IL-23 production in human macrophages is regulated negatively by tumor necrosis factor α-induced protein 3 and positively by specificity protein 1 after stimulation of the toll-like receptor 7/8 signaling pathway.

The IL-23/IL-17 axis plays an important role in the development of autoimmune diseases, but the mechanism regulating IL-23 production is mainly unknown. We investigated how TNFAIP3 and Sp1 affect IL-23 production by human macrophages after exposure to resiquimod, a TLR7/8 agonist. IL-23 production was significantly upregulated by resiquimod but only slightly by LPS (a TLR4 agonist). Interestingly, IL-23 levels were significantly attenuated after sequential stimulation with LPS and resiquimod, but IL-12p40 and IL-18 levels were not. TLR4-related factors induced by LPS may regulate IL-23 expression via TLR7/8 signaling. LPS significantly enhanced TNFAIP3 and IRAK-M levels but reduced Sp1 levels. After exposure to resiquimod, RNA interference of TNFAIP3 upregulated IL-23 significantly more than siRNA transfection of IRAK-M did. In contrast, knockdown of Sp1 by RNA interference significantly attenuated IL-23 production. Transfection with siRNA for TNFAIP3 enhanced IL-23 expression significantly. After stimulation with resiquimod, GW7647-an agonist for PPARα (an inducer of NADHP oxidase)-and siRNA for UCP2 (a negative regulator of mitochondrial ROS generation) enhanced TNFAIP3 and reduced IL-23. siRNA for p22phox and gp91phox slightly increased Sp1 levels. However, after exposure to resiquimod siRNA-mediated knockout of DUOX1/2 significantly enhanced Sp1 and IL-23 levels, and decreased TNFα-dependent COX-2 expression. Concomitantly, TNFAIP3 levels was attenuated by DUOX1/2 siRNA. TNFAIP3 and Sp1 levels are reciprocally regulated through ROS generation. In conclusion, after stimulation of the TLR7/8 signaling pathway IL-23 production in human macrophages is regulated negatively by TNFAIP3.

TRIM28/TIF1ß and Fli-1 negatively regulate peroxynitrite generation via DUOX2 to decrease the shedding of membrane-bound fractalkine in human macrophages after exposure to substance P.

The chemokine fractalkine is synthesized as a membrane-bound protein, but studies have shown that serum levels of soluble fractalkine are elevated in inflammatory and autoimmune diseases. Patients with autoimmune diseases also have increased serum levels of neuropeptide substance P (SP). The shedding activity of the ADAM family is induced by peroxynitrite, but that of SP is unclear. Treatment of human macrophages with SP upregulated levels of membrane-bound fractalkine. Interestingly, small interfering RNA (siRNA) for DUOX2 further increased membrane-bound fractalkine but decreased soluble fractalkine compared with cells treated with SP alone. SP induced nitric oxide 2/inducible nitric oxide synthase (NOS2/iNOS) mRNA and increased levels of nitrotyrosine, a biomarker of peroxynitrite, whereas transfection with DUOX2 siRNA blunted upregulation of nitrotyrosine. Most importantly, N(ω)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) decreased protein levels of nitrotyrosine and concomitantly increased expression of membrane-bound fractalkine after exposure to SP. As for the signaling pathway of TGFß1 (an inhibitor of iNOS mRNA expression), silencing of RNA for TAK-1 upregulated membrane-bound fractalkine, but silencing of RNA for the Smad family did not. Interfering RNA of transcription factor specificity protein 1 (Sp1) upregulated protein levels of TGFß1/LAP. Most importantly, double transfection with siRNA for Sp1 and TRIM28/TIF1ßor Fli-1 led to a significant increase in TGFß1/LAP levels and a corresponding reduction of NOS2/iNOS, which inhibited the shedding of membrane-bound fractalkine. In conclusion, TRIM28/TIF1ß and Fli-1 negatively regulate TGFß1 expression to upregulate the generation of peroxynitrite, leading to increased shedding of membrane-bound fractalkine induced by SP.

Di-(2-Ethylhexyl) Phthalate Promotes Release of Tissue Factor-Bearing Microparticles From Macrophages via the TGFß1/Smad/PAI-1 Signaling Pathway.

