fMLP-induced Chemotaxis Research Articles

Erianin alleviates LPS-induced acute lung injury via antagonizing P-selectin-mediated neutrophil adhesion function

Ethnopharmacological relevanceDendrobium officinale Kimura et Migo, known as “Tiepi Shihu” in traditional Chinese medicine, boasts an extensive history of medicinal use documented in the Chinese Pharmacopoeia. “Shen Nong Ben Cao Jing” records D. officinale as a superior herbal medicine for fortifying “Yin” and invigorating the five viscera. Erianin, a benzidine compound, emerges as a prominent active constituent derived from D. officinale, with the pharmacological efficacy of D. officinale closely linked to the anti-inflammatory properties of erianin. Aim of the studyAcute lung injury (ALI) is a substantial threat to global public health, while P-selectin stands out as a promising novel target for treating acute inflammatory conditions. This investigation aims to explore the therapeutic potential of erianin in ALI treatment and elucidate the underlying mechanisms. Experimental designThe effectiveness of erianin in conferring protection against ALI was investigated through comprehensive histopathological and biochemical analyses of lung tissues and bronchoalveolar lavage fluid (BALF) in an in vivo model of LPS-induced ALI in mice. The impact of erianin on fMLP-induced neutrophil chemotaxis was quantitatively assessed using the Transwell and Zigmond chamber, respectively. To determine the therapeutic target of erianin and elucidate their binding capability, a series of sophisticated assays were employed, including drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and molecular docking analyses. ResultsErianin demonstrated a significant alleviation of LPS-induced acute lung injury, characterized by reduced total cell and neutrophil counts and diminished total protein contents in BALF. Moreover, erianin exhibited a capacity to decrease proinflammatory cytokine production in both lung tissues and BALF. Notably, erianin effectively suppressed the activation of NF-κB signaling in the lung tissues of LPS- challenged mice; however, it did not exhibit in vitro inhibitory effects on inflammation in LPS-induced human pulmonary microvascular endothelial cells (HPMECs). Additionally, erianin blocked the adhesion and rolling of neutrophils on HPMECs. While erianin did not influence endothelial P-selectin expression or cytomembrane translocation, it significantly reduced the ligand affinity between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). ConclusionsErianin inhibits P-selectin-mediated neutrophil adhesion to activated endothelium, thereby alleviating ALI. The present study highlights the potential of erianin as a promising lead for ALI treatment.

A novel diselenide attenuates the carrageenan-induced inflammation by reducing neutrophil infiltration and the resulting tissue damage in mice

Selenium-containing compounds have emerged as promising treatment for redox-based and inflammatory diseases. This study aimed to investigate the in vitro and in vivo anti-inflammatory activity of a novel diselenide named as dibenzyl[diselanediyIbis(propane-3-1diyl)] dicarbamate (DD). DD reacted with HOCl (k = 9.2 x 107 M−1s−1), like glutathione (k = 1.2 x 108 M−1s−1), yielding seleninic and selenonic acid derivatives, and it also decreased HOCl formation by activated human neutrophils (IC50=4.6 μM) and purified myeloperoxidase (MPO) (IC50=3.8 μM). However, tyrosine, MPO-I and MPO-II substrates, did not restore HOCl formation in presence of DD. DD inhibited the oxidative burst in dHL-60 cells with no toxicity up to 25 µM for 48h. Next, an intraperitoneal administration of 25, 50, and 75 mg/kg DD decreased total leukocyte, neutrophil chemotaxis, and inflammation markers (MPO activity, lipid peroxidation, albumin exudation, nitrite, TNF-α, IL-1β, CXCL1/KC, and CXCL2/MIP-2) on a murine model of carrageenan-induced peritonitis. Likewise, 50 mg/kg DD (i.p.) decreased carrageenan-induced paw edema over 5h. Histological and immunohistochemistry analyses of the paw tissue showed decreased neutrophil count, edema area, and MPO, carbonylated, and nitrated protein staining. Furthermore, DD treatment decreased the fMLP-induced chemotaxis of human neutrophils (IC50=3.7 μM) in vitro with no toxicity. Lastly, DD presented no toxicity in a single-dose model using mice (50 mg/kg, i.p.) over 15 days and in Artemia salina bioassay (50 to 2000 µM), corroborating findings from in silico toxicological study. Altogether, these results demonstrate that DD attenuates carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting damage from MPO-mediated oxidative burst.

