Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

2',3-dihydroxy-5-methoxybiphenyl suppresses fMLP-induced superoxide anion production and cathepsin G release by targeting the ß-subunit of G-protein in human neutrophils.

This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between ß subunit of G-protein (Gß) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gß-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC50:2.57 ±â€¯0.22 µM), cathepsin G release (IC50:18.72 ±â€¯3.76 µM) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gß-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47ph°x and P40ph°x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gß-protein with PLCß2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47ph°x and P40ph°x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gß-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47ph°x and p40phox.

Isolation of Terpenoids from the Stem of Ficus aurantiaca Griff and their Effects on Reactive Oxygen Species Production and Chemotactic Activity of Neutrophils.

Three new triterpenoids; namely 28,28,30-trihydroxylupeol (1); 3,21,21,26-tetrahydroxy-lanostanoic acid (2) and dehydroxybetulinic acid (3) and seven known compounds; i.e., taraxerone (4); taraxerol (5); ethyl palmitate (6); herniarin (7); stigmasterol (8); ursolic acid (9) and acetyl ursolic acid (10) were isolated from the stem of Ficus aurantiaca Griff. The structures of the compounds were established by spectroscopic techniques. The compounds were evaluated for their inhibitory effects on polymorphonuclear leukocyte (PMN) chemotaxis by using the Boyden chamber technique and on human whole blood and neutrophil reactive oxygen species (ROS) production by using a luminol-based chemiluminescence assay. Among the compounds tested, compounds 1-4, 6 and 9 exhibited strong inhibition of PMN migration towards the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) with IC50 values of 6.8; 2.8; 2.5; 4.1; 3.7 and 3.6 µM, respectively, comparable to that of the positive control ibuprofen (6.7 µM). Compounds 2-4, 6, 7 and 9 exhibited strong inhibition of ROS production of PMNs with IC50 values of 0.9; 0.9; 1.3; 1.1; 0.5 and 0.8 µM, respectively, which were lower than that of aspirin (9.4 µM). The bioactive compounds might be potential lead molecules for the development of new immunomodulatory agents to modulate the innate immune response of phagocytes.

Limonoids from the Seeds of Swietenia macrophylla and Their Anti-Inflammatory Activities.

A new limonoid, swietemacrophin (1), was isolated from the seeds of Swietenia macrophylla, together with five known compounds 2-6. The structure of 1 was determined through extensive 1D/2D-NMR and mass-spectrometric analyses. Swietemacrophin (1), humilinolide F (2), 3,6-O,O-diacetylswietenolide (3), 3-O-tigloylswietenolide (4), and swietemahonin E (5) exhibited inhibition (IC50 values≤45.44 µM) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Compounds 1, 4, 5, and swietenine (6) showed potent inhibition with IC50 values≤36.32 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation.

Splitomicin inhibits fMLP-induced superoxide anion production in human neutrophils by activate cAMP/PKA signaling inhibition of ERK pathway.

Splitomicin, is a cell-permeable lactone derived from naphthol and known to be a potent selective inhibitor of Sir2 (silent information regulator 2). Previous studies have demonstrated that naphtholic compounds possess an inhibitory effect on neutrophils. Here, we present our investigation on the inhibitory effects of splitomicin in human neutrophils. The primary goal of our study was to locate a possible candidate on inflammatory reactions and to hopefully develop a novel anti-inflammatory therapy. Neutrophils were prepared following standard procedures. Neutrophils induced by either fMLP (1 µM) or PMA (100 nM) were observed using a flow cytometer and the intracellular production of superoxide anions was investigated at different splitomicin concentrations. The cytosolic Ca(++) influx concentration was measured using a fluorescence spectrophotometer, and Mac-1 expression was detected with a flow cytometer. The MAP kinases were measured using western blotting. Our results showed that splitomicin inhibited superoxide anion production by fMLP (1 µM) and NaF (20mM) in a concentration-dependent manner (37.5-450 µM). Splitomicin (300 and 450 µM) also suppressed fMLP-induced intracellular calcium ion mobilization and extracellular-signal regulated kinase (ERK) phosphorylation. Moreover, splitomicin could inhibit fMLP-induced Mac-1 expression and increase cAMP levels in human neutrophils. Our data demonstrated that splitomicin exhibits a noticeable inhibitory effect on superoxide anion production in human neutrophils. This negative effect was well-correlated with increased cAMP levels via PKA activity and the subsequent inhibition of ERK (p42/p44) phosphorylation to decrease superoxide anion production.

