Mechanism of Nanoformulated Graphene Oxide-Mediated Human Neutrophil Activation.

Understanding the molecular mechanisms of graphene oxide (GO)-based biomaterials is important for logical biomedical applications. Previous studies have revealed biointeractions between GO and immune effector cells, but the effects on neutrophils, crucial cells in the immune system, have not been thoroughly discussed. In this study, GO nanoformulations were synthesized with different functional groups, including GO, GO-carboxylated (GO-COOH), and PEGylated GO (GO-PEG), with different surface features, which were elucidated using imaging methods and surface-sensitive quantitative spectroscopic techniques, including atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoemission spectroscopy (XPS). The GO-based nanoformulations elicited reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation in human neutrophils. Nanoformulated GO stimulates NET development via the formation of ROS. An endocytosis study revealed that nanoformulated GO facilitated internalization by neutrophils via macropinocytosis and actin-dependent phagocytosis. Importantly, calcium mobilization and phosphorylation proteins such as mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) and AKT were involved in the activation of neutrophils. These findings offer the first verification that nanoformulated GO exhibits direct effects on human neutrophils.

Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium.

The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 µg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton's tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca(2+) levels ([Ca(2+)]i), whereas PP2 prolonged the time required for [Ca(2+)]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca(2+).

Osthol attenuates neutrophilic oxidative stress and hemorrhagic shock-induced lung injury via inhibition of phosphodiesterase 4.

Oxidative stress caused by neutrophils is an important pathogenic factor in trauma/hemorrhagic (T/H)-induced acute lung injury (ALI). Osthol, a natural coumarin found in traditional medicinal plants, has therapeutic potential in various diseases. However, the pharmacological effects of osthol in human neutrophils and its molecular mechanism of action remain elusive. In this study, our data showed that osthol potently inhibited the production of superoxide anion (O2(•-)) and reactive oxidants derived therefrom as well as expression of CD11b in N-formylmethionylleucylphenylalanine (FMLP)-activated human neutrophils. However, osthol inhibited neutrophil degranulation only slightly and it failed to inhibit the activity of subcellular NADPH oxidase. FMLP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) was inhibited by osthol. Notably, osthol increased the cAMP concentration and protein kinase A (PKA) activity in activated neutrophils. PKA inhibitors reversed the inhibitory effects of osthol, suggesting that these are mediated through cAMP/PKA-dependent inhibition of ERK and Akt activation. Furthermore, the activity of cAMP-specific phosphodiesterase (PDE) 4, but not PDE3 or PDE7, was significantly reduced by osthol. In addition, osthol reduced myeloperoxidase activity and pulmonary edema in rats subjected to T/H shock. In conclusion, our data suggest that osthol has effective anti-inflammatory activity in human neutrophils through the suppression of PDE4 and protects significantly against T/H shock-induced ALI in rats. Osthol may have potential for future clinical application as a novel adjunct therapy to treat lung inflammation caused by adverse circulatory conditions.

A novel immunomodulatory effect of ugonin U in human neutrophils via stimulation of phospholipase C.

Neutrophils have a crucial role in the immune system and are the first line of defense against pathogenic invaders. Neutrophil activation is required for their defensive function and can be induced by diverse stimuli, through either binding to cell surface receptors or direct intracellular target molecule stimulation. In this study, we found that 4″a,5″,6″,7″,8″,8″a-hexahydro-5,3',4'-trihydroxy-5″,5″,8″a-trimethyl-4H-chromeno [2″,3″:7,6]flavone (ugonin U), a flavonoid isolated from Helminthostachys zeylanica (L) Hook, significantly induced superoxide production and release in a time- and concentration-dependent manner. A series of experiments was performed to dissect the mechanism of ugonin U-induced respiratory burst in human neutrophils. Our results demonstrated that ugonin U induced a slow increase in intracellular Ca(2+), which was necessary for ugonin U-stimulated superoxide release. Use of a formyl peptide receptor (FPR) blocker, G protein inhibitor, and protein tyrosine kinase (PTK) inhibitor proved that FPR, G proteins, and PTKs were not associated with ugonin U-induced respiratory burst. Additionally, immunoblotting results revealed that ugonin U did not affect the phosphorylation of mitogen-activated protein kinases and protein tyrosine. Nevertheless, a phospholipase C (PLC) inhibitor and an inositol triphosphate (IP3) receptor antagonist considerably suppressed ugonin U-stimulated Ca(2+) mobilization and subsequent superoxide release. Ugonin U also induced an increase in intracellular IP3 formation, which could be blocked using a PLC inhibitor. In conclusion, our study reveals that ugonin U represents the first identified natural flavonoid compound to directly stimulate PLC. Moreover, ugonin U induces respiratory burst via the PLC/IP3/Ca(2+) pathway in human neutrophils.

