Rabbit Neutrophils Research Articles

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

Antimicrobial α-defensins as multi-target inhibitors against amyloid formation and microbial infection.

Amyloid aggregation and microbial infection are considered as pathological risk factors for developing amyloid diseases, including Alzheimer's disease (AD), type II diabetes (T2D), Parkinson's disease (PD), and medullary thyroid carcinoma (MTC). Due to the multifactorial nature of amyloid diseases, single-target drugs and treatments have mostly failed to inhibit amyloid aggregation and microbial infection simultaneously, thus leading to marginal benefits for amyloid inhibition and medical treatments. Herein, we proposed and demonstrated a new “anti-amyloid and antimicrobial hypothesis” to discover two host-defense antimicrobial peptides of α-defensins containing β-rich structures (human neutrophil peptide of HNP-1 and rabbit neutrophil peptide of NP-3A), which have demonstrated multi-target, sequence-independent functions to (i) prevent the aggregation and misfolding of different amyloid proteins of amyloid-β (Aβ, associated with AD), human islet amyloid polypeptide (hIAPP, associated with T2D), and human calcitonin (hCT, associated with MTC) at sub-stoichiometric concentrations, (ii) reduce amyloid-induced cell toxicity, and (iii) retain their original antimicrobial activity upon the formation of complexes with amyloid peptides. Further structural analysis showed that the sequence-independent amyloid inhibition function of α-defensins mainly stems from their cross-interactions with amyloid proteins via β-structure interactions. The discovery of antimicrobial peptides containing β-structures to inhibit both microbial infection and amyloid aggregation greatly expands the new therapeutic potential of antimicrobial peptides as multi-target amyloid inhibitors for better understanding pathological causes and treatments of amyloid diseases.

Evaluation of Platelet-Rich Plasma and Neutrophil Antimicrobial Extract as Two Autologous Blood-Derived Agents

The platelet-rich plasma (PRP) and antimicrobial peptides neutrophil extract (AMP extract) were prepared from rabbit neutrophils as two autologous blood-derived preparations, which could be applied locally to enhance healing process of tissues. Both preparations were analyzed using the MALDI TOF method for accurate qualitative assay. Growth factors (PDGF and VEGF) and microbicidal protein were reported in PRP. In AMP extract α-defensins, namely; NP-1, -2, -3a, -3b, -4, and -5 and cathelicidins represented among other by 15-kDa antibacterial protein (p15s) were detected. In the second part of our study the influence of antimicrobial extract on macrophages in vitro was tested. Then, degranulation of neutrophils in vitro and generation of reactive intermediates by these cells under the influence of AMP extract were assessed. As estimated, the addition of AMP extract into cultures of macrophages decreased superoxide anion generation after 5 days of incubation. Furthermore, AMP extract inhibited degranulation and respiratory burst in neutrophils, therefore in this regard it suppress proinflammatory effect of two studied populations of leukocytes.

Effects of neutrophils peptide-1 transgenic Chlorella ellipsoidea on the gut microbiota of male Sprague-Dawley rats, as revealed by high-throughput 16S rRNA sequencing.

Rabbit neutrophils peptide-1 (NP-1) is a type of defensin that possesses a broad spectrum of antimicrobial activity. Chlorella ellipsoidea is a new eukaryotic expression system for exogenously producing NP-1. The NP-1 transgenic C. ellipsoidea can be directly added into feed as antimicrobial agent without any purification procedure for the NP-1 peptide. However, the effects of C. ellipsoidea and NP-1 on the host gut microbiota should be explored before application. In this study, diets containing different concentrations (1.25, 2.5, and 5%) of C. ellipsoidea and NP-1 transgenic C. ellipsoidea were administered to male Sprague-Dawley rats. Compared with the chow diet control group, none of the experimental groups showed any significant differences in their growth indices, and no histopathological damage was observed. The phylotypes of gut microbiota in the control group, the 5% C. ellipsoidea diet group and the 5% NP-1 transgenic C. ellipsoidea diet group were determined by 16S rRNA sequencing. The results showed that both 5% experimental groups had shifted community memberships of gut microbiota. In particular, the 5% NP-1 transgenic C. ellipsoidea diet exhibited an increased abundance of most Gram-positive bacterial taxa and a reduced abundance of most Gram-negative bacterial taxa, and it promoted the growth of some lactic acid bacterial genera. Lactic acid bacteria, especially the Bifidobacterium and Lactobacillus, have been widely reported to be benefic effects on the host. Thus NP-1 transgenic C. ellipsoidea is promising feed additive and gut regulator, as it have the potential to increase the abundance of Bifidobacterium and Lactobacillus in gut microbiota of animal.