A role for the low-affinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils.

The formation of adenosine dampens inflammation by inhibiting most cells of the immune system. Among its actions on neutrophils, adenosine suppresses superoxide generation and regulates chemotactic activity. To date, most evidence implicates the G(s) protein-coupled A(2A) adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A(2B)AR is now known to be expressed in neutrophils and that it is a G(s) protein-coupled receptor, we examined in this study whether it signals to suppress neutrophil activities by using 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY 60-6583), a new agonist for the human A(2B)AR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A(2B)AR. We found that treating mouse neutrophils with low concentrations (10(-9) and 10(-8) M) of BAY 60-6583 inhibited formylated-methionine-leucine-phenylalanine (fMLP)-stimulated superoxide production by either naive neutrophils, tumor necrosis factor-α-primed neutrophils, or neutrophils isolated from mice treated systemically with lipopolysaccharide. This inhibitory action of BAY 60-6583 was confirmed to involve the A(2B)AR in experiments using neutrophils obtained from A(2B)AR gene knockout mice. It is noteworthy that BAY 60-6583 increased fMLP-stimulated superoxide production at higher concentrations (>1 µM), which was attributed to an AR-independent effect. In a standard Boyden chamber migration assay, BAY 60-6583 alone did not stimulate neutrophil chemotaxis or influence chemotaxis in response to fMLP. These results indicate that the A(2B)AR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A(2A)AR in regulating the proinflammatory actions of neutrophils.

Appraisal of the antichemotactic activity of flavonoids on polymorphonuclear neutrophils.

Flavonoids are polyphenols that are ubiquitous in plants and frequently consumed in the diet. They are suggested to have many beneficial actions on human health, including anti-inflammatory activity. Their properties have been studied in a number of cell types, but little is known about their effects on neutrophil biology. Consequently, we selected 25 flavonoids with different structural features to evaluate their in vitro inhibition of rat polymorphonuclear neutrophil (PMN) chemotaxis, employing a modified Boyden chamber. Migratory activity was measured towards a chemotactic stimulant, formyl-Met-Leu-Phe or lipopolysaccharide. Furthermore, the cytotoxic effect of flavonoids on PMNs was determined by the release of cytosolic lactate dehydrogenase (LDH). Ten flavonoids significantly retarded the migration of PMNs with at least one of the concentrations tested in a range between 0.625 and 100 µM; the best antichemotactic agents were flavone, flavonol, quercetin and rutin. None of the flavanones evaluated presented any significant inhibition of migration in this assay. Our findings indicated that non-hydroxylated flavones possess a better antichemotactic activity when compared to flavones with hydroxy groups. The presence of a sugar moiety in rutin did not produce any increase in this effect, when compared to the respective aglycone analogue. Finally, none of the flavonoids exhibited cell toxicity and for many of these flavonoids this is the first report of the inhibition of PMN chemotaxis.

Guinea pig ileum motility stimulation elicited by N-formyl-Met-Leu-Phe (fMLF) involves neurotransmitters and prostanoids.

