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.     

Anti-inflammatory and analgesic effects of the sesquiterpene lactone budlein A in mice: inhibition of cytokine production-dependent mechanism

The anti-inflammatory activities of some medicinal plants are attributed to their contents of sesquiterpene lactones. In the present study, the anti-inflammatory and anti-nociceptive activity of a sesquiterpene lactone isolated from Viguiera robusta, budlein A in mice was investigated. The treatment with budlein A dose--(1.0-10.0 mg/kg, p.o., respectively) dependently inhibited the carrageenan-induced: i. neutrophil migration to the peritoneal cavity (2-52%), ii. neutrophil migration to the paw skin tissue (32-74%), iii. paw oedema (13-74%) and iv. mechanical hypernociception (2-58%) as well as the acetic acid-induced writhings (0-66%). Additionally, budlein A (10.0 mg/kg) treatment inhibited the mechanical hypernociception-induced by tumour necrosis factor (TNF-alpha, 36%), Keratinocyte-derived chemokine (KC, 37%) and Interleukin-1beta (IL-1beta, 28%), but not of prostaglandin E(2) or dopamine. Budlein A also inhibited the carrageenan-induced release of TNF-alpha (52%), KC (70%) and IL-1beta (59%). Furthermore, an 8 days treatment with budlein A inhibited Complete Freund's adjuvant (10 microl/paw)-induced hypernociception, paw oedema and paw skin myeloperoxidase activity increase while not affecting the motor performance or myeloperoxidase activity in the stomach. Concluding, the present data suggest that budlein A presents anti-inflammatory and antinociceptive property in mice by a mechanism dependent on inhibition of cytokines production. It supports the potential beneficial effect of orally administered budlein A in inflammatory diseases involving cytokine-mediated nociception, oedema and neutrophil migration.     

Anti-inflammatory effects of red pepper (Capsicum baccatum) on carrageenan- and antigen-induced inflammation

Inflammation is a pivotal component of a variety of diseases, such as atherosclerosis and tumour progression. Various naturally occurring phytochemicals exhibit anti-inflammatory activity and are considered to be potential drug candidates against inflammation-related pathological processes. Capsicum baccatum L. var. pendulum (Willd.) Eshbaugh (Solanaceae) is the most consumed species in Brazil, and its compounds, such as capsaicinoids, have been found to inhibit the inflammatory process. However, the anti-inflammatory effects of C. baccatum have not been characterized. Thus, this study was designed to evaluate the effects of C. baccatum juice in animal models of acute inflammation induced by carrageenan and immune inflammation induced by methylated bovine serum albumin. Pretreatment (30 min) of rats with pepper juice (0.25-2.0 g kg(-1)) significantly decreased leucocyte and neutrophil migration, exudate volume and protein and LDH concentration in pleural exudates of a pleurisy model. This juice also inhibited neutrophil migration and reduced the vascular permeability on carrageenan-induced peritonitis in mice. C. baccatum juice also reduced neutrophil recruitment and exudate levels of pro-inflammatory cytokines TNF-alpha and IL-1beta in mouse inflammatory immune peritonitis. Furthermore, we demonstrated that the main constituent of C. baccatum juice, as extracted with chloroform, is capsaicin. In agreement with this, capsaicin was able to inhibit the neutrophil migration towards the inflammatory focus. To our knowledge, this is the first demonstration of the anti-inflammatory effect of C. baccatum juice and our data suggest that this effect may be induced by capsaicin. Moreover, the anti-inflammatory effect induced by red pepper may be by inhibition of pro-inflammatory cytokine production at the inflammatory site.     