Effect of mutated defensin NP-1 on sciatic nerve regeneration after transection—A pivot study

Defensins are small cationic peptides that constitute the first line of defense against pathogens and are involved in immune regulation. In this study, their role in peripheral nerve regeneration was investigated. Rat sciatic nerves were transected and the two nerve stumps were bridged by a chitin conduit with a gap of 5mm between the stumps. The animals were injected intramuscularly with mutated rabbit neutrophil peptide 1 (defensin mNP-1), the positive control nerve growth factor (NGF) or the negative control saline, for 7 consecutive days after repair. After 6 weeks, the sciatic functional index (SFI), MNCV (motor nerve conductive velocity) and morphological parameters including myelinated fiber amounts, fiber diameter, axon diameter, myelin thickness and G-ratio were measured. Compared to the SFI of saline group, the NGF and mNP-1 groups had an increase of 18.3% and 18.8%, respectively. The numbers of myelinated fibers in the distal nerve of NGF and mNP-1 groups were 1.45- and 1.32-fold higher than in the saline group. The MNCVs of NGF and mNP-1 groups were 7.3 and 4.4 times of that of saline group. Fiber diameter, axon diameter, myelin thickness and G-ratio in the NGF and mNP-1 groups were also significantly higher than those of saline group. Our results demonstrate that, like NGF, the defensin mNP-1 can promote regeneration after a peripheral nerve cut.

Cloning of the Human C5a Anaphylatoxin Receptor, and More.

Cloning of the Human C5a Anaphylatoxin Receptor, and More.

The flavonols quercetin, myricetin, kaempferol, and galangin inhibit the net oxygen consumption by immune complex-stimulated human and rabbit neutrophils.

Stimulated human neutrophils exhibit increased net oxygen consumption (NOC) due to the conversion of O2 into the superoxide anion by the NADPH oxidase enzymatic complex during the respiratory burst. In several inflammatory diseases, overproduction of these oxidants causes tissue damage. The present study aims to: (a) optimize the experimental conditions used to measure the NOC in serum-opsonized zymosan (OZ)- and insoluble immune complex (i-IC)-stimulated human and rabbit neutrophils; and (b) compare the effect of four flavonols (quercetin, myricetin, kaempferol, and galangin) on this activity. We used a Clark-type oxygen electrode to measure the NOC of stimulated neutrophils. Eliciting the neutrophil respiratory burst with OZ and i-IC yielded similar maximum O2 uptake levels within the same species, but the human neutrophil NOC was almost four times higher than the rabbit neutrophil NOC. The optimal experimental conditions established for both cell types were 4 x 10(6) neutrophils mL(-1), 2 mg mL(-1) OZ, and 240 microg mL(-1) i-IC. Upon stimulation with OZ or i-IC, the tested flavonols reduced the human and rabbit neutrophil NOC in the same order of potency--quercetin and galangin were the most and the least potent, respectively. These compounds were around four times more effective in inhibiting the rabbit as compared to the human neutrophil NOC, respectively. The four flavonols were not toxic to human or rabbit neutrophils. The experimental conditions used are suitable for both the determination of human and rabbit neutrophil NOC and for the assessment of the modulatory effects of natural compounds on these activities. The relationship between the level of NOC and the inhibitory potency of the flavonols suggests that rabbit neutrophils can be useful experimental models to predict the effect of drugs on immune complex-stimulated human neutrophils.

