Current synthetic inhibitors of human neutrophil elastase in 2005

The human neutrophil elastase (HNE)-related diseases, such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), are a serious problem worldwide. To prevent excessive production of HNE, which is a major pathogenic factor of these disorders, and to restore the appropriate balance between HNE and its inhibitors, research and development of potent synthetic HNE inhibitors has been conducted for > 30 years worldwide. At present, the following four agents can be described as the most successful: the acyl-enzyme inhibitors, ONO-5046 and MR-889, and the transition-state inhibitors, ONO-6818 and AE-3763. Additionally, protein inhibitors such as DX-890 (EPI-hNE-4), recombinant α1-antitrypsin, elafin and secretory leukocyte protease inhibitor (SLPI) are also being developed and actively researched in 2005. This review focuses upon the major synthetic HNE inhibitors, including those patented in 2004.     

Effects of lecithinized superoxide dismutase and a neutrophil elastase inhibitor (ONO-5046) on hyperoxic lung injury in rat

Reactive oxygen and neutrophil metabolites have been implicated in the development of hyperoxic lung injury. We determined the protective effects of either a superoxide dismutase or neutrophil elastase inhibitor and the combination of both agents on the development of hyperoxic lung injury in rats. Two drugs (lecithinized superoxide dismutase and ONO-5046) were used in the present study. Lecithinized superoxide dismutase, a lecithin derivative bound to recombinant CuZn superoxide dismutase, has a higher affinity for cells such as polymorphonuclear leukocytes and endothelial cells than recombinant human superoxide dismutase. N-[2-[4-2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl]? aminoacetic acid (ONO-5046), a specific neutrophil elastase inhibitor, which was developed as a low-molecular weight inhibitor, showed protective effects against various lung injuries. Rats were exposed to over 90% oxygen for 72 h, and bronchoalveolar lavage was performed to evaluate the permeability and neutrophil accumulation in the lungs. Rats were treated with lecithinized superoxide dismutase (30,000 U/day, intravenously n=7) or ONO-5046 (10 mg/kg, intramuscularly twice a day, n=7) or a combination of both drugs (n=7). Albumin concentration and neutrophil counts in bronchoalveolar lavage fluid were compared between animals with and without drug treatment. Either lecithinized superoxide dismutase or ONO-5046 treatment significantly decreased albumin concentration and neutrophil counts in bronchoalveolar lavage fluid compared to those in the animals of the hyperoxia-alone group (n=9). However, albumin leakage and neutrophil accumulation in the rat lung treated with combined agents were identical to that of either the lecithinized superoxide dismutase or ONO-5046 treatment. These findings suggest that lecithinized superoxide dismutase and ONO-5046 are useful drugs to protect against hyperoxic lung injury in rats. However, there were no additive effects by the combination in preventing hyperoxic lung injury.     

Current synthetic inhibitors of human neutrophil elastase

The imbalance between human neutrophil elastase (HNE, EC 3.4.21.37) and endogenous antiproteinases is considered to cause various HNE-mediated inflammatory disorders, such as adult respiratory distress syndrome, cystic fibrosis, pulmonary emphysema and rheumatoid arthritis. To realise the optimum therapeutic approach and investigate accurate pathogenic mechanism of these diseases, new synthetic HNE inhibitors have been under development. Through the long-time intensive research by many pharmaceutical industries and institutes, the most promising strategies at present may result in the application of two-types of small-molecular-weight HNE inhibitors: acyl-enzyme inhibitors (ONO-5046, L-694458 and MR-889) or transition-state inhibitors (ONO-6818, AE-3763 and FK-706). This review mainly focuses on the experimental and clinical evaluation of recent synthetic HNE inhibitors, including 14 current patent reports claimed since 1998.     

Tumour necrosis factor-alpha and leukotriene B(4) mediate the neutrophil migration in immune inflammation.