NDP-MSH inhibits neutrophil migration through nicotinic and adrenergic receptors in experimental peritonitis

Melanocortin is a potent anti-inflammatory molecule. However, little is known about the effect of melanocortin on acute inflammatory processes such as neutrophil migration. In the present study, we investigated the ability of [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP-MSH), a semisynthetic melanocortin compound, in the inhibition of neutrophil migration in carrageenin-induced peritonitis model. Herein, subcutaneous pretreatment with NDP-MSH decreased neutrophil trafficking in the peritoneal cavity in a dose-dependent manner. NDP-MSH inhibited vascular leakage, leukocyte rolling, and adhesion and reduced peritoneal macrophage inflammatory protein 2, but not TNF-alpha, IL-1beta, IL-10, and keratinocyte-derived chemokine production. In addition, the effect on neutrophil migration was reverted by the pretreatment with both propranolol (a nonselective beta-adrenergic antagonist) and mecamylamine (a nonselective nicotinic antagonist) but not by splenectomy surgery. Moreover, NDP-MSH intracerebroventricular administration inhibited neutrophil migration, indicating participation of the central nervous system. Our results propose that the NDP-MSH effect may be due to a spleen-independent neuro-immune pathway that efficiently regulates excessive neutrophil recruitment to tissues.     

Redundant function of macrophage inflammatory protein-2 and KC in tumor necrosis factor-alpha-induced extravasation of neutrophils in vivo

Tumor necrosis factor-alpha (TNF-alpha) stimulates the expression CXC chemokines, i.e. macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), and neutrophil extravasation. However, the individual role of MIP-2 and KC in the recruitment process of neutrophils in vivo remains elusive. By use of intravital microscopy in the mouse cremaster muscle, we analyzed the effect of specific inhibition of CXC chemokines, alone and together, on TNF-alpha-induced leukocyte rolling, firm adhesion and recruitment. After stimulation with TNF-alpha, the mRNA levels of both MIP-2 and KC were increased. Notably, separate administration of antibodies directed against MIP-2 and KC had no effect on TNF-alpha-induced neutrophil extravasation. In contrast, combined injection of anti-MIP-2 and anti-KC antibodies markedly inhibited extravascular migration of neutrophils. Moreover, MIP-2 and KC dose-dependently increased neutrophil recruitment, however, no synergistic effect of combined stimulation with MIP-2 and KC on the neutrophil response was found. Taken together, these data suggest that MIP-2 and KC are functionally redundant in TNF-alpha-induced neutrophil accumulation and that neutralization of both MIP-2 and KC may be necessary in order to reduce accumulation of neutrophils in cytokine-activated tissues.     

Role of neutrophil elastase in LTB4-induced neutrophil transmigration in vivo assessed with a specific inhibitor and neutrophil elastase deficient mice

BACKGROUND AND PURPOSE: The serine protease neutrophil elastase (NE) appears to regulate inflammatory responses at multiple levels but its role in leukocyte transmigration in vivo remains unclear. The present study aimed to address this issue by using both an NE inhibitor (ONO-5046) and NE deficient (NE(-/-)) mice. EXPERIMENTAL APPROACH: A number of inflammatory mediators (LTB(4), KC and PAF) were investigated in vitro for their ability to stimulate the release and the surface expression of NE by neutrophils. In addition, the role of NE in leukocyte migration elicited by topical LTB(4) was investigated in vivo in mouse cremasteric venules as observed by intravital microscopy. KEY RESULTS: Amongst the mediators tested in vitro, LTB(4) was found to be a highly potent and efficacious inducer of NE cell surface expression on murine neutrophils. Furthermore, in wild-type mice (WT), LTB(4)-induced leukocyte transmigration was reduced by intravenous ONO-5046 (66% inhibition), an effect that appeared to occur at the level of the perivascular basement membrane. Interestingly, LTB(4)-induced responses were normal in NE(-/-) mice and, while ONO-5046 had no inhibitory effect in these animals, the broad-spectrum serine protease inhibitor aprotinin suppressed leukocyte transmigration in both WT and NE(-/-) mice. CONCLUSIONS AND IMPLICATIONS: The findings demonstrate the potent ability of LTB(4) to induce cell-surface expression of NE and provide evidence for the involvement of NE in LTB(4)-induced neutrophil transmigration in vivo. The results also suggest the existence of compensatory mechanisms in NE(-/-) mice, highlighting the added value of investigating pharmacological blockers in parallel with genetic deletion.     