Panton-Valentine leucocidin and pneumonia

Laura Shallcross and colleagues con cluded on the basis of their meta-analyses that individuals with pneumonia were less likely to be infected with a PantonValentine leucocidin (PVL)-positive Staphylococcus aureus strain than are those with skin and soft-tissue infection. While PVL-positive strain might have a lesser propensity to cause pneumonia compared with skin and soft-tissue infection, these meta-analyses cannot be used to make any inference regarding whether or not PVL contributes to the pathogenesis and outcomes of pneumonia. Such inference could be made on the basis of comparison of outcomes in patients with PVLpositive pneumonia versus those with PVL-negative pneumonia. However, Shallcross and colleagues did not do a meta-analysis on outcomes of PVL-positive and PVL-negative pneu monia, but instead provide qualitative summary of pneumonia outcomes from seven studies that used diff erent study designs and cannot be combined. Gillet and colleagues’ study addressing this issue was unfortunately excluded from their systematic review. Gillet and colleagues’ study showed that median survival was 4 days in patients with PVL-positive pneumonia compared with 25 days in patients with PVL-negative pneumonia, although overall mortality rates were not different between groups at 60 days after hospital admission. The rapidly fatal course of infection caused by the PVL-positive strains was associated with acute respiratory distress syndrome and characterised by severe hypoxaemia, leucopenia, haemoptysis, alveolar haemorrhage, and necrosis. An animal model is needed to investi gate molecular mechanisms by which PVL induces the rapidly fatal course of haemorrhagic necrotising pneumonia. In this regard, Shallcross and colleagues’ review failed to identify another study that elucidated mechanisms by which PVL rapidly induces severe hypoxaemia, leucopenia, lung necrosis, pulmonary oedema, alveolar haemorrhage, haemoptysis, and death in a rabbit model of necrotising pneumonia. This rabbit model showed that PVL causes lung infl ammation b y activating and recruiting neutrophils into the lungs and then lysing them to release granule enzymes and reactive oxygen metabolites that damage the lungs. In contrast with the fact that rodent and monkey neutrophils are resistant to cytotoxic eff ects of PVL, the similar susceptibilities of rabbit and human neutrophils to PVL indicate that the rabbit model of necrotising pneumonia could be used for preclinical development and evaluation of anti-PVL therapeutic approaches. Ultimately, the question of whether or not PVL has a role in disease severity could be settled by a clinical trial testing effi cacy of anti-PVL therapy (eg, specifi c monoclonal antibody that neutralises PVL) in protecting against rapidly progressive necrotising pneumonia in human beings.

English

Defensin is a small, cysteine-rich, cationic peptides family with antimicrobial and cytotoxic properties. Among all known defensins, neutrophil peptide-1 (NP-1), which is expressed mainly in rabbit neutrophils, has a broad resistance spectrum to pathogens such as Treponema pallidum , many Grampositive bacteria, Gram-negative bacteria, fungi, viruses. Besides, it has the same striking inhibition or toxic effect on some nausea and tumor cells. Due to the broad antibacterial spectrum and special mechanism of microbial inhibition, rabbit defensin has been transformed into some plants and expressed via genetic engineering. And it plays an important role in genetic engineering of anti-disease plants and plants species’ improvement. This article reviewed and discussed the advantages and research progress of the rabbit defensin genetic engineering of plant in recent years, and also focuses on the existing problems and new strategies in this area. Key words: Rabbit defensin (NP-1), structure, bioactivity, genetic engineering of plant.

Panton-Valentine Leukocidin Does Play a Role in the Early Stage of Staphylococcus aureus Skin Infections: A Rabbit Model

Despite epidemiological data linking necrotizing skin infections with the production of Panton-Valentine leukocidin (PVL), the contribution of this toxin to the virulence of S. aureus has been highly discussed as a result of inconclusive results of in vivo studies. However, the majority of these results originate from experiments using mice, an animal species which neutrophils - the major target cells for PVL - are highly insensitive to the action of this leukocidin. In contrast, the rabbit neutrophils have been shown to be as sensitive to PVL action as human cells, making the rabbit a better experimental animal to explore the PVL role. In this study we examined whether PVL contributes to S. aureus pathogenicity by means of a rabbit skin infection model. The rabbits were injected intradermally with 108 cfu of either a PVL positive community-associated methicillin-resistant S. aureus isolate, its isogenic PVL knockout or a PVL complemented knockout strain, and the development of skin lesions was observed. While all strains induced skin infection, the wild type strain produced larger lesions and a higher degree of skin necrosis compared to the PVL knockout strain in the first week after the infection. The PVL expression in the rabbits was indirectly confirmed by a raise in the serum titer of anti-LukS-PV antibodies observed only in the rabbits infected with PVL positive strains. These results indicate that the rabbit model is more suitable for studying the role of PVL in staphylococcal diseases than other animal models. Further, they support the epidemiological link between PVL producing S. aureus strains and necrotizing skin infections.