Larixol inhibits fMLP-induced superoxide anion production and chemotaxis by targeting the βγ subunit of Gi-protein of fMLP receptor in human neutrophils

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein βγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 μM)-induced superoxide anion production (IC50:1.98 ± 0.14 μM), the release of cathepsin G (IC50:2.76 ± 0.15 μM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the βγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCβ by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the βγ subunits of Gi-protein with downstream signaling of the fMLP receptor.

Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

Toll-like receptors are innate immune receptors that play a critical role in pathogen-associated molecular pattern recognition. TLR10 was recently identified and very limited data are available on its expression, mechanisms that regulate its expression, and its role in primary immune cells. To study the expression pattern of TLR10 in primary immune cells, we examined TLR10 protein expression in naive and Escherichia coli lipopolysaccharide (LPS)-activated human neutrophils. Human neutrophils challenged with LPS showed a decrease in total and surface TLR10 expression at 90 min. TLR10 in LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, to suggest its endocytosis. There was increased colocalization of TLR10 with TLR4 at LPS 60 min followed by decrease at later LPS treatment times. Treatment with TLR4 neutralizing antibody decreased cytoplasmic localization of TLR10 in LPS-treated neutrophils. Reactive oxygen species (ROS) depletion and neutralization of p65 subunit of NF-κB in LPS-treated neutrophils decreased TLR10 expression. Live cell imaging of LPS-activated neutrophils showed TLR10 translocation in the leading edge and TLR10 knockdown in neutrophils reduced their fMLP-induced chemotaxis and the number of neutrophils with pseudopodia but without affecting the expression of key proteins of actin nucleation process, ARP-3 and Diap1. Taken together, our findings show that neutrophil activation alters TLR10 expression through ROS production and NF-κB regulation, and TLR10 knockdown reduced neutrophil chemotaxis.

Larixol Inhibits Fmlp-Induced Superoxide Anion Production and Chemotaxis by Targeting the Βγ Subunit of Gi-Protein of Fmlp Receptor in Human Neutrophils

Larixol Inhibits Fmlp-Induced Superoxide Anion Production and Chemotaxis by Targeting the Βγ Subunit of Gi-Protein of Fmlp Receptor in Human Neutrophils

Prolyl-Isomerase Pin1 Controls Key fMLP-Induced Neutrophil Functions.

Neutrophils are key cells of the innate immune and inflammatory responses. They are the first blood cells to migrate to the infection site where they release high amounts of reactive oxygen species (ROS) and several peptides and enzymes required for microbial killing. However, excessive neutrophil activation can induce tissue injury participating in inflammation, thus the characterization of the enzymes involved in neutrophil activation could help to identify new pharmacological targets to treat inflammation. The prolyl-isomerase Pin1 is a ubiquitous enzyme involved in several functions, however, its role in neutrophil functions is less known. In this study, we show that the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP or fMLF), a G-protein coupled receptor (GPCR) agonist-induced Pin1 activation in human neutrophils. PiB and juglone, two Pin1 inhibitors inhibited Pin1 activity in neutrophils and consequently inhibited fMLP-induced chemotaxis and -degranulation of azurophil and specific granules as measured by myeloperoxidase and neutrophil gelatinase-associated lipocalin (NGAL) release respectively. We also showed that PiB inhibited TNFα + fMLP-induced superoxide production, confirming the effect of juglone. These data show that inhibitors of Pin1 impaired key pro-inflammatory neutrophil functions elicited by GPCR activation and suggest that Pin1 could control neutrophil inflammatory functions.

Anti-Inflammatory and Healing Activity of the Hydroalcoholic Fruit Extract of Solanum diploconos (Mart.) Bohs