BACKGROUND: Plasminogen activator inhibitor type 1 promotes formation of endothelial microparticles with procoagulant activity. However, it remains unclear whether di-(2-ethylhexyl) phthalate, a peroxisome proliferator-activated receptor α agonist, influences microparticle formation. MATERIALS AND METHODS: The effect of di-(2-ethylhexyl) phthalate on release of tissue factor-bearing microparticles was investigated using human M1 macrophages. RESULTS: Exposure of M1 macrophages to di-(2-ethylhexyl) phthalate significantly upregulated expression of plasminogen activator inhibitor type 1, whereas incubation of macrophages with small interfering RNA for peroxisome proliferator-activated receptor α attenuated it. Di-(2-ethylhexyl) phthalate significantly increased the tissue factor protein level in culture supernatants of M1 macrophages, but not M2 macrophages. After purification of proteins by centrifugal filtration, western blotting detected 2 high molecular weight bands of tissue factor-bearing microparticles in culture supernatants of M1 macrophages. The upper band showed binding to factor VIIa and tissue factor pathway inhibitor, unlike the lower band. This suggested heterogeneity of the procoagulant activity of tissue factor-bearing microparticles, presumably dependent upon encryption/decryption of tissue factor. Phosphatidylserine contributes to tissue factor decryption, and western blotting revealed that the density of phosphatidylserine was reduced in the upper tissue factor band compared with the lower band. Di-(2-ethylhexyl) phthalate also upregulated transforming growth factor-ß1 protein production by M1 macrophages. Moreover, silencing of Smad2, Smad3 or Smad4 attenuated plasminogen activator inhibitor type 1 expression and tissue factor-release from macrophages after di-(2-ethylhexyl) phthalate stimulation. CONCLUSIONS: Di-(2-ethylhexyl) phthalate promotes formation of tissue factor-bearing microparticles in human M1 macrophages via the transforming growth factor-ß1/Smad/ plasminogen activator inhibitor type 1 signaling pathway.

Cross-talk between the transcription factor Sp1 and C/EBPß modulates TGFß1 production to negatively regulate the expression of chemokine RANTES.

RANTES is a key chemokine for atherosclerosis, and obesity is associated with progression of atherosclerosis. Substance P (SP) increases glucose uptake and accumulation of lipids in adipocytes, and SP may upregulate RANTES expression. This study investigated the mechanism of RANTES expression by human M1 macrophages stimulated with SP. SP upregulated RANTES protein expression, whereas aprepitant (an NK1R antagonist) blunted this response. Pretreatment of macrophages with BIRB796 (a combined p38γ/p38δ inhibitor) led to a significant decrease of RANTES expression. Next, we investigated the effect of several NK1R internalization factors on RANTES expression, including GRK2, ß-arrestin 2, dynamin, ROCK, and TGFß1. Exposure of macrophages to SP upregulated TGFß1 expression. Silencing of ß-arrestin 2 or GRK2 significantly enhanced the RANTES protein level after stimulation by SP, whereas TGFß1/2/3 siRNA or dynasore (a dynamin inhibitor) decreased RANTES and Y-27632 (a ROCK inhibitor) had no effect. Surprisingly, silencing of transcription factor specificity protein 1 (Sp1) or inhibition of Sp1 activity by mithramycin led to significant upregulation of TGFß1 protein and corresponding enhancement of RANTES expression (by ELISA or western blotting), whereas siRNA for C/EBPß attenuated expression of both TGFß1 and RANTES. Next, we investigated transcriptional cross-talk among Sp1 and C/EBPß, TIF1ß, or Fli-1 in relation to RANTES expression. Compared with TIF1ß or Fli-1 siRNA, C/EBPß siRNA showed significantly stronger inhibition of RANTES production by Sp1 siRNA-transfected macrophages after stimulation with SP. In conclusion, transcription factor Sp1 engages in cross-talk with C/EBPß and modulates TGFß1 production to negatively regulate RANTES expression in macrophages stimulated with SP. In conclusion, cross-talk between the transcription factor Sp1 and C/EBPß modulates TGFß1 production to negatively regulate expression of the atherogenic chemokine RANTES in SP-stimulated macrophages, while RANTES is upregulated by SP via the p38γδMAPK/C/EBPß/TGFß1 signaling pathway.

Transcription factor specificity protein 1 modulates TGFß1/Smad signaling to negatively regulate SIGIRR expression by human M1 macrophages stimulated with substance P.