Evidence that endogenous interleukin-1 is involved in leukocyte migration in acute experimental inflammation in rats and mice.

As a putative mediator of inflammation interleukin-1 has been implicated in the recruitment of leukocytes during the early stages of the inflammatory reaction. In the present report we have investigated the release of endogenous IL-1 in the rat zymosan pleurisy and in the mouse zymosan peritonitis. In both cases the release of the cytokine was maximal 4 hours after zymosan injection and appeared to be time-related to neutrophil migration into the inflammatory site. The effect of in vivo treatment with dexamethasone in rat pleurisy and with polyclonal anti-murine IL-1 beta antibody in mouse peritonitis was also assessed. The steroid reduced both cell migration and the release of IL-1-like activity as well as the formation of exudate and the release of eicosanoids. The anti-IL-1 beta serum inhibited selectively the number of neutrophil that migrated to the inflamed site (approximately 40%) and the IL-1 activity recovered in (approximately 70%) the exudate. In vitro incubation of the inflammatory exudate with polyclonal anti-murine IL-1 alpha or anti-murine IL-1 beta sera allowed the identification of the IL-1 species present. In the rat pleurisy IL-1 biological activity was mainly due to the alpha species, whereas IL-1 beta was the only species apparently present in the mouse peritoneal exudate.     

Delayed polymorphonuclear leukocyte infiltration is an important component of Thalassophryne maculosa venom pathogenesis

Thalassophryne maculosa fish envenomation is characterized by severe pain, dizziness, fever, edema and necrosis. Here, the dynamic of cellular influx, activation status of phagocytic cells, and inflammatory modulator production in the acute inflammatory response to T. maculosa venom was studied using an experimental model. Leukocyte counting was performed (2 h to 21 days) after venom injection in BALB/c mice footpads. Our results showed an uncommon leukocyte migration kinetic after venom injection, with early mononuclear cell recruitment followed by elevated and delayed neutrophil influx. The pattern of chemokine expression is consistent with the delay in neutrophil recruitment to the footpad: T. maculosa venom stimulated an early production of IL-1beta, IL-6, and MCP-1, but was unable to induce an effective early TNF-alpha and KC release. Complementary to these observations, we detected a marked increase in soluble KC and TNF-alpha in footpad at 7 days post-venom injection when a prominent influx of neutrophils was also detected. In addition, we demonstrated that bone marrow-derived macrophages and dendritic cells were strongly stimulated by the venom, showing up-regulated ability to capture FITC-dextran. Thus, the reduced levels of KC and TNF-alpha in footpad of mice concomitant with a defective accumulation of neutrophils at earlier times provide an important clue to uncovering the mechanism by which T. maculosa venom regulates neutrophil movement.     

Angiopoietin-1 inhibits endothelial permeability,neutrophil adherence and IL-8 production

1 Angiopoietin-1 (Ang1) is an angiogenic growth factor that binds to the Tie2 receptor on vascular endothelium, promoting blood vessel maturation and integrity. In the present study, we have investigated whether Ang1 also possesses anti-inflammatory properties by determining its effects on endothelial barrier function, neutrophil (PMN) adherence to endothelial cells (EC) and production of the PMN chemotactic factor interleukin-8 (IL-8). 2 Pretreatment of endothelial monolayers with Ang1 attenuated the permeability increase induced by thrombin in both lung microvascular cells and a human endothelial cell line. Similarly, Ang1 prevented the permeability-inducing effects of platelet-activating factor, bradykinin and histamine. 3 Pretreatment of EC with Ang1 also reduced the adherence of PMN to EC stimulated by thrombin. In contrast to its ability to counteract the increase in monolayer permeability brought about by various inflammatory agents, Ang1 did not affect the ability of histamine, PAF, or tumor necrosis factor-alpha to stimulate PMN adherence to EC. 4 In addition to its ability to inhibit PMN adherence, Ang1 diminished IL-8 production from EC challenged with thrombin in a concentration-dependent manner. 5 When EC were preincubated with the specific Rho kinase (ROCK) inhibitor Y-27632, we observed a reduction in PMN adherence in response to thrombin, as well as a decrease in thrombin-stimulated IL-8 production. Coincubation of monolayers with Y-27632 and Ang1 did not further attenuate the above-mentioned responses. However, Ang-1 failed to inhibit the activation of RhoA in response to thrombin, suggesting that inhibition of EC adhesiveness for PMN and IL-8 production by Ang1 does not result from reduced ROCK activation. 6 We conclude that Ang1 can counteract several aspects of the inflammatory response, including endothelial permeability, PMN adherence to EC as well as inhibition of IL-8 production by EC.     