Background Solanum diploconos (Mart.) Bohs is a native Brazilian plant belonging to the Solanaceae family, popularly known as “tomatinho do mato” and poorly investigated. Herein, we presented for the first time evidence for the anti-inflammatory and wound healing activities of S. diploconos fruit hydroalcoholic extract. Material and Methods. In vitro fMLP-induced chemotaxis, LPS-induced inflammatory mediator levels (cytokines by ELISA and NO release by Griess reaction), and adhesion molecule expression (CD62L, CD49d, and CD18, by flow-cytometry) were assessed in neutrophils treated with different concentrations of the extract. Inflammation resolution was measured by the efferocytosis assay and the healing activity by in vivo and in vitro assays. The air pouch model of carrageenan-induced inflammation in Swiss mice was used to investigate the in vivo anti-inflammatory effects of the extract. Leukocyte influx (by optical microscopy) and cytokine release were quantified in the pouch exudates. Additionally, the acute and subacute toxic and genotoxic effects of the extract were evaluated. Results In vitro, the extract impaired neutrophil chemotaxis and its ability to produce and/or release cytokines (TNFα, IL-1β, and IL-6) and NO upon LPS stimuli (p < 0.01). LPS-treated neutrophils incubated with the extract presented increased CD62L expression (p < 0.01), indicating a reduced activation. An enhanced efferocytosis of apoptotic neutrophils by macrophages was observed and accompanied by higher IL-10 and decreased TNFα secretion (p < 0.01). In vivo, similar results were noted, including reduction of neutrophil migration, protein exudation, and cytokine release (p < 0.01). Also, the extract increased fibroblast proliferation and promoted skin wound healing (p < 0.01). No signs of toxicity or genotoxicity were observed for the extract. Conclusion S. diploconos fruit extract is anti-inflammatory by modulating neutrophil migration/activation as well macrophage-dependent efferocytosis and inflammatory mediator release. It also indicates its potential use as a healing agent. Finally, the absence of acute toxic and genotoxic effects reinforces its possible use as medicinal product.

Abstract 1436: Mechanistic investigation of NAP1051, a lipoxin A4 biomimetic, in treating colorectal cancer

Abstract Purpose: Resolving tumor-associated inflammation in the tumor microenvironment (TME) can restore the immune response against malignant tissue. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. We developed a LXA4 biomimetic, NAP1051, with increased stability and longer half-life, and demonstrated its properties using in vitro assays and in vivo xenograft tumor models and explored the underlying mechanism of action (MoA). Methods: Differentiated HL-60 and THP-1 induced by DMSO and PMA, respectively, were used as models. dHL-60 was used to determine the effect of NAP1051 on fMLP-induced neutrophil chemotaxis. NAP1051-induced dTHP-1 efferocytosis of apoptotic dHL-60 was measured using fluorescent microscopy. Concentration escalation and time of NAP1051 exposure were used to probe the molecular mechanism using Western Blot and RT-PCR. The efficacy of oral NAP1051 was evaluated in CT26 (CRC) xenograft model established in immunocompetent Balb/c mice with a dosage escalation design. Results: NAP1051 inhibited neutrophil chemotaxis towards fMLP by &amp;gt; 40% at 1, 10 and 100 nM (p &amp;lt; 0.05). NAP1051 dose-dependently promoted dTHP-1 efferocytosis (p &amp;lt; 0.05) and was equipotent to ATLA4. In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT from 10 nM to 1 µM. When compared to ATLA4 and W-peptide, NAP1051 caused strong phosphorylation at both S473 and T308 of AKT. The NAP1051-induced p-ERK1/2 and p-AKT were in parallel and in a dose-dependent and time-dependent way. MoA studies revealed that such p-ERK1/2 was mediated by MEK1/2. However, NAP1051 led to p-AKT at both S473 and T308 despite PI3K inhibition, indicating a PI3K-independent pathway was involved. In the mouse xenograft CRC model, we demonstrated that NAP1051 significantly inhibit tumor growth when given p.o. at 5 mg/kg/day. Flow cytometry on splenic cells showed that NAP1051 reduced splenic neutrophil and MDSC populations, which correlated to the tumor sizes. IHC analyses revealed that NAP1051 decreased the intratumoral Ly6G+ neutrophils most significantly at 5 mg/kg/day (p&amp;lt;0.1). In combination with low-dose 5-FU and CTX, NAP1051 significantly decreased splenic neutrophils and MDSCs compared to chemotherapies alone (p&amp;lt;0.05). Conclusions: NAP1051 has LXA4-like activity including inhibiting fMLP-mediated neutrophil chemotaxis and promoting macrophage efferocytosis of apoptotic cells. The molecular MoA of NAP1051 involves ERK1/2 and AKT signaling pathways which are interrelated closely. A PI3K-independent pathway may be activated and resulted in AKT activation. NAP1051 inhibited tumor growth significantly at 5 mg/kg/day in a CRC model with significant changes in splenic and intratumoral immune cell populations. With these properties, NAP1051 may be an effective component in the treatment of CRCs by modulating the inflammatory immune cells in the TME. Citation Format: Tiange Dong, Priyal Dave, Brandon Ebright, Kabir Ahluwalia, Eugene Zhou, Isaac Asante, Malika Salimova, Hua Pei, Tracey Lin, Andrew Mead, Zeyang Li, Nicos Petasis, Stan Louie. Mechanistic investigation of NAP1051, a lipoxin A4 biomimetic, in treating colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1436.