1. We investigated the mediators responsible for neutrophil migration induced by ovalbumin (OVA) in immunized mice and the mechanisms involved in their release. 2. OVA administration promoted dose- and time-dependent neutrophil migration in immunized, but not in non-immunized mice, which was mediated by leukotriene B(4) (LTB(4)) and tumour necrosis factor (TNF)alpha, since it was inhibited by LTB(4) synthesis inhibitor (MK 886) or by LTB(4) receptor antagonist (CP 105,696), by dexamethasone and by antiserum to TNFalpha (82, 85, 63 and 87%, respectively). Confirming TNFalpha involvement, OVA challenge in immunized p55 TNF receptor deficient mice (p55(-/-)) did not promote neutrophil migration (control: 2.90 +/- 0.68; p55(-/-): 0.92+/-0.23 x 10(6) neutrophils cavity(-1)). 3. OVA-stimulated peritoneal cells from immunized mice released a neutrophil chemotactic factor which mimicked, in naive mice, neutrophil migration induced by OVA. 4. Supernatant chemotactic activity is due to TNFalpha and LTB(4), since its release was inhibited by MK 886 (93%) and dexamethasone (90%), and significant amounts of these mediators were detected. 5. TNFalpha and LTB(4) released by OVA challenge seem to act through a sequential mechanism, since MK 886 inhibited (88%) neutrophil migration induced by TNFalpha. Moreover, peritoneal cells stimulated with TNFalpha released LTB(4). 6. CD(4)(+) T cells are responsible for TNFalpha release, because the depletion of this subset prevented the release of TNFalpha (control: 400 +/- 25; immunized: 670 +/- 40; CD(4)(+) depleted: 435 +/- 18 pg ml(-1)). 7. In conclusion, neutrophil migration induced by OVA depends on TNFalpha released by CD(4)(+) cells, which acts through an LTB(4)-dependent mechanism.     

Neutrophil elastase inhibitor, ONO-5046 suppresses ozone-induced airway mucus hypersecretion in guinea pigs

To investigate the role of neutrophil elastase in ozone-induced airway hypersecretion, we measured goblet cell secretion by using a semiquantitative morphometric technique in guinea pigs. The magnitude of mucus discharge was estimated from the mucus score, which is inversely related to the degree of mucus discharge in histological sections of trachea stained for mucus glycoprotein with periodic acid Schiff/Alcian blue. Mucus hypersecretion of goblet cells was induced by ozone exposure and persisted for up to 5 h after exposure. Pretreatment with N-[2-?4-(2,2-dimethyl-propionyloxy) phenyl-sulfonylamino? benzoyl] aminoacetic acid (ONO-5046), a specific neutrophil elastase inhibitor (200 mg/kg, intraperitoneally), significantly inhibited goblet cell hypersecretion both just after and 5 h after ozone-exposure, but the latter inhibition was not complete. In bronchoalveolar lavage fluid, ozone exposure significantly increased the number of neutrophils just after and 5 h after exposure, while ONO-5046 significantly inhibited the increase in neutrophils only 5 h after ozone-exposure. These results indicate that neutrophil elastase may play an important role in the ozone-induced tracheal goblet cell hypersecretion and influx of neutrophils.     

Thromboxane A2 up-regulates neutrophil elastase release in Syrian hamsters with trinitrobenzene sulfonic acid-induced colitis

Neutrophil elastase (NE) is a factor that aggravates colitis. We investigated the influence of thromboxane A2 (TXA2) and leukotriene B4 (LTB4) on NE release in Syrian hamsters with trinitrobenzene sulfonic acid-induced colitis. Colonic specimens with colitis were incubated with U-46619 (a TXA2 analogue) or LTB4 in vitro and NE release was examined. As a result, U-46619 increased NE release, while LTB4 had no effect. The NE release induced by U-46619 was inhibited by a TP-receptor antagonist. To demonstrate that TXA2 caused NE release in vivo as well, while LTB4 did not, colitis animals were treated with nordihydroguaiaretic acid (NDGA), a dual inhibitor of cyclooxygenase/lipoxygenase; and colonic luminal TXB(A)2 and LTB4 levels and NE activity were determined. The TXB(A)2 level was significantly correlated with NE activity, while no correlation was found between LTB4 and NE activity. An inhibitory effect of NDGA on the ulcer area was also observed, and NE activity was significantly correlated with the ulcer area. The suppression of TXA2 production by NDGA may result in the inhibition of NE release so that colonic tissue damage becomes less severe. Regulation of NE release is a new biological action of TXA2 that has not been reported before.     