Lonchocarpus sericeus lectin decreases leukocyte migration and mechanical hypernociception by inhibiting cytokine and chemokines production

In this study, we tested the potential use of a lectin from Lonchocarpus sericeus seeds (LSL), to control neutrophil migration and inflammatory hypernociception (decrease of nociceptive threshold). Pretreatment of the animals intravenously (15 min before) with LSL inhibited neutrophil migration to the peritoneal cavity in a dose-dependent fashion confirmed by an inhibition of rolling and adhesion of leukocytes by intravital microscopy. We also tested the ability of the pretreatment with LSL to inhibit neutrophil migration on immunised mice, and it was observed that a strong inhibition of neutrophil migration induced by ovoalbumin in immunized mice. Another set of experiments showed that pretreatment of the animals with LSL, inhibited the mechanical hypernociception in mice induced by the i.pl. injection of OVA in immunized mice and of carrageenan in naïve mice, but not that induced by prostaglandin E₂ (PGE₂) or formalin. This anti-nociceptive effect correlated with an effective blockade of neutrophil influx, as assessed by the hind paw tissue myeloperoxidase levels. In addition, we measured cytokines (TNF-α and IL-1β) and chemokines (MIP-1α [CCL3] and KC [CXCL1]) from the peritoneal exudates and i.pl. tissue. Animals treated with LSL showed inhibition of cytokines and chemokines release in a dose-dependent manner. In conclusion, we demonstrated that the inhibitory effects of LSL on neutrophil migration and mechanical inflammatory hypernocicepetion are associated with the inhibition of the production of cytokines and chemokines.     

Systemic administration of interleukin-2 inhibits inflammatory neutrophil migration: role of nitric oxide

1. Interleukin-2 (IL-2) has proinflammatory properties that limit its therapeutic use. Its side effects are mainly explained by the induction of a vascular leakage syndrome. Cytokines, as TNF-alpha and IL-1beta, and nitric oxide (NO) generated by IL-2-activated leukocytes play a role in this defect. 2. As the systemic release of these mediators inhibits neutrophil migration to a specific inflammatory site, we investigated now whether IL-2 administrated systemically inhibits the neutrophil recruitment to the inflamed peritoneum. The involvement of NO in the process was also addressed. 3. Using peritoneal neutrophils, we show that the intravenous treatment of the mice with IL-2 inhibits the neutrophil migration induced by carrageenin, LPS or fMLP. In confirmation, IL-2-treated mice showed a significant reduction in leukocyte rolling and adhesion in mesenteric microcirculation evaluated after carrageenin, LPS and fMLP injections. Aminoguanidine prevented the inhibitory effect of IL-2 on carrageenin-induced neutrophil migration, rolling and adhesion. In contrast, IL-2 failed to reduce the lung leukocyte infiltration induced by LPS. Therefore, IL-2 inhibition of neutrophil migration is organ specific. 4. Our results indicate that IL-2 administered systemically inhibits neutrophil recruitment to some inflammatory sites through a mechanism dependent on NO. The results also reinforce the needs to determine the mechanism by which patients treated with IL-2 show increased risks of infection.     