Pharmacological profiling of JME-173, a novel mexiletine derivative combining dual anti-inflammatory/anti-spasmodic functions and limited action in Na+ channels

Existing evidence suggests that the local anaesthetic mexiletine can be beneficial for patients with asthma. However, caution is required since anaesthesia of the airways inhibits protective bronchodilator neuronal reflexes, limiting applications in conditions of hyperirritable airways. Here, we describe the synthesis of a new series of mexiletine analogues, which were screened for reduced activity in Na+ channels and improved smooth muscle relaxant effects, that were evaluated using the patch-clamp technique and an isolated tracheal organ bath, respectively. JME-173 (1-(4-bromo-3,5-dimethylphenoxy)propan-2-amine) was the most effective among the four mexiletine analogues investigated. JME-173 was then studied in vivo using a murine model of lung inflammation induced by cigarette smoke (CS) and in vitro using neutrophil chemotaxis and mast cell degranulation assays. Finally, the JME-173 pharmacokinetic profile was assessed using HPLC-MS/MS bioanalytical method. JME-173 directly inhibited IL-8 (CXCL8)- and FMLP-induced human neutrophil chemotaxis and allergen-induced mast cell degranulation. After oral administration 1 h before CS exposure, JME-173 (50 mg/kg) strongly reduced the increased number of macrophages and neutrophils recovered in the bronchoalveolar effluent without altering lymphocyte counts. Pharmacokinetic experiments of JME-173 (10 mg/kg, orally) showed values of maximum concentration (Cmax), maximum time (Tmax), area under the blood concentration-time curve (AUC0-t) and area under the blood concentration-time curve from 0–Inf (AUC0-inf) of 163.3 ± 38.3 ng/mL, 1.2 ± 0.3 h, 729.4 ± 118.3 ng*h/ml and 868.9 ± 117.1 ng*h/ml (means ± S.E.M.), respectively. Collectively, these findings suggest that JME-173 has the potential to be an effective oral treatment for diseases associated with bronchoconstriction and inflammation.

Mycobacterial cord factor enhances migration of neutrophil-like HL-60 cells by prolonging AKT phosphorylation.

Trehalose 6,6'-dimycolate (TDM), or cord factor, is a crucial stimulus of immune responses during Mycobacterium tuberculosis infection. Although TDM has immuno-stimulatory properties, including adjuvant activity and the ability to induce granuloma formation, the mechanisms underlying these remain unknown. We hypothesized that TDM stimulates transendothelial migration of neutrophils, which are the first immune cells to infiltrate the tissue upon infection. In this study, it was shown that TDM enhances N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis and transendothelial movement by prolonging AKT phosphorylation in human neutrophils. TDM induced expression of macrophage-inducible C-type lectin, a receptor for TDM, and induced secretion of pro-inflammatory cytokines and chemokines in differentiated HL-60 cells. In 2- and 3-D neutrophil migration assays, TDM-stimulated neutrophils showed increased fMLP-induced chemotaxis and transendothelial migration. Interestingly, following fMLP stimulation of TDM-activated neutrophils, AKT, a crucial kinase for neutrophil polarization and chemotaxis, showed prolonged phosphorylation at serine 473. Taken together, these data suggest that TDM modulates transendothelial migration of neutrophils upon mycobacterial infection through prolonged AKT phosphorylation. AKT may therefore be a promising therapeutic target for enhancing immune responses to mycobacterial infection.

Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo

Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.

Carbon Monoxide–Releasing Molecule-401 Suppresses Polymorphonuclear Leukocyte Migratory Potential by Modulating F-Actin Dynamics

PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMNwith CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P<0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distributionand fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P<0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophilmigratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics.

Endotoxin promotes neutrophil hierarchical chemotaxis via the p38-membrane receptor pathway.