In guinea-pig ileum (GPI), the chemotactic peptide N-formyl-Met-Leu-Phe-OH (fMLF) possesses spasmogenic properties through the activation of formyl peptide receptors (FPRs). Despite this, the mediators involved remain to be elucidated. fMLF (1nM-1µM) induced a dose-dependent contraction of GPI (EC(50)=24nM), that is blocked by pre-treatment with the FPRs antagonist Boc(2). The pre-treatment with tetrodotoxin (TTX) atropine or with SR140333 reduced the fMLF-induced contraction, whereas with hexamethonium, MEN10627, SB222200, mepyramine, cimetidine, thioperamide or methysergide did not produce any effect. With DuP697 pre-treatment, but not with piroxicam, reduced the fMLF-induced contraction. After stimulation with 24nM fMLF, a strong increase in the PGE(2) levels was observed. Finally, the concomitant blocking of the NK(1) receptor, the muscarinic receptors and COX-2 abolished the GPI contractions induced by fMLF. fMLF induced a concentration-dependent contraction of guinea-pig jejunum (EC(50)=11nM), proximal colon (EC(50)=3.5nM) and distal colon (EC(50)=2.2nM), with a time-course similar to that observed in GPI. In these preparations as well, the co-administration of atropine, SR140333 and DuP697 abolished the contractions induced by fMLF. Intraperitoneal injection of fMLF (0.1 or 1µmol/kg) enhanced the gastrointestinal motility in mice, abolished by the co-administration of atropine, SR140333 and DuP697. In conclusion, we showed that fMLF exerts spasmogenic actions on guinea-pig intestine both in vitro and in vivo through the release of acetylcholine and substance P from myenteric motorneurons and through prostanoids, probably from the inflammatory cells of the enteric immune system.

1-Methyl and 1-(2-hydroxyalkyl)-5-(3-alkyl/cycloalkyl/phenyl/naphthylureido)-1H-pyrazole-4-carboxylic acid ethyl esters as potent human neutrophil chemotaxis inhibitors.

In this paper we report the synthesis and the chemotaxis inhibitory activity of a number of 1H-pyrazole-4-carboxylic acid ethyl esters 2 functionalized in N1 with a methyl group or different hydroxyalkyl chains and in position 5 with a series of 3-substituted urea groups. These compounds were designed as development of previous pyrazole-urea derivatives that resulted potent IL8-induced neutrophil chemotaxis inhibitors in vitro. Most of the new compounds revealed a potent inhibition of both IL8- and fMLP-OMe-stimulated neutrophil chemotaxis. The most active compounds in the fMLP-OMe induced chemotaxis test showed IC(50) in the range 0.19 nM-2 microM; but we observed a very strong inhibition in the IL8-induced chemotaxis test, having the most active compounds IC(50) at pM concentrations. In vivo compounds 2e and 2f, although to a lesser extent, at 50mg/kg os decreased granulocyte infiltration in zymosan-induced peritonitis in mice.

Evaluation of the Role of FMLF chemotaxic peptide receptors in umbilical cord blood granulocytes from newborns at risk of infectious inflammatory diseases.

We studied the role of receptors with high and low affinity for fMLF chemotaxic peptide in the generation of active oxygen species by umbilical cord blood granulocytes from newborns with normal neonatal period, born after normal or complicated gestation, in children with manifestations of bacterial infection born after complicated pregnancy, and in granulocytes of non-pregnant women with normal reproductive function. Granulocytes of children born after complicated pregnancy exhibited high reactivity in induction of respiratory burst in a wide range of fMLF concentrations. The presentation of receptors with high and low affinity on granulocytes during initiation of the respiratory burst differs in children born after complicated pregnancy and in healthy babies born after normal gestation. Presumably, the detected differences result from high expression of receptors with low affinity for fMLF and disorders or immaturity of mechanisms responsible for receptor inactivation.

Blockade of cytosolic phospholipase A(2) and 5-lipoxygenase activation in neutrophils by a natural isoflavanquinone abruquinone A.