Impaired host defense to Klebsiella pneumoniae infection in mice treated with the PDE4 inhibitor rolipram

The increase in levels of cAMP in leukocytes by selective inhibitors of PDE4 may result in reduction of inflammation, and may be useful in the treatment of pulmonary inflammatory disorders in humans. Here, we have assessed whether oral treatment with the prototype PDE4 inhibitor, rolipram, interfered with the antibacterial host response following pulmonary infection of mice with Klebsiella pneumoniae. K. pneumoniae infection induced a marked increase in the recruitment of neutrophils to the lungs and the production of proinflammatory cytokines and chemokines, including tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC), in bronchoalveolar (BAL) fluid and lung tissue. There were also detectable amounts of interleukin-10 (IL-10) and significant lethality. Treatment with rolipram (3-30 mg kg-1) was associated with earlier lethality and significant inhibition of the TNF-alpha production. This was associated with enhanced production of IL-10 in lung tissue of rolipram-treated animals. Rolipram treatment did not affect KC expression and the recruitment of neutrophils in the lung tissue. Over 70% of neutrophils that migrated into the BAL fluid following K. pneumoniae infection ingested bacteria. Treatment with rolipram inhibited the percentage of neutrophils undergoing phagocytosis of K. pneumoniae in a dose-dependent manner. Maximal inhibition (62%) occurred at doses equal to or greater than 10 mg kg-1. Thus, treatment of mice with the PDE4 inhibitor rolipram is accompanied by earlier lethality, enhanced bacterial load and decreased capacity of the responding host to produce TNF-alpha and of neutrophils to phagocytose bacteria. It will be important to investigate whether the shown ability of PDE4 inhibitors to inhibit neutrophil phagocytosis and control experimental bacterial infection will translate into an inhibition of the ability of neutrophils to deal with infectious microorganisms in the clinical setting.     

CXC chemokines, MIP-2 and KC, induce P-selectin-dependent neutrophil rolling and extravascular migration in vivo

The purpose of this study was to examine the impact of CXC chemokines, i.e. macrophage inflammatory protein-2 (MIP-2) and KC, on leukocyte-endothelium interactions in detail and to evaluate the role of P-selectin by use of intravital microscopy in the mouse cremaster muscle. Administration of MIP-2 and KC provoked a dose (5 - 500 ng)- and time (0 - 4 h)-dependent increase in leukocyte rolling, adhesion and tissue recruitment. Neutrophils comprised more than 92% of the leukocyte response. Pretreatment with an antibody directed against P-selectin (RB40.34) significantly inhibited MIP-2- and KC-induced leukocyte rolling by more than 96%. This marked decrease in rolling abolished firm adhesion and extravascular accumulation of neutrophils (>89% reduction), suggesting that CXC chemokines induce P-selectin-dependent rolling, which in turn apparently is a precondition for the subsequent stationary adhesion and extravasation of neutrophils. Moreover, the extravascular recruitment of leukocytes was evaluated in whole-mounts of the cremaster muscle without preceding intravital microscopy. Using this approach, it was again observed that MIP-2- and KC-induced neutrophil accumulation was completely dependent onP-selectin function. In contrast to the CXC chemokines, administration of the classical chemoattractant formyl-methionyl leucyl phenylalanine (fMLP) did not provoke extravascular tissue accumulation of neutrophils. We could not detect gene expression of CXCR2 in murine endothelial cells, whereas neutrophils were positive, indicating that the stimulatory effect of CXC chemokines on leukocyte-endothelium interactions is not a direct effect on the endothelium but rather an indirect effect via activation of an intermediary tissue cell. However, challenge with MIP-2 and KC did not increase the number of degranulated mast cells. In conclusion, our data demonstrate that CXC chemokines induce all steps in the extravasation process of leukocytes, including rolling, adhesion and transmigration in vivo. Moreover, these results show that P-selectin plays a critical role in MIP-2 and KC provoked neutrophil recruitment as a critical mediator of initial leukocyte rolling. Additionally, our study suggest that a restricted action of MIP-2 and KC on neutrophils is far too simplistic to explain the complex mechanisms of action of CXC chemokines on neutrophil infiltration in vivo.     