Neutrophils are the most abundant leukocytes in peripheral blood and play critical a role in bacterial infection, tumor immunity and wound repair. Clarifying the process of neutrophil chemotaxis to target sites of immune activity has been a focus of increased interest within the past decade. In bacterial infectious foci, neutrophils migrate toward the bacterial-derived chemoattractant N-formyl-Met-Leu-Phe (fMLP) and ignore other intermediary chemoattractants to arrive at the area of infection. Using an under agarose chemotaxis assay, we observed that the bacterial fMLP-induced neutrophil chemotaxis signal overrode interleukin 8 (IL-8)- and leukotriene B4 (LTB4)-induced chemotaxis signals. Moreover, in the presence of bacterial lipopolysaccharide (LPS), the fMLP-induced hierarchical chemotaxis signal was enhanced. Further studies revealed that LPS increased the membrane expression of the fMLP receptor, formyl peptide receptor 1 (FPR1). However, expression levels of the membrane receptors for IL-8 and LTB4 were decreased by LPS administration. A human Phospho-mitogen-activated protein kinase (MAPK) proteome array showed that the p38 pathway was significantly activated by LPS stimulation. Moreover, p38 was responsible for the altered expression of neutrophil membrane chemoattractant receptors. Inhibition of neutrophil p38 restored LPS-improved hierarchical chemotaxis. Taken together, these data indicate that endotoxin promotes neutrophil hierarchical chemotaxis via the p38-membrane receptor pathway.

English

Neutrophils are leukocytes that are actively recruited to sites of tissue infection and/or injury by directed movement (chemotaxis). In vitro assessment of inhibition of neutrophil chemotaxis is a physiologic indicator of anti-inflammatory potential. To identify nontoxic, anti-inflammatory agents, plant-derived compounds (curcumin, resveratrol, rosmarinic acid and piperine) were assessed for effects on in vitro neutrophil movement. Effects were determined on directed migration (chemotaxis) towards the potent chemoattractant of bacterial cell wall origin, f-met-leu-phe (fMLP). Curcumin significantly inhibited neutrophil chemotaxis in a concentration-dependent manner with statistically significant inhibition at 50 and 100 &mu;M. Similarly, resveratrol (25, 50, 100 &mu;M) and rosmarinic acid (100 &mu;M) significantly inhibited fMLP-induced chemotaxis in concentration-dependent manners. Piperine had no effect on neutrophil chemotaxis. These results indicate that curcumin, resveratrol and rosmarinic acid have the potential to elicit anti-inflammatory effects. &nbsp; Key words: Neutrophils, phytochemicals, chemotaxis,&nbsp; inflammation.

Am80, a retinoic acid receptor agonist, ameliorates murine vasculitis through the suppression of neutrophil migration and activation

Vasculitis is characterized by leukocyte infiltration in the vessel walls, with destructive damage to mural structures. Retinoids are compounds that bind to retinoic acid receptors and exert biologic activities similar to those of vitamin A, including modulatory effects on cell proliferation and differentiation. This study was undertaken to examine the therapeutic effects of a synthetic retinoid, Am80, in a murine model of vasculitis induced by Candida albicans water-soluble fraction (CAWS). Vasculitis was induced in BALB/c mice by intraperitoneal injection of CAWS. Neutrophils were depleted by injection of antineutrophil antibody-positive serum. Am80 was administered orally once daily. Vasculitis was evaluated histologically. Migration of labeled adoptively transferred cells was quantified. Chemotaxis was assessed by cell mobility analysis. Production of reactive oxygen species (ROS) and phosphorylation of MAPKs were measured by flow cytometry. Concentrations of elastase were measured by enzyme-linked immunosorbent assay. Administration of CAWS induced vasculitis in the coronary arteries and aortic root, with abundant neutrophil infiltration. Depletion of neutrophils reduced CAWS-induced vasculitis. Treatment with Am80 led to a significant attenuation of the vasculitis score and inhibition of the migration of transferred neutrophils into the site of vasculitis. In vitro, Am80 suppressed fMLP-induced chemotaxis of human peripheral blood neutrophils. ROS production and elastase release by stimulated neutrophils were reduced by AM80 treatment, and Am80 also inhibited phosphorylation of ERK-1/2 and p38 in neutrophils stimulated with fMLP plus lipopolysaccharide. Am80 significantly suppressed CAWS-induced vasculitis. This effect was presumably exerted via inhibition of neutrophil migration and activation.

