IL-8 mRNA, neither lymphotactin-β nor fraktalkine mRNA was detected. Interestingly, expression of MCP-1 mRNA was decreased significantly after exposure to HNE (85×10(3)μM/L), suggesting the induction of nuclear factor-kappa B repressing factor. SIGNIFICANCE Adipocytes from subjects with a BMI >30kg/m(2) or <30kg/m(2) have different chemokine profiles. Only adipocytes from subjects with a BMI >30kg/m(2) express lymphotactin-β and fractalkine mRNA. Differential chemokine profiles of visceral adipocytes contribute to infiltration of adipose tissue by adaptive immune cells. Neutrophil activation is involved in induction of nuclear factor-kappa B repressing factor, resulting in regulation of immune cell trafficking.

Di-(2-ethylhexyl) phthalate suppresses IL-12p40 production by GM-CSF-dependent macrophages via the PPARα/TNFAIP3/TRAF6 axis after lipopolysaccharide stimulation

Activation of peroxisome proliferator–activated receptor α (PPARα) by di-(2-ethylhexyl) phthalate (DEHP) has an anti-inflammatory effect. This study investigated the potential combined influence of PPARα, tumor necrosis factor α-induced protein 3 (TNFAIP3/A20), and tumor necrosis factor receptor–associated factor 6 (TRAF6) on interleukin (IL)-12p40 production by macrophages exposed to DEHP and stimulated with lipopolysaccharide (LPS). LPS upregulated IL-12p40 expression by granulocyte-macrophage colony-stimulating factor–dependent macrophages (on day 9 of culture), whereas adding DEHP to cultures significantly attenuated the response of IL-12p40 to LPS stimulation. PPARα protein was also reduced by DEHP. Interestingly, transfection of macrophages with small interfering RNA (siRNA) duplexes for PPARα, TNFAIP3/A20, or dual oxidase 2 restored the response of IL-12p40 protein to LPS stimulation in the presence of DEHP. siRNAs for various protein kinase Cs (PKCs) (α, β, γ, or δ) also restored IL-12p40 production by macrophages exposed to LPS and DEHP. While LPS upregulated both IL-12p40 and TNFAIP3/A20 production, adding DEHP to cultures dramatically reduced IL-12p40 and TNFAIP3/A20 levels. Silencing of PKCα reduced TNFAIP3/A20 production, whereas PKCγ siRNA (but not PKCβ or δ siRNA) significantly increased TNFAIP3/A20. TRAF6 was also attenuated by macrophages with DEHP. The PPARα/TNFAIP3/TRAF6 axis may have an important role in the mechanism through which DEHP reduces IL-12p40 production by LPS-stimulated macrophages.