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.     

The effects of (R)-N-(1-methyl-2-phenylethyl) adenosine (L-PIA), a standard A1-selective adenosine agonist on rat acute models of inflammation and neutrophil function.

L-PIA, a standard A1-selective adenosine agonist, was evaluated orally in carrageenan (CRG)- and reverse passive arthus-pleurisy. White blood cell (WBC) and exudate accumulation were assessed four hours after induction of the inflammatory response. L-PIA inhibited WBC accumulation in both models with ID50's of 4.37 and 4.42 mg/kg, respectively. In contrast, exudate was inhibited by L-PIA only in the CRG pleurisy model (ID50 = 1.01 mg/kg). In mechanistic studies, L-PIA reversed the drop in circulating neutrophil count which occurred within 15 minutes after CRG injection, suggesting that L-PIA may inhibit adhesion of the cells to the endothelium. The effects of L-PIA on several parameters of rat neutrophil function were determined. Enzyme release, O2-, TXB2, and LTB4 production were monitored in response to FMLP and opsonized zymosan (SOZ) stimulation. At high concentrations, L-PIA had a mild inhibitory effect on O2- release in response to FMLP and had a moderate effect on arachidonic acid metabolite production in response to both stimuli. The other response were unaffected. These results suggest that L-PIA may prevent diapedisis or neutrophil adhesion to the endothelium, but has a minimal effect on enzyme release, O2-, LTB4 and TXB2 production.     

黄芪总甙的抗炎作用及其作用机制探索

目的探索黄芪总甙(AST)的抗炎作用及其作用机制。方法采用大鼠角叉菜胶气囊炎症模型，测定渗出液量、中性白细胞游出数、蛋白质、PGE2、IL-8、NO、PLAa含量以及O2的生成量。结果AST40、80mg·kg-1可使角叉菜胶诱导大鼠气囊炎症的渗出液量、中性白细胞游出数、蛋白质含量显著减少，降低渗出液及中性白细胞中PLA2活性，减少渗出液中IL-8含量及中性白细胞Oi的生成。AST也可明显减少渗出液中PGF2、NO的含量。结论AST的抗炎作用机理与其降低血管通透性和抑制白细胞游出、降低PLA2活性、减少IL-8、PGE2、NO等炎症介质的产生与抑制氧自由基生成有关。     

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.     

Mechanisms underlying the inhibitory effects of tachykinin receptor antagonists on eosinophil recruitment in an allergic pleurisy model in mice

The activation of tachykinin NK receptors by neuropeptides may induce the recruitment of eosinophils in vivo. The aim of the present study was to investigate the effects and underlying mechanism(s) of the action of tachykinin receptor antagonists on eosinophil recruitment in a model of allergic pleurisy in mice. Pretreatment of immunized mice with capsaicin partially prevented the recruitment of eosinophils after antigen challenge, suggesting the potential contribution of sensory nerves for the recruitment of eosinophils Local (10-50 nmol per pleural cavity) or systemic (100-300 nmol per animal) pretreatment with the tachykinin NK1 receptor antagonist SR140333 prevented the recruitment of eosinophils induced by antigen challenge of immunized mice. Neither tachykinin NK2 nor NK3 receptor antagonists suppressed eosinophil recruitment. Pretreatment with SR140333 failed to prevent the antigen-induced increase of interleukin-5 concentrations in the pleural cavity. Similarly, SR140333 failed to affect the bone marrow eosinophilia observed at 48 h after antigen challenge of immunized mice. SR140333 induced a significant increase in the concentrations of antigen-induced eotaxin at 6 h after challenge. Antigen challenge of immunized mice induced a significant increase of Leucotriene B4 (LTB4) concentrations at 6 h after challenge. Pretreatment with SR140333 prevented the antigen-induced increase of LTB4 concentrations. Our data suggest an important role for NK1 receptor activation with consequent LTB4 release and eosinophil recruitment in a model of allergic pleurisy in the mouse. Tachykinins appear to be released mainly from peripheral endings of capsaicin-sensitive sensory neurons and may act on mast cells to facilitate antigen-driven release of LTB4.     