Increased neutrophil migration in smokers with or without chronic obstructive pulmonary disease

The number of airway neutrophils is increased in chronic obstructive pulmonary disease (COPD), and this may have a central pathophysiological role in the disease. In addition, activation of neutrophils increases their migration into sites of injury. We hypothesize that circulating neutrophils are activated in smokers. Peripheral blood neutrophils were isolated from healthy non-smokers (n = 15), and smokers with (n = 15) or without COPD (n = 15), who were matched with regard to cumulative tobacco exposure, and chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8, CXCL8) and leukotriene B(4) (LTB(4)) were assessed using the ChemoTx System (Neuro Probe Inc., Gaithersburg, MD, USA). Serum tumour necrosis factor-α (TNF-α) concentrations were measured by ELISA. Surface expression of the neutrophil activation marker, CD11b, was measured by flow cytometry. The chemotactic response to CXCL8 was increased in smokers with or without COPD (P < 0.05). Migration towards LTB(4) was increased in smokers without COPD compared with non-smokers (P < 0.05), whereas there was no difference in fMLP-induced chemotaxis between the groups. There was a correlation between serum TNF-α levels and migration induced by IL-8 (Rho = 0.442; P = 0.038) and LTB(4) (Rho = 0.428; P = 0.044) in the smokers. Furthermore, there was a tendency towards higher CD11b expression in the COPD group (P = 0.057). Chemotaxis of circulating neutrophils towards CXCL8, and partly towards LTB(4), is increased in smokers, indicating a systemic influence of smoking on cell activation, irrespective of the presence of airflow limitation. The relationship between TNF-α and chemotactic response suggests that TNF-α is involved in neutrophil activation, resulting in enhanced migration.

Anti-neutrophilic inflammatory activity of ASP3258, a novel phosphodiesterase type 4 inhibitor

Neutrophil-dominant pulmonary inflammation is an important feature of chronic obstructive pulmonary disease (COPD). Here, we evaluated the in vitro and in vivo anti-neutrophilic inflammatory activities of ASP3258, a novel, orally active, and selective phosphodiesterase (PDE) 4 inhibitor with anti-inflammatory potency comparable to that of second-generation compound roflumilast but with lower emetic activity in vivo. In in vitro experiments using human peripheral blood neutrophils, PDE4 inhibitors ASP3258, cilomilast, and roflumilast inhibited fMLP-induced superoxide production in a concentration-dependent manner with IC50 values of 5.0, 96, and 4.7 nM, respectively. ASP3258, cilomilast, and roflumilast also attenuated fMLP-induced neutrophil chemotaxis in a concentration-dependent manner with IC30 values of 18, 270, and 9.7 nM, respectively. In contrast, the glucocorticoid prednisolone inhibited neither superoxide production nor chemotaxis up to 1 μM. In a rat model of lipopolysaccharide (LPS)-induced lung inflammation, orally administered ASP3258, cilomilast, roflumilast, and prednisolone (at 10 or 30 mg/kg) dose-dependently attenuated pulmonary accumulation of neutrophils. The inhibitory effect of ASP3258 was more potent than cilomilast and almost the same as roflumilast and prednisolone. Treatment with ASP3258 inhibited the elevation of TNF-α in the bronchoalveolar lavage fluid following LPS instillation. Histological examination revealed significant inhibition of neutrophil and macrophage infiltration into alveoli by ASP3258. Overall, these findings suggest that ASP3258 has therapeutic potential for treating neutrophilic inflammation such as COPD, partly through direct inhibition of neutrophil activation as well as possibly through inhibition of the TNF-α-mediated pathway.

Protective effects of luteolin against lipopolysaccharide-induced acute lung injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils

To investigate whether luteolin, the major polyphenolic components of Lonicera japonica, has beneficial effects against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to determine whether the protective mechanism involves anti-inflammatory effects on neutrophils. ALI was induced with intratracheal instillation of LPS in mice. The level of ALI was determined by measuring the cell count and protein content in bronchoalveolar lavage (BAL) fluid. Neutrophils were stimulated with formyl-Met-Leu-Phe (fMLP) or LPS in vitro. Chemotaxis and superoxide anion generation were measured to evaluate neutrophil activation. The potential involvement of intracellular signaling molecules in regulating neutrophil activation was analyzed by using Western blot. LPS induced ALI in mice, as evidenced with leukocyte infiltration and protein leakage into the lungs. Luteolin attenuated LPS-induced leukocyte infiltration and protein extravasation. In cell studies, luteolin attenuated the fMLP-induced neutrophil chemotaxis and respiratory burst (IC(50) 0.2+/-0.1 micromol/L and 2.2+/-0.8 micromol/L, respectively), but had a negligible effect on superoxide anion generation during phorbol myristate acetate stimulation. Furthermore luteolin effectively blocked MAPK/ERK kinase 1/2 (MEK), extracellular signal-regulated kinase (ERK), and Akt phosphorylation in fMLP- and LPS-stimulated neutrophils. These results indicate that luteolin has beneficial effects against LPS-induced ALI in mice, and the attenuation of neutrophil chemotaxis and respiratory burst by luteolin involves the blockade of MEK-, ERK-, and Akt-related signaling cascades.