Urinary trypsin inhibitor protects against systemic inflammation induced by lipopolysaccharide

Urinary trypsin inhibitor (UTI), a serine protease inhibitor, has been widely used as a drug for patients with acute inflammatory disorders such as disseminated intravascular coagulation, shock, and pancreatitis in Japan. Recent studies have demonstrated that serine protease inhibitors may play an anti-inflammatory role beyond merely an inhibitory action on neutrophil elastase at the site of inflammation at least in vitro. To clarify the direct contributions of UTI to inflammatory condition in vivo, we analyzed its roles in experimental systemic inflammatory response induced by intraperitoneal administration of lipopolysaccharide (LPS) using UTI deficient (-/-) mice and corresponding wild-type (WT) mice. After LPS (1 mg/kg) challenge, UTI (-/-) mice revealed a significant elevation of plasma fibrinogen and fibrinogen/fibrin degradation products and a decrease in white blood cell counts compared with WT mice. LPS treatment induced more severe neutrophilic inflammation in the lung and the kidney obtained from UTI (-/-) mice than in those from WT mice, which was confirmed by histological examination. The protein levels of proinflammatory mediators, such as macrophage chemoattractant protein (MCP)-1 in the lungs, MCP-1 and keratinocyte chemoattractant (KC) in the kidneys, and interleukin-1beta, macrophage inflammatory protein-2, MCP-1, and KC in the liver, were significantly greater in UTI (-/-) mice than in WT mice after LPS challenge. Our results suggest that UTI protects against systemic inflammatory response and subsequent organ injury induced by bacterial endotoxin, at least partly through the inhibition of the enhanced expression of proinflammatory cytokines and chemokines.     

Hepatic Clearance of ONO-5046, a Novel Neutrophil Elastase Inhibitor,in Rats and in the Rat Perfused Liver

The hepatic clearance of ONO-5046 (N-[2-[4-(2,2-dimethylpropionyloxy)phenylsulphonyl-amino]benzoyl]aminoacetic acid), a low-molecular-weight neutrophil elastase inhibitor, has been investigated in rats and in the rat perfused liver. This ester was easily hydrolysed to its inactive metabolite EI-601 (N-[2-[(4-hydroxy-phenyl)sulphonylamino]benzoyl]aminoacetic acid) in liver homogenate and in erythrocytes suspension in-vitro. On the other hand, it was stable in biological media such as plasma and whole blood, which contain plasma proteins. Scatchard plot analysis of ONO-5046 binding to bovine serum albumin (BSA) in-vitro indicated that the association constant (K) and number of binding sites (n) were 6.91 times 10⁴ (M^?1) and 4.33, respectively. Thus, ONO-5046 (100 μM) would bind to plasma proteins to an extent >99% at physiological plasma-protein concentrations. The total plasma clearance of ONO-5046 in rats was constant (approximately 9 mL min^?1 kg^?1) under different steady-state plasma concentrations (5–50 μM) a value equivalent to the hepatic clearance. In the rat perfused liver, the hepatic extraction ratio of ONO-5046 was significantly reduced by adding BSA to the dosing solution. Thus, the relatively low hepatic clearance of ONO-5046, which has an ester linkage in its structure and is naturally susceptible to enzymatic hydrolysis, was found to be because of the extremely high protein-binding of the compound.     

Role of leukotrienes in the regulation of human granulocyte behaviour: dissociation between agonist-induced activation and retardation of apoptosis