Effects of inhibition of PDE4 and TNF-alpha on local and remote injuries following ischaemia and reperfusion injury

1. The effects of phosphodiesterase (PDE)4 and TNF-alpha inhibition were assessed on the local and remote injuries following intestinal ischaemia and reperfusion (I/R) injury in rats. 2. The PDE4 inhibitor rolipram dose-dependently (1 - 10 mg kg(-1)) suppressed the local (intestine) and remote (lung) increases in vascular permeability and neutrophil recruitment following mild I/R injury. SB207499 (ariflo), a structurally-distinct PDE4 inhibitor, also suppressed the injuries following mild I/R injury. 3. In a severe model of I/R injury, treatment with rolipram (10 mg kg(-1)) partially reversed the local and remote increases in vascular permeability, neutrophil recruitment, intestinal haemorrhage and intestinal LTB(4) concentrations. The anti-TNF-alpha anti-serum was more effective than rolipram at inhibiting local and remote injuries and prevented the lethality associated with severe I/R. 4. Rolipram and anti-TNF-alpha prevented the increase in the concentrations of TNF-alpha in the lung and intestine, but rolipram only partially inhibited the elevation of this cytokine in serum. Rolipram had little effect on the increases of IL-1 beta concentrations in lung and serum, whereas treatment with anti-TNF-alpha markedly increased the concentration of this cytokine. Concentrations of IL-10 rose significantly in the lung and serum and these increases were blocked by rolipram or anti-TNF-alpha. 5. The capacity of PDE4 inhibitors to block the recruitment of neutrophils into tissues, the production of LTB(4) and of the pro-inflammatory cytokines TNF-alpha, IL-1 beta and IL-6 appear to underlie their anti-inflammatory effects in our model of I/R injury. Overall, PDE4 inhibition was less effective than inhibition of TNF-alpha for protection against I/R injury.     

Evaluation of time course and inter-relationship of inflammatory mediators in experimental inflammatory reaction

Inflammatory signs, such as heat, redness, swelling and pain, have been described from the Greek era. In these phenomena various endogenous active substances, i.e., inflammatory mediators, could cause and manifest vascular dilatation, a vascular permeability increase and sensitization of pain receptors, etc. In order to evaluate the roles of inflammatory mediators, we have studied the time courses of inflammatory reaction along with detection of various active substances directly or indirectly in the experimental animal model of pleurisy, such as rat carrageenin-induced, and zymosan-induced pleurisy. These pleurisies showed almost similar time courses of pleural exudate accumulation and neutrophil migration. However, mediators detected in the exudates of such pleurisies were different; in carrageenin-induced pleurisy bradykinin and prostacyclin (PGI2) caused exudate formation, while zymosan-induced pleurisy showed early degradation of mast cells and activation of complements, followed by an increase in platelet activating factor (PAF). In both pleurisies TNF alpha, IL-1, IL-6 and CINC (cytokine-induced neutrophil chemoattractant) appeared similarly in the exudates to cause chemoattractant for neutrophils. TNF alpha and IL-1 could stimulate to produce IL-6 and IL-8. While prostaglandins may regulate cytokine production via a cellular cAMP-dependent mechanism. Thus one should consider the time for application of anti-inflammatory drugs, such as cyclooxygenase inhibitor, indomethacin, since it causes increases in TNF alpha and IL-1 production by reducing PGI2 and prostaglandin E2 (PGE2) levels. In conclusion, inflammatory reaction has its own automatic regulation mechanism through complex cross talks between inflammatory mediators.