Validating 123I-metaiodobenzylguanidine as a platelet marker for non-invasive imaging in rabbits

Introduction Recent in vitro studies in our laboratory have demonstrated that platelets are necessary for leukocyte recruitment and airway remodelling in models of allergic airway inflammation, and also migrate to lung tissues in response to anti-IgE or relevant allergens in allergic asthma. Non-invasive imaging of platelet migration in vivo would provide a further insight into the roles of platelets in inflammatory diseases such as asthma, and metaiodobenzylguanidine (MIBG) was considered as a suitable platelet marker. Methods The kinetics of MIBG uptake into rabbit platelets, the effect of MIBG on platelet function and the effect of platelet activation on MIBG uptake and retention were investigated. MIBG-labelled platelets were administered intravenously into rabbits and the time course of radioactivity in the lung and blood was monitored as a function of stimulation. Results Following a 4 h incubation of MIBG in rabbit PRP, a near maximal MIBG uptake (52.4 ± 20.2%) in platelets occurred. This time point was chosen for subsequent in vitro studies. In vitro platelet function studies showed that MIBG has no effect on ADP or PAF-induced platelet aggregation, PAF-induced thromboxane production or fMLP-induced platelet chemotaxis. However, serotonin showed a significant effect on MIBG uptake and retention, but only at high concentrations. Stimulation of rabbit platelets with ADP and PAF caused a significant release of stored MIBG in vitro. Following i.v. administration of MIBG labelled platelets, the response to i.v. ADP and PAF stimulation was small but significant. Discussion The release of MIBG from platelets in vivo, particularly following stimulation, leads to high background levels. Therefore, MIBG may have limited utility as a label for imaging platelets in vivo using PET. However, it may be a useful marker in detecting pathological conditions where platelet migration is involved.

Flavonols attenuate the immediate and late-phase asthmatic responses to aerosolized-ovalbumin exposure in the conscious guinea pig

We previously reported that quercetin and rutin have potent, anti-asthmatic activity, but the structure-activity relationships of flavonoids and anti-asthmatic agents are still poorly understood. In the current study, the effects of kaempferol, fisetin, and morin on the immediate-phase response (IAR) and late-phase response (LAR) caused by exposure to aerosolized-ovalbumin (OA) in OA-sensitized guinea pigs were evaluated by determining the specific airway resistance (sRaw), recruitment of leukocytes and chemical mediators in bronchoalveolar lavage fluid (BALF), histopathological surveys, and determination of neutrophil chemotaxis. Fisetin and kaempherol (30 mg/kg, p.o.) significantly ( P < 0.01) inhibited sRaw by 47.93% and 30.05% in IAR, and 54.45% and 40.50% in LAR, when compared to vehicle control, respectively. Furthermore, all three studied flavonols (30 mg/kg, p.o.) significantly ( P < 0.05) inhibited the recruitment of total, as well as subtypes of, leukocytes into the lung BALF. This recruitment inhibition corresponded to the inhibition of leukocyte infiltration, particularly of eosinophils and neutrophils, into the lung in pathological surveys and formly-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil chemotaxis studies. Kaempferol inhibited FMLP-induced neutrophil chemotaxis in a concentration-dependent manner in a tested range of 1–100 μM. Fisetin inhibited histamine content and peroxidase (EPO) activity in BALF in a dose-dependent manner. All three tested flavonols significantly ( P < 0.01) inhibited histamine content at 10 mg/kg, and phospholipase A 2 (PLA 2) and EPO activities at 30 mg/kg (p.o.) in BALF. Kaempherol had a greater anti-asthmatic effect than other flavonols. Fisetin demonstrated the greatest inhibition of sRaw, whereas morin had lesser effects. These results indicate that the lower the molecular weight, the greater the anti-asthmatic activities of these compounds.