The stimuli inducing expression of single immunoglobulin IL-1-related receptor (SIGIRR) and the relevant regulatory mechanisms are not well defined. Transforming growth factor ß1 (TGFß1) delays internalization of neurokinin-1 receptor (NK1R) and subsequently enhances cellular signaling. This study investigated the effect of TGFß1 on SIGIRR protein production by human M1 macrophages in response to stimulation with substance P (SP). SP caused upregulation of SIGIRR expression in a concentration-dependent manner, whereas aprepitant (an NK1R inhibitor) blunted this response. Silencing p38γMAPK or TAK-1 partially attenuated the response to SP stimulation, while TGFß1/2/3 siRNA dramatically diminished it. SP induced much greater SIGIRR protein production than either lipopolysaccharide (a TLR4 agonist) or resiquimod (a TLR7/8 agonist). Unexpectedly, silencing of transcription factor specificity protein 1 (Sp1) led to significant upregulation of SIGIRR expression after SP stimulation, while KLF2 siRNA only partially enhanced it and Fli-1 siRNA reduced it. SP also upregulated TGFß1 expression, along with a corresponding increase of SIGIRR protein, whereas silencing TGFß1/2/3 blunted these responses. Sp1 siRNA or mithramycin (a gene-selective Sp1 inhibitor) significantly enhanced the expression of TGFß1 and SIGIRR by macrophages after SP stimulation. Importantly, this effect of Sp1 siRNA on TGFß1 and SIGIRR was blunted by siRNA for Smad2, Smad3, or Smad4, but not by TAK-1 siRNA. Next, we investigated the influence of transcription factor cross-talk on SIGIRR expression in response to SP. Co-transfection of macrophages with Sp1 siRNA and C/EBPß or TIF1ß siRNA attenuated the upregulation of SIGIRR by SP, while a combination of Sp1 siRNA and Fli-1 siRNA dramatically diminished it. In conclusion, TGFß1 may be an intermediary between SP/NK1R activation and SIGIRR expression in Sp1 siRNA-transfected macrophages. In addition, Sp1 modulates TGFß1/Smad signaling and negatively regulates SIGIRR protein production by macrophages after SP stimulation.

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

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.

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 α (SIRPα), which acts as an inhibitory receptor, and this SP-D/SIRPα 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 SIRPα expression by macrophages. PD98059 (an extracellular signal-regulated kinase [ERK] inhibitor) blunted induction of SIRPα expression by GM-CSF. SP-D significantly attenuated IL-12p40 production by macrophages after stimulation with LPS. Silencing of SIRPα/ß/γ significantly reversed this inhibitory effect of SP-D. In contrast, neither SB023580 (a p38α/ß MAPK inhibitor) nor BIRB796 (a p38γ/δ 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 SIRPα/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.

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 ß-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.

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

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 ß-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-ß, 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-ß 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-ß 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.

Mechanism of interferon-gamma production by monocytes stimulated with myeloperoxidase and neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell-cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFN-gamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signal-regulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase.

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.

Mechanism of tissue factor production by monocytes stimulated with neutrophil elastase.

BACKGROUND: Monocytes and neutrophils are activated during disseminated intravascular coagulation. Tissue factor, the main initiator of coagulation, is expressed by monocytes, while elastase is released by neutrophils. AIMS: This study investigated tissue factor production by peripheral monocytes after stimulation with human neutrophil elastase. METHODS: Tissue factor mRNA levels were investigated by the reverse transcriptase-polymerase chain reaction and tissue factor protein production was assessed by western blotting when monocytes were exposed to neutrophil elastase with or without preincubation using various inhibitors. RESULTS: Neutrophil elastase upregulated tissue factor mRNA and protein levels in monocytes. Both U73122 (phospholipase C inhibitor) and TMB-8 (intracellular calcium antagonist) prevented the upregulation of tissue factor mRNA. SB203580 (p38 mitogen-activated protein kinase inhibitor) suppressed this response, but PD98059 (extracellular signal-regulated kinase inhibitor) did not. Ro-318425 (ATP-competitive and selective protein kinase C (PKC) inhibitor) and Go 6976 (inhibitor of conventional PKCs and PKCµ) blocked the upregulation of tissue factor mRNA expression. Go 6983 (broad-spectrum PKC inhibitor) and CGP 4125 (staurosporine analog) partially attenuated it, as did a PKC theta/delta inhibitor. CONCLUSIONS: Neutrophil elastase mainly enhances tissue factor production by monocytes via the phospholipase C/conventional PKC/p38 MAPK pathway, although a novel PKC is also involved.