1 Since most inflammatory mediators that stimulate granulocyte responsiveness also delay apoptosis, it is often assumed that activation and longevity are causally related. Using isolated human peripheral blood neutrophils and eosinophils, we examined this association by exploiting the proinflammatory lipid mediators, the leukotrienes (LTs), and investigated granulocyte function and apoptosis. 2 LTB(4) induced elevation of intracellular free Ca(2+) concentration ([Ca(2+)](i)), cell polarisation and retardation of neutrophil apoptosis, although the antiapoptotic effect occurred only at concentrations > or =300 nM. LTB(4)-induced activation was attenuated by CP-105,696, a BLT1-specific antagonist suggesting classical LTB(4) receptor BLT1 involvement. 3 Despite demonstrating the presence of the neutrophil intracellular LTB(4) receptor peroxisome-proliferator activator receptor-alpha (PPARalpha) in neutrophils, the selective PPARalpha agonist WY-14,643 did not mimic LTB(4)-induced prosurvival effects. 4 LTB(4)-induced survival, however, also appeared to be mediated by BLT1 since CP-105,696 inhibited the LTB(4)-mediated antiapoptotic effect. Furthermore, based on studies with CP-105,696 and 5-lipoxygenase inhibitors, lipopolysaccharide (LPS)-, granulocyte-macrophage colony-stimulating factor (GM-CSF)-, dexamethasone- and dibutyryl-cAMP (db-cAMP)-induced delay of neutrophil apoptosis did not involve autocrine production of LTB(4). 5 Although LTB(4) and LTD(4) induced human eosinophil [Ca(2+)](i) elevation and polarization, these LTs did not influence eosinophil apoptosis. Furthermore, LTB(4)- and LTD(4)-induced eosinophil activation was attenuated by CP-105,696 and the Cys-LT(1) receptor antagonist montelukast, respectively, highlighting specific receptor dependency. 6 Thus, mediator-triggered granulocyte activation and antiapoptotic pathways are distinct events that can be differentially regulated.     

Neutrophil elastase inhibitors as treatment for COPD

Chronic obstructive pulmonary disease, characterised by a slowly progressive, irreversible airways limitation, is a major worldwide cause of chronic morbidity and mortality. The imbalance between human neutrophil elastase and endogenous antiproteases may cause excess human neutrophil elastase in pulmonary tissues, which may be considered a major pathogenic factor in chronic obstructive pulmonary disease. Great effort has been devoted to finding a method to restore the balance, resulting in the discovery of potent two-typed small-molecular-weight human neutrophil elastase inhibitors. In the application of chronic obstructive pulmonary disease therapy, the human neutrophil elastase inhibitors mainly focused upon include ONO-5046, MR-889, L-694,458, CE-1037, GW-311616 and TEI-8362 as the acyl-enzyme inhibitors; and ONO-6818, AE-3763, FK-706, ICI-200,880, ZD-0892 and ZD-8321 as the transition-state inhibitors. In this review, various problems that remain to be solved in the clinical use of human neutrophil elastase inhibitors are discussed.     

Neutrophil elastase inhibitors as treatment for COPD

Chronic obstructive pulmonary disease, characterised by a slowly progressive, irreversible airways limitation, is a major worldwide cause of chronic morbidity and mortality. The imbalance between human neutrophil elastase and endogenous antiproteases may cause excess human neutrophil elastase in pulmonary tissues, which may be considered a major pathogenic factor in chronic obstructive pulmonary disease. Great effort has been devoted to finding a method to restore the balance, resulting in the discovery of potent two-typed small-molecular-weight human neutrophil elastase inhibitors. In the application of chronic obstructive pulmonary disease therapy, the human neutrophil elastase inhibitors mainly focused upon include ONO-5046, MR-889, L-694,458, CE-1037, GW-311616 and TEI-8362 as the acyl-enzyme inhibitors; and ONO-6818, AE-3763, FK-706, ICI-200,880, ZD-0892 and ZD-8321 as the transition-state inhibitors. In this review, various problems that remain to be solved in the clinical use of human neutrophil elastase inhibitors are discussed.     

Niacin inhibits carrageenan-induced neutrophil migration in mice

Several emerging lines of evidence support an anti-inflammatory role for nicotinic acid (niacin); however, its role in the regulation of leukocyte migration in response to inflammatory stimuli has not been elucidated until now. Herein, we have examined the effect of nicotinic acid on neutrophil recruitment in experimentally induced inflammation. We demonstrated that nicotinic acid treatment inhibited interleukin (IL)-8-induced, leukotriene (LT)B₄-induced, and carrageenan-induced neutrophil migration into the pleural cavity of BALB/c mice and reduced neutrophil rolling and adherence in a mouse cremaster muscle preparation. Surprisingly, nicotinic acid treatment increased the level of the neutrophil chemoattractant KC in response to carrageenan. These results suggest that nicotinic acid plays an important role in the regulation of inflammation due to its ability to inhibit the actions of the neutrophil chemoattractants IL-8 and LTB₄. Further inhibition of chemoattractants leads to impairment of leukocyte rolling and adherence to the vascular endothelium in the microcirculation of inflamed tissues.     

In vivo pharmacologic profile of ONO-1078: a potent, selective and orally active peptide leukotriene (LT) antagonist.

