Airway epithelial phosphoinositide 3-kinase-δ contributes to the modulation of fungi-induced innate immune response.

BACKGROUND: Respiratory fungal exposure is known to be associated with severe allergic lung inflammation. Airway epithelium is an essential controller of allergic inflammation. An innate immune recognition receptor, nucleotide-binding domain, leucine-rich-containing family, pyrin-domain-containing-3 (NLRP3) inflammasome, and phosphoinositide 3 kinase (PI3K)-δ in airway epithelium are involved in various inflammatory processes. OBJECTIVES: We investigated the role of NLRP3 inflammasome in fungi-induced allergic lung inflammation and examined the regulatory mechanism of NLRP3 inflammasome, focusing on PI3K-δ in airway epithelium. METHODS: We used two in vivo models induced by exposure to Aspergillus fumigatus (Af) and Alternaria alternata (Aa), as well as an Af-exposed in vitro system. We also checked NLRP3 expression in lung tissues from patients with allergic bronchopulmonary aspergillosis (ABPA). RESULTS: Assembly/activation of NLRP3 inflammasome was increased in the lung of Af-exposed mice. Elevation of NLRP3 inflammasome assembly/activation was observed in Af-stimulated murine and human epithelial cells. Similarly, pulmonary expression of NLRP3 in patients with ABPA was increased. Importantly, neutralisation of NLRP