Bioactive secondary metabolites of a marine Bacillus sp. inhibit superoxide generation and elastase release in human neutrophils by blocking formyl peptide receptor 1.

It is well known that overwhelming neutrophil activation is closely related to acute and chronic inflammatory injuries. Formyl peptide receptor 1 (FPR1) plays an important role in activation of neutrophils and may represent a potent therapeutic target in inflammatory diseases. In the present study, we demonstrated that IA-LBI07-1 (IA), an extract of bioactive secondary metabolites from a marine Bacillus sp., has anti-inflammatory effects in human neutrophils. IA significantly inhibited superoxide generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated neutrophils, but failed to suppress the cell responses activated by non-FPR1 agonists. IA did not alter superoxide production and elastase activity in cell-free systems. IA also attenuated the downstream signaling from FPR1, such as the Ca2+, MAP kinases and AKT pathways. In addition, IA inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analogue of FMLP, to FPR1 in human neutrophils and FPR1-transfected HEK293 cells. Taken together, these results show that the anti-inflammatory effects of IA in human neutrophils are through the inhibition of FPR1. Also, our data suggest that IA may have therapeutic potential to decrease tissue damage induced by human neutrophils.

Propofol inhibits superoxide production, elastase release, and chemotaxis in formyl peptide-activated human neutrophils by blocking formyl peptide receptor 1.

Neutrophils play a critical role in acute and chronic inflammatory processes, including myocardial ischemia/reperfusion injury, sepsis, and adult respiratory distress syndrome. Binding of formyl peptide receptor 1 (FPR1) by N-formyl peptides can activate neutrophils and may represent a new therapeutic target in either sterile or septic inflammation. Propofol, a widely used i.v. anesthetic, has been shown to modulate immunoinflammatory responses. However, the mechanism of propofol remains to be established. In this study, we showed that propofol significantly reduced superoxide generation, elastase release, and chemotaxis in human neutrophils activated by fMLF. Propofol did not alter superoxide generation or elastase release in a cell-free system. Neither inhibitors of γ-aminobutyric acid receptors nor an inhibitor of protein kinase A reversed the inhibitory effects of propofol. In addition, propofol showed less inhibitory effects in non-FPR1-induced cell responses. The signaling pathways downstream from FPR1, involving calcium, AKT, and ERK1/2, were also competitively inhibited by propofol. These results show that propofol selectively and competitively inhibits the FPR1-induced human neutrophil activation. Consistent with the hypothesis, propofol inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analog of fMLF, to FPR1 in human neutrophils, differentiated THP-1 cells, and FPR1-transfected human embryonic kidney-293 cells. To our knowledge, our results identify, for the first time, a novel anti-inflammatory mechanism of propofol by competitively blocking FPR1 in human neutrophils. Considering the importance of N-formyl peptides in inflammatory processes, our data indicate that propofol may have therapeutic potential to attenuate neutrophil-mediated inflammatory diseases by blocking FPR1.

YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages.

Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E1 (a stable PGE2 analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE1- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE1 significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE1-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE1 also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE1-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway.

2-(2-Fluorobenzamido)benzoate ethyl ester (EFB-1) inhibits superoxide production by human neutrophils and attenuates hemorrhagic shock-induced organ dysfunction in rats.

Neutrophil activation after trauma-hemorrhagic shock (T/H) has been implicated in the development of multiple organ dysfunction (MOD). In this study, we report that a small chemical compound, 2-(2-fluorobenzamido)benzoic acid ethyl ester (EFB-1), exhibited a potent inhibitory effect on the formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced superoxide anion (O2•-) release and CD11b expression by human neutrophils. Additionally, administration of EFB-1 in rats subjected to T/H caused a significant improvement in MOD. EFB-1 treatment induced an increase in cAMP formation and protein kinase (PK) A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. FMLP-induced phosphorylation of protein kinase B (AKT), but not calcium mobilization, was reduced by EFB-1. The inhibitory effects of EFB-1 on O(2•-) production, CD11b expression, and AKT phosphorylation were reversed by PKA inhibitors (H89 and KT5720). Significantly, administration of EFB-1 (1 mg/kg body wt) attenuated the myeloperoxidase activity of the intestines, lungs, and liver and reduced the wet/dry weight ratio of the intestines and lungs and plasma alanine aminotransferase and aspartate aminotransferase levels in Sprague-Dawley rats after T/H. Therefore, EFB-1 is a new inhibitor of cAMP-specific PDE that potently suppresses O(2•-) release and CD11b expression by human neutrophils and attenuates T/H-induced MOD in rats.

DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide anion production in activated human neutrophils and attenuates hemorrhagic shock-induced lung injury in rats.

Neutrophils are activated following hemorrhagic shock and the accumulation of neutrophils in the lung is associated with lung injury. This research investigated the effects of a semisynthetic 2-benzoylaminobenzoic acid derivative, methyl 2-(2-fluorobenzamido)benzoate (DSM-RX78), on superoxide anion (O(2)(-)) production in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils, and on lung injury in Sprague-Dawley rats subjected to trauma-hemorrhage. DSM-RX78 concentration-dependently inhibited O(2)(-) production, but not elastase release, in FMLP-activated human neutrophils. DSM-RX78 displayed no superoxide-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. Significantly, DSM-RX78 increased cAMP formation and protein kinase (PK)A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. These results show that DSM-RX78 is a new inhibitor of cAMP-specific PDE. Moreover, DSM-RX78 reduced FMLP-induced phosphorylation of protein kinase B (Akt), but not calcium mobilization. The inhibitory effects of DSM-RX78 on O(2)(-) production and Akt phosphorylation were reversed by PKA inhibitors, suggesting that DSM-RX78 regulates O(2)(-) production of human neutrophils by promoting cAMP/PKA-dependent inhibition of Akt activation. On the other hand, administration of DSM-RX78 significantly attenuated the increase in myeloperoxidase activity and edema in the lung, as well as protein concentrations in bronchoalveolar lavage fluid in rats after trauma-hemorrhagic shock. In summary, these results strongly suggest that DSM-RX78 exerts anti-inflammatory effects, which result from the elevation of cAMP levels and PKA activity through its inhibition of cAMP-specific PDE. Also, our findings show that DSM-RX78 attenuates hemorrhagic shock-induced lung injury in rats.

Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigo naturalis is used by traditional Chinese medicine to treat various inflammatory diseases. AIM OF THE STUDY: Topical indigo naturalis ointment showed efficacy in treating psoriasis in our previous clinical studies. In this study, we investigated the anti-inflammatory effects of the extract of indigo naturalis (QD) and its main components indirubin, indigo, and tryptanthrin in human neutrophils. MATERIALS AND METHODS: Superoxide anion (O2(.-)) generation and elastase release were measured by spectrophotometry. Some important signals including mitogen-activated protein kinase (MAPK), cAMP, and calcium were studied by Western blot analysis, an enzyme immunoassay, and spectrofluorometry. RESULTS: QD significantly inhibited O2(.-) generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion, while neither indirubin, indigo, nor tryptanthrin produced a comparable result. QD attenuated the FMLP-induced phosphorylation of extracellular regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Furthermore, QD inhibited calcium mobilization caused by FMLP. However, QD did not affect cellular cAMP levels. On the other hand, neither indirubin, indigo, nor tryptanthrin produced similar changes in human neutrophils. CONCLUSIONS: Taken collectively, these findings indicate that QD, but not indirubin, indigo, or tryptanthrin, inhibited O2(.-) generation and elastase release in FMLP-induced human neutrophils, which was at least partially mediated by the inhibition of MAPK activation and regulation of calcium mobilization.

Suppression of superoxide anion and elastase release by C18 unsaturated fatty acids in human neutrophils.

The structure-activity relationship of 18-carbon fatty acids (C(18) FAs) on human neutrophil functions and their underlying mechanism were investigated. C(18) unsaturated (U)FAs potently inhibited superoxide anion production, elastase release, and Ca(2+) mobilization at concentrations of <10 microM in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils. However, neither saturated FA nor esterified UFAs inhibited these neutrophil functions. The inhibitory potencies of C(18) UFAs decreased in the following order: C(18):1 > C(18):2 > C(18):3 > C(18):4. Notably, the potency of attenuating Ca(2+) mobilization was closely correlated with decreasing cellular responses. The inhibitions of Ca(2+) mobilization by C(18) UFAs were not altered in a Ca(2+)-containing Na(+)-deprived medium. Significantly, C(18) UFAs increased the activities of plasma membrane Ca(2+)-ATPase (PMCA) in neutrophils and isolated cell membranes. In contrast, C(18) UFAs failed to alter either the cAMP level or phosphodiesterase activity. Moreover, C(18) UFAs did not reduce extracellular Ba(2+) entry in FMLP- and thapsigargin-activated neutrophils. In summary, the inhibition of neutrophil functions by C(18) UFAs is attributed to the blockade of Ca(2+) mobilization through modulation of PMCA. We also suggest that both the free carboxy group and the number of double bonds of the C(18) UFA structure are critical to providing the potent anti-inflammatory properties in human neutrophils.