We investigated the in vivo antagonistic activity of ONO-1078 against peptide leukotrienes (LTs) in guinea pigs. ONO-1078, when administered p.o. (0.3-3 mg/kg), caused a dose-dependent reduction of LTC4-, LTD4- and LTE4-induced bronchoconstriction, LTD4-induced airway microvascular leakage and LTD4-induced increase in cutaneous vascular permeability. When administered intravenously, ONO-1078 (3-30 micrograms/kg) inhibited these responses approximately 200-600 fold more potently than FPL55712. When guinea pigs were treated with indomethacin to examine the antagonism of ONO-1078 on the direct action against peptide LTs, intravenous (3-30 micrograms/kg) and oral (0.3-3 mg/kg) administration of ONO-1078 also inhibited LTC4- and LTD4-induced bronchoconstriction, and its activity was approximately 300-500 fold more potent than that of FPL55712. ONO-1078 (10 mg/kg, i.v.) had no inhibitory effect on bronchoconstrictions induced by histamine, acetylcholine, serotonin, arachidonic acid, LTB4, prostaglandin (PG) F2 alpha, PGD2, 9 alpha, 11 beta-PGF2, a stable thromboxane A2 mimetic agent and platelet activating factor. Furthermore, oral administration of ONO-1078 (1-10 mg/kg) inhibited slow-reacting substance of anaphylaxis mediated bronchoconstriction induced by antigen in a dose-dependent manner. These results indicate that ONO-1078 is an extremely potent, selective and orally active peptide LT antagonist and that oral administration of ONO-1078 antagonizes not only exogenously administered peptide LTs but also endogenous peptide LTs.     

Anti-inflammatory effects of the partially purified extract of radix Stephaniae tetrandrae: comparative studies of its active principles tetrandrine and fangchinoline on human polymorphonuclear leukocyte functions

We hypothesized that prevention of neutrophil from activation may underlie the myocardial protective effect of the specially processed extract of radix Stephaniae tetrandrae (SPRST). Inflammatory responses in isolated peripheral human neutrophils were studied in the presence or absence of SPRST. SPRST (1-10 microg/ml) concentration-dependently prevented N-formyl-methionyl-leucyl-phenylalanine (fMLP)- or leukotriene B(4) (LTB(4))-induced neutrophil adhesion and transmigration. Comparable results were also observed in neutrophils pretreated with fangchinoline (Fan) or tetrandrine (Tet), two active components in SPRST. It has been reported that neutrophil adhesion/transmigration is mainly Mac-1 (CD11b/CD18)-dependent and could be modulated by reactive oxygen species (ROS) production. SPRST, Tet, and Fan diminished fMLP- or LTB4-induced Mac-1 up-regulation and ROS production. SPRST, Fan, Tet, and verapamil impaired fMLP-induced rapid intracellular alkalization, an essential mechanism for neutrophil ROS production, and [Ca(2+)](i) increment, suggesting that a calcium dependent pathway might be involved. Direct G protein activation by AlF(4)(-) also triggered [Ca(2+)](i) increment and adhesion that could be abolished by pertussis toxin and were partially reversed by SPRST, Fan, and Tet. These results reveal that inhibition of neutrophil adhesion and transmigration may account for SPRST's myocardial protective effect. This effect of SPRST may be mediated by component(s) in addition to Tet and Fan because combination of 0.1 microg/ml of Tet and Fan did not mimic the effect of SPRST. We conclude that SPRST exerts anti-inflammatory effects by interfering with ROS production and Ca(2+) influx through G protein modulation to prevent Mac-1 up-regulation in neutrophil activation.     