Induction of neurite-outgrowth in PC12 cells by alpha-toxin from Clostridium perfringens

Alpha-toxin-induced phosphorylation of PDK1 via the tyrosine kinase A (TrkA) receptor signaling pathway plays an important role in the activation of rabbit neutrophils. The relation between the toxin and TrkA, however, remains poorly understood. Here, we show that the toxin-induced phosphorylation of TrkA is closely related to the induction of neurite-outgrowth in PC12 cells. The toxin induced neurite-outgrowth and phosphorylation of TrkA in the cells in a dose-dependent manner. K252a, a TrkA inhibitor, and shRNA for TrkA inhibited the toxin-induced neurite-outgrowth, and phosphorylation of TrkA and ERK1/2. PD98059, an inhibitor of the ERK1/2 cascade, inhibited phosphorylation of ERK1/2 and the neurite-outgrowth induced by alpha-toxin. The wild-type toxin induced the formation of diacylglycerol, and neurite-outgrowth, but H148G, a variant toxin which binds to cell membranes and has lost the enzymatic activity did not. We demonstrated that the phosphorylation of TrkA through the phospholipid metabolism induced by the toxin synergistically play a key role in neurite-outgrowth.

Construction and expression of rabbit neutrophil peptide-1 gene in Escherichia coli

Rabbit neutrophil peptide-1 (NP-1) is a prototypic rabbit α-defensin with a broad antimicrobial spectrum. The coding sequence of NP-1 was amplified and cloned into pET-31b(+) to construct an expression vector, pET31-NP1, which was transformed into E. coli BLR(DE3)pLysS for expressing the fusion NP-1 protein (fNP-1). The fNP-1 is downstream of a ketosteroid isomerase (KSI) and upstream of a (His)6-Tag, as KSI-NP1-His6. The optimal condition, cultivation in enriched LB medium and induction with 0.5 mM IPTG for 6 h, was determined for fNP-1 production. The fNP-1 was purified by Ni-NTA resin and cleaved by cyanogen bromide to release matured NP-1 peptide. The matured NP-1 peptide showed significant antimicrobial activities against clinical bacteria, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The application of this expression approach represents a potential method to produce NP-1 by fusion protein expressed in E. coli without cytotoxicity.

Staphylococcus aureus Panton-Valentine Leukocidin Is a Very Potent Cytotoxic Factor for Human Neutrophils

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.

CC16 Inhibits the Migration of Eosinophils Towards the Formyl Peptide fMLF but not Towards PGD2

Clara cell 16-kDa (CC16) is an anti-inflammatory protein chiefly produced in the lung epithelium. CC16 has been shown to inhibit the migration of rabbit neutrophils and human monocytes toward the formyl peptide N-formyl-methionine-leucin-phenylalanin (fMLF). Eosinophils migrate towards prostaglandin D2 (PGD(2)) and CC16 has been shown to bind to PGD(2). Therefore we investigated if CC16 could inhibit the migration of human eosinophils and neutrophils towards fMLF and/or PGD(2). Migration of eosinophils and neutrophils was assessed in a microplate migration system using specific ligands and receptor antagonists. CC16 inhibited the migration of eosinophils and neutrophils toward fMLF, which is likely to result from the interaction of CC16 with members of the formyl-peptide receptor family. However, CC16 did not inhibit eosinophil migration towards PGD(2). We therefore propose that CC16 may down-modulate the entry of human eosinophils and neutrophils into the airways during inflammation in the lung.

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens α-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Effects of defensin and lactoferrin on functional activity of endothelial cells in vitro

We studied the effects of antibacterial peptides and proteins (defensins and lactoferrins) on functional activity of endothelial cells in vitro: proliferative activity and adhesion of human endothelial ECV-304 cells to the matrix were evaluated, alpha-Defensin (NP-2) from rabbit neutrophils, total alpha-defensin (HNP 1-3) from human neutrophils, and lactoferrins from porcine neutrophils and human milk were studied. Defensins stimulated and lactoferrin in doses of 1-10 microg/ml inhibited proliferation and adhesion of endothelial cell. The stimulatory effect of defensins on proliferation and adhesion was reproduced in fibroblast culture. Lactoferrins did not modify proliferation of fibroblasts, but suppressed their adhesion. These data suggest that antibiotic proteins and peptides are prospective objects for the creation of drugs regulating angiogenesis.