Abruquinone A, a natural isoflavanquinone, suppressed A23187- and formyl-Met-Leu-Phe (fMLP)-induced production of thromboxane B(2) and leukotriene B(4) from rat neutrophils. This compound failed to inhibit the enzymatic activity of ram seminal vesicles cyclooxygenase (COX) and human recombinant 5-lipoxygenase (5-LO) in cell-free systems. Abruquinone A diminished the arachidonic acid release from [(3)H]arachidonic acid-loaded neutrophils stimulated with either fMLP or A23187, whereas it had no inhibitory effect on the cytosolic phospholipase A(2) (cPLA(2)) activity of neutrophil cytosolic fraction. Based on the Western blot analysis, the nuclear membrane recruitment of cPLA(2) and 5-LO was inhibited by abruquinone A in A23187- as well as in fMLP-stimulated cells. Moreover, the phosphorylation of both cPLA(2) and extracellular signal regulated kinases (ERKs) induced by fMLP and A23187 was attenuated by abruquinone A in a parallel concentration-dependent manner. Abruquinone A attenuated both fMLP- and ionomycin-mediated [Ca(2+)](i) elevation in a concentration range that inhibited the recruitment of cPLA(2) to nuclear membrane. These results indicate that the blockade of leukotriene B(4) production by abruquinone A implicates the attenuation of 5-LO membrane translocation. Inhibition of thromboxane B(2) production by abruquinone A is due to the attenuation of cPLA(2) membrane recruitment and/or cPLA(2) phosphorylation through the blockade of [Ca(2+)](i) elevation and ERK activation, respectively.

(2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl)butyrolactone suppresses fMLP-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils.

This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 microM), PMA (100 nM) or leukotriene B(4) (LTB(4); 1 microM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC(50) value of 0.3+/-0.1 microM. Intracellular signaling caused by fMLP, PMA or LTB(4) were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB(4) or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC(50) of 1.5+/-0.2 microM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.

2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives: new potent inhibitors of fMLP-induced neutrophil chemotaxis.

It is well known that both acute and chronic autoimmune inflammatory disorders arise following a breakdown in control of neutrophil activation and recruitment. In the search for new anti-inflammatory agents, we synthesized some new 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives and tested them in vitro in order to evaluate their ability to interfere with human neutrophil functions. All tested compounds showed strong inhibition of fMLP-OMe-induced chemotaxis, although they appeared unable to block degranulation and the fMLP-OMe-induced respiratory burst, and were inactive in binding experiments.

Inhibitory effects of N-acetylcysteine on the functional responses of human eosinophils in vitro.

BACKGROUND: Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. OBJECTIVE: The aim of this work was to study the effects of the clinically used antioxidant N-acetyl-L-cysteine (NAC) on the functional responses of human-isolated eosinophils. METHODS: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca(2+) signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47(phox)-p67(phox) translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay). RESULTS: NAC (0.1-1 mm) inhibited the extracellular generation of oxygen species induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with -logIC(50) values of 3.61+/-0.03 and 3.36+/-0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5-1 mm). NAC (1 mm) did not alter the fMLP-induced Ca(2+) signal but augmented the eosinophil content of reduced GSH and inhibited p47(phox)-p67(phox) translocation. NAC inhibited the release of ECP ( approximately 90% inhibition at 1 mm) from fMLP-activated eosinophils. CONCLUSION: Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation.

Adiponectin inhibits superoxide generation by human neutrophils.

Adiponectin (Ad), a member of the adipocytokine family, has been reported to possess antiinflammatory properties. We investigated the effects of full-length human Ad (hAd) on phorbol 12-myristate 13-acetate (PMA)-induced O2-* generation by human neutrophils. hAd, even at the lowest tested concentration of 0.001 microg/ml, after 30-min pretreatment of cells, significantly inhibited O2-* generation by neutrophils stimulated with PMA (100 nM). However, no relation between the dose of hAd and extent of inhibition of PMA-induced O2-* generation was observed with increasing the concentration of hAd up to 1 microg/ml. hAd also significantly inhibited neutrophil O2-* generation stimulated by N-formyl-methionyl-leucyl-phenylalanine (100 microM) and diacylglycerol (500 nM), as well as the PMA-induced neutrophil nitroblue tetrazolium reduction and H2O2 formation. Pretreatment of neutrophils with pronase-digested hAd failed to inhibit the PMA-induced O2-* generation. For the first time, this study revealed that Ad inhibited O2-* generation by neutrophils, possibly through regulation of NADPH oxidase.

S100A8 triggers oxidation-sensitive repulsion of neutrophils.

The inflammatory response to tissue injury is a multi-faceted process. During this process, neutrophils migrate in the extravascular spaces, directed to the site of injury by chemical gradients generated by chemotactic molecules. S100A8, a protein associated with a wide variety of inflammatory conditions, is heavily over-expressed in association with inflammation. We hypothesized that human S100A8 possesses neutrophil-repelling properties that result in an anti-inflammatory effect in vivo. The chemotactic activity of S100A8 on neutrophils was tested in Transwell chemotaxis assays. Analysis of the data indicates that S100A8 causes a repulsion of peripheral neutrophils, an activity that S100A8 loses upon its oxidation. Using a mutant of S100A8 resistant to oxidation and consistent with the in vitro findings, we demonstrated that S100A8 causes a strong anti-inflammatory effect in the rat air-pouch model of inflammation in vivo. These data highlight a naturally occurring novel anti-inflammatory pathway and provide potential molecular targets for the development of novel anti-inflammatory therapeutics. Abbrevations: ethylene diamine tetraacetic acid (EDTA); limulus amoebocyte lysate assay (LAL); pertussis toxin (PTX); forward scatter (FSC); Interleukin-8 (IL-8); formyl-Met-Leu-Phe (fMLP); monocyte chemotactic protein 1 (MCP1).

The effect of morphine on mast cell-mediated mucosal permeability.

BACKGROUND: The late phase of post-traumatic multisystem organ failure is associated with sepsis from organisms that normally reside within the gut's lumen. Morphine, a commonly employed analgesic in injured patients, is associated with intestinal stasis, bacterial overgrowth, and translocation when administered to rats. N-formyl-methionyl-leucyl-phenylalanine (FMLP), a toxic product of gram-negative organisms, provokes an increase in mucosal permeability when infused into the ileal lumen of this species. The current study was designed to examine the effects of morphine on FMLP perturbation of the mucosal barrier of the ileum of rats and mice to an impermeant macromolecule, dextran 4400. The potential role of mucosal mast cells in the response to either agent alone or in combination was examined. METHODS: Intact and isolated segments of distal ileum of naïve and sensitized (Trichinella spiralis and egg albumin) Sprague-Dawley rats were exposed to FMLP with or without morphine or doxantrazole, a mast cell-stabilizing agent. Isolated segments of distal ileum of mast cell-deficient mice also were studied. RESULTS: Mucosal exposure of distal ileal mucosa (intact and isolated, and naive and sensitized) to FMLP was associated with an increase in permeability to dextran 4400, which was completely ablated by morphine and doxantrazole. Sensitization was associated with a prolongation of the FMLP response. Ilea of mast cell-deficient mice (but not their wild type litter mates) were unresponsive to FMLP. CONCLUSIONS: Morphine antagonizes the provocative effect of FMLP on the mucosal barrier to dextran 4400 of the ilea of rats and mice. Intestinal mucosal mast cells play a central role in the process.

Differential effects of short-acting beta2-agonists on human granulocyte functions.

BACKGROUND: beta2-Adrenergic agonists play a pivotal role in the management of bronchial asthma. Although the major effect of short-acting beta2-agonists on the airway is relaxation of smooth muscles, they may also have several effects on surrounding immunomodulatory cells. METHODS: We examined whether widely used short-acting beta2-agonists differ in their ability to modulate granulocyte functions, such as superoxide anion (O2-) production and degranulation. RESULTS: Procaterol (PC), a full agonist, significantly inhibited both O2- production by granulocytes (neutrophils and eosinophils) and their degranulation at the clinically relevant concentrations, whereas salbutamol and tulobuterol (partial agonists) showed smaller effects. PC inhibited N-formyl methionyl-leucyl-phenylalanine-induced O2- production and peroxidase release, but failed to inhibit responses induced by phorbol 12-myristate 13-acetate and/or opsonized zymosan. Exposure to 5 x 10(-8)M PC for 120 min resulted in approximately 50% inhibition of O2- production and degranulation of neutrophils. The effects of beta2-agonists were more obvious in neutrophils than in eosinophils. A selective beta2-receptor antagonist, ICI-118551, reversed the inhibitory effect of beta2-agonists (PC, salbutamol, tulobuterol B) on N-formyl methionyl-leucyl-phenylalanine-induced O2- production. CONCLUSIONS: These results suggest that beta2-agonists had an inhibitory effect on granulocyte functions, mainly mediated viareceptors and their efficacy. Our observations support that beta2-agonists with a rapid onset of action and high intrinsic efficacy (short-acting and full agonists) may be optimal for the rescue therapy against acute asthma attack and sedation of its airway inflammation in an early phase.

Dapsone suppresses human neutrophil superoxide production and elastase release in a calcium-dependent manner.

BACKGROUND: Dapsone (4,4'-diaminodiphenyl sulphone) is a powerful therapeutic tool in many skin diseases including neutrophilic dermatoses. The drug has an outstanding therapeutic efficacy against many skin diseases characterized by neutrophil-rich infiltrates; however, mechanisms of its action are poorly understood. OBJECTIVES: We investigated the effects of dapsone on respiratory and secretory functions of human neutrophils triggered by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the physiological agonist C5a, and phorbol myristate acetate (PMA). METHODS: Human neutrophils were isolated from venous blood obtained from healthy donors. We detected extracellular production of superoxide (O(2) (-)) by cytochrome C reduction assay, and intracellular production of O(2) (-) by flow cytometry. Neutrophil elastase release was measured by the cleavage of the specific elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. Measurement of cytosolic free calcium concentration was performed using the calcium-reactive fluorescence probe, Fluo-3. RESULTS: Dapsone suppressed intra- and extracellular production of O(2) (-) and elastase release triggered by fMLP and C5a, but not by PMA. Both fMLP and C5a signalled the above pathways by inducing calcium influx, but PMA functions bypassed calcium influx. Dapsone was capable of antagonizing the induction of calcium influx. CONCLUSIONS: These findings suggest that one mechanism of the anti-inflammatory action of dapsone is inhibition of calcium-dependent functions of neutrophils including release of tissue-damaging oxidants and proteases in the affected skin.

Lysophosphatidylcholine modulates neutrophil oxidant production through elevation of cyclic AMP.

Lysophosphatidylcholine (LPC) is an oxidized phospholipid present in micromolar concentrations in blood and inflamed tissues. The effects of LPC on neutrophil functions remain incompletely understood, because conflicting reports exist for its stimulatory and inhibitory roles. We report in this study that LPC inhibits superoxide generation in fMLP- and PMA-stimulated neutrophils without affecting fMLP-induced Ca(2+) mobilization and cell viability. This effect was observed with LPC dissolved in ethanol, but not with LPC stock solutions prepared in water or in BSA-containing aqueous solution with sonication. Under the same experimental conditions, platelet-activating factor primed neutrophils for superoxide generation. The inhibitory effect of LPC was observed within 30 s after its application and was maximal at LPC concentrations between 0.1 and 1 muM. Inhibition of superoxide generation was accompanied by a 2.5-fold increase in the intracellular cAMP concentration. In addition, LPC reduced fMLP-stimulated phosphorylation of ERK and Akt and membrane translocation of p67(phox) and p47(phox). The protein kinase A inhibitors H-89 and adenosine 3'5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP) partially restored superoxide production in LPC-treated neutrophils, indicating involvement of protein kinase A in LPC-mediated inhibition. Using an ex vivo mouse lung perfusion model that measures lung weight change and capillary filtration coefficient, we found that LPC prevented lung vascular injury mediated by fMLP-activated neutrophils. Taken together, these results suggest that LPC-induced elevation of intracellular cAMP is partially responsible for its inhibition of neutrophil NADPH oxidase activation. A similar mechanism of inhibition may be used for the control of neutrophil-mediated tissue injury.