The contractile action of leukotriene B4 in the guinea-pig lung involves a vascular component

Leukotriene B4 (LTB4) is a potent leukocyte chemoattractant, acting on specific receptors, BLT receptors. The aim of this study was to examine the mechanism of action of LTB4 in the guinea-pig lung, using strips of lung parenchyma (GPLP), spirals of trachea (GPT) and bronchus (GPB) and rings of pulmonary artery (GPPA). Mechanical responses were studied in organ baths, and mediator release was assessed using enzyme immuno assay. LTB4 induced similar contractions of GPLP and GPPA, whereas LTB4 had only small contractile effects in GPT and GPB. In addition, the contractile response to LTB4 was reproduced in the human pulmonary artery. In the GPLP, the unselective BLT receptor antagonist ONO-4057 abolished the contractions induced by LTB4, whereas the selective BLT1 receptor antagonist U-75302 only partly inhibited the LTB4-induced contractions. In the GPPA, both antagonists abolished the response to LTB4. The effect of LTB4 in GPPA and GPLP was indirect and mediated by the release of thromboxane A2 and histamine, as supported by selective pharmacologic interventions and measurements of thromboxane B2 and histamine in the organ baths. In conclusion, the results indicate a new biological function of LTB4, namely to constrict isolated pulmonary arteries. Moreover, the findings suggest that the LTB4-induced contractions of GPPA were mediated by a BLT1 receptor, whereas BLT2 receptor activation accounted for a major part of the contraction of GPLP, making the latter preparation a suitable assay for BLT2 receptors.British Journal of Pharmacology (2004) 141, 449-456. doi:10.1038/sj.bjp.0705641     

Investigation of vascular endothelial growth factor effects on pulmonary endothelial monolayer permeability and neutrophil transmigration.

This study sought to determine whether vascular endothelial growth factor (VEGF)-induced permeabilisation of pulmonary endothelium to macromolecules could be related to a permissive role for neutrophil-derived VEGF in neutrophil transmigration. Treatment of human pulmonary artery endothelial cell (HPAEC) monolayers with 1, 10 or 100 ng/ml VEGF for 15 min or 1, 10 ng/ml for 90 min significantly increased endothelial permeability to trypan blue-labelled albumin (TB-BSA). These increases were correlated with changes in the cellular distribution of F-actin, as visualised by rhodamine-phalloidin staining: increased stress fibre formation, cellular elongation and formation of intercellular gaps after 15 min; at 90 min, there was also evidence of microspike formation and extension of spindle processes from the cell surface. Treatment of human neutrophil suspensions with 200 nM phorbol myristyl acetate (PMA), n-formyl-methionyl leucylphenylalanine (fMLP, 10 nM), interleukin-8 (IL-8, 10 nM) (but not with leukotriene B(4) (LTB(4)) 100 nM), for 30 min caused significant extracellular release of neutrophil VEGF stores. A permissive role for neutrophil-derived VEGF in facilitating migration across HPAEC monolayers was assessed in experiments using a functional blocking antihuman VEGF antibody. In the presence of this antibody (10 microg/ml), neutrophil migration in response to fMLP (10 nM), IL-8 (10 nM) or LTB(4) (100 nM) was not significantly different to that in the absence of antibody. We conclude that neutrophil-derived VEGF does not play a functional role in facilitating neutrophil migration across pulmonary vascular endothelium, despite its ability to induce cytoskeletal changes and enhance endothelial macromolecular permeability.     

Neutrophil recruitment is inhibited by nicotinamide in experimental pleurisy in mice

Several emerging lines of evidence support an anti-inflammatory role for nicotinamide and other vitamin B components. However, the mechanisms underlying their activity remain unclear. In the present study, we investigated the ability of nicotinamide to inhibit both neutrophil recruitment in IL-8-, LTB(4)- or carrageenan-induced pleurisy in mice and the rolling and adherence of neutrophils. Nicotinamide inhibited IL-8-, LTB(4)- and carrageenan-induced neutrophil migration, KC production and carrageenan-induced neutrophil rolling and adherence. We propose that the effects of nicotinamide in inhibiting neutrophil recruitment in carrageenan-induced pleurisy may be due to the ability of nicotinamide to inhibit the action of IL-8 and LTB(4), decrease KC production, and inhibit early events that regulate leukocyte migration from blood vessels into tissue.     

Mast cell-derived tumour necrosis factor-alpha mediates macrophage inflammatory protein-2-induced recruitment of neutrophils in mice

Recent studies have indicated that mast cells play an intermediate role in chemokine-induced neutrophil recruitment in vivo. The aim of the present investigation was to determine the role of tumour necrosis factor-alpha (TNF-alpha) in neutrophil recruitment provoked by the CXC chemokine macrophage inflammatory protein-2 (MIP-2). For this purpose, we used mast cell- and TNF-alpha-deficient mice and studied neutrophil adhesion to endothelial cells in vitro and neutrophil recruitment in the mouse cremaster muscle in vivo. In contrast to the classical chemoattractant formyl-methionine-leucine-phenylalanin (fMLP), MIP-2 dose dependently increased neutrophil accumulation in vivo. This MIP-2-regulated neutrophil recruitment was abolished in mast cell-deficient mice. TNF-alpha increased E-selectin mRNA expression in both wild-type (WT) and mast cell-deficient mice. In contrast, MIP-2 challenge increased gene expression of E-selectin in WT but not in mast cell-deficient animals. Moreover, MIP-2-provoked extravascular accumulation of neutrophils was reduced by 78% in mice lacking TNF-alpha. In order to better define the role of mast cell-derived TNF-alpha in neutrophil responses to MIP-2, we used an in vitro endothelial cell adhesion assay with and without mast cells. Interestingly, MIP-2-induced neutrophil adhesion to endothelial cells was decreased by 58% using TNF-alpha-deficient compared to WT mast cells. Moreover, mast cell secretion of TNF-alpha increased by more than 71% in response to challenge with MIP-2. Taken together, our results suggest that MIP-2-induced neutrophil recruitment is mediated by TNF-alpha released from local mast cells. These findings help to explain the complex molecular interactions between chemokines, mast cell activation and neutrophil infiltration in vivo.     

Thearubigin,the major polyphenol of black tea,ameliorates mucosal injury in trinitrobenzene sulfonic acid-induced colitis

Inflammatory bowel disease is characterized by oxidative and nitrosative stress, leukocyte infiltration and upregulation of proinflammatory cytokines. The aim of the present study was to examine the protective effects of thearubigin, an anti-inflammatory and anti-oxidant beverage derivative, on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice, a model for inflammatory bowel disease. Intestinal lesions (judged by macroscopic and histological score) were associated with neutrophil infiltration (measured as increase in myeloperoxidase activity in the mucosa), increased serine protease activity (may be involved in the degradation of colonic tissue) and high levels of malondialdehyde (an indicator of lipid peroxidation). Both nitric oxide (NO) and O(2)(-) were increased with concomitant upregulation in the mRNA expression of proinflammatory cytokine response and inducible NO synthase (iNOS). Dose-response studies revealed that pretreatment of mice with thearubigin (40 mg kg(-1) day(-1), i.g. for 10 days) significantly ameliorated the appearance of diarrhoea and the disruption of colonic architecture. Higher dose (100 mg kg(-1)) had comparable effects. This was associated with a significant reduction in the degree of both neutrophil infiltration and lipid peroxidation in the inflamed colon as well as decreased serine protease activity. Thearubigin also reduced the levels of NO and O(2)(-) associated with the favourable expression of T-helper 1 cytokines and iNOS. Consistent with these observations, nuclear factor kappa B (NF-kappa B) activation in colonic mucosa was suppressed in thearubigin-treated mice. The results of this study suggest that thearubigin, the most predominant polyphenol of black tea, exerts beneficial effects in experimental colitis and may, therefore, be useful in the treatment of inflammatory bowel disease.     

Pharmacological profile of a specific neutrophil elastase inhibitor,Sivelestat sodium hydrate

Imbalance between neutrophil elastase (NE) and its endogenous protease inhibitors has been considered to be one of possible mechanisms by which NE causes lung tissue destruction. It has been shown that the amount and/or activity of NE is increased in blood and bronchoalveolar lavage fluid in patients with acute lung injury. Accordingly, animals undergoing acute lung injury have increased NE activity such as in blood and bronchoalveolar lavage fluid. Sivelestat sodium hydrate (Sivelestat) is a synthetic inhibitor of NE with highly specificity to NE. Many studies have indicated that Sivelestat treatment improves inflammatory and edematous changes of lungs and survival as well as increased NE activity in several animal models of acute lung injury. Clinical studies have demonstrated that Sivelestat improves this injury that is associated with systemic inflammatory response syndrome. As compared with endogenous protease inhibitors that have high molecular mass, Sivelestat, a synthetic and low molecular weight elastase inhibitor, may be delivered to the inflammatory sites more easily and effectively and is considred to improve typical symptoms of acute lung injury. Clinical use of Sivelestat would further clarify the usefulness of this compound in clinical acute lung injury.