Effects of chitooligosaccharides on rabbit neutrophils in vitro

The effects of chitooligosaccharides(COS) on resting and PMA-activated neutrophils were estimated. MTT assays, NO estimation and superoxide detection revealed that chitooligosaccharides at concentrations of 25, 50, 75, and 100 μg/ml increased the viability, ability to produce reactive oxygen intermediates and nitrogen intermediates of resting neutrophils. Superoxide detection, degranulation assay and adhesion assay suggested that chitooligosaccharides at concentrations from 50 to 150 μg/ml reduced PMA-induced activation of neutrophils.

Licofelone, a Balanced Inhibitor of Cyclooxygenase and 5-Lipoxygenase, Reduces Inflammation in a Rabbit Model of Atherosclerosis

Licofelone, a dual anti-inflammatory drug that inhibits 5-lipoxygenase (LOX) and cyclooxygenase (COX) enzymes, may have a better cardiovascular profile that cycloxygenase-2 inhibitors due to cycloxygenase-1 blockade-mediated antithrombotic effect and a better gastrointestinal tolerability. We examined the anti-inflammatory effect of licofelone on atherosclerotic lesions as well as in isolated neutrophils from whole blood of rabbits compared with a selective inhibitor of COX-2, rofecoxib. We also assessed the antithrombotic effect of licofelone in rabbit platelet-rich plasma. For this purpose, 30 rabbits underwent injury of femoral arteries, and they were randomized to receive 10 mg/kg/day licofelone or 5 mg/kg/day rofecoxib or no treatment during 4 weeks with atherogenic diet in all cases. Ten healthy rabbits were used as controls. Neutrophils and platelets were isolated from peripheral blood of rabbits for ex vivo studies. Licofelone reduced intima/media ratio in injured arteries, the macrophages infiltration in the neointimal area, monocyte chemoattractant protein-1 (MCP-1) gene expression, and the activation of nuclear factor-kappaB in rabbit atheroma. Moreover, licofelone inhibited COX-2 and 5-LOX protein expression in vascular lesions. Rofecoxib only diminished COX-2 protein expression and MCP-1 gene expression in vascular atheroma. Prostaglandin E(2) in rabbit plasma was attenuated by both drugs. Licofelone almost abolished 5-LOX activity by inhibiting leukotriene B4 generation in rabbit neutrophils and prevented platelet thromboxane B2 production from whole blood. Licofelone reduces neointimal formation and inflammation in an atherosclerotic rabbit model more markedly than rofecoxib. This effect, together with the antiplatelet activity of licofelone, suggests that this drug may have a favorable cardiovascular profile.

Signal Transduction Mechanism Involved inClostridium perfringensAlpha-Toxin-Induced Superoxide Anion Generation in Rabbit Neutrophils

Clostridium perfringens alpha-toxin induces the generation of superoxide anion (O2(-)) via production of 1,2-diacylglycerol (DG) in rabbit neutrophils. The mechanism of the generation, however, remains poorly understood. Here we report a novel mechanism for the toxin-induced production of O2(-) in rabbit neutrophils. Treatment of the cells with the toxin resulted in tyrosine phosphorylation of a protein of about 140 kDa. The protein reacted with anti-TrkA (nerve growth factor high-affinity receptor) antibody and bound nerve growth factor. Anti-TrkA antibody inhibited the production of O2(-) and binding of the toxin to the protein. The toxin induced phosphorylation of 3-phosphoinositide-dependent protein kinase 1 (PDK1). K252a, an inhibitor of TrkA receptor, and LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), reduced the toxin-induced production of O2(-) and phosphorylation of PDK1, but not the formation of DG. These inhibitors inhibited the toxin-induced phosphorylation of protein kinase C theta (PKCtheta). U73122, a phospholipase C (PLC) inhibitor, and pertussis toxin inhibited the toxin-induced generation of O2(-) and formation of DG, but not the phosphorylation of PDK1. These observations show that the toxin independently induces production of DG through activation of endogenous PLC and phosphorylation of PDK1 via the TrkA receptor signaling pathway and that these events synergistically activate PKCtheta in stimulating an increase in O2(-). In addition, we show the participation of mitogen-activated protein kinase-associated signaling events via activation of PKCtheta in the toxin-induced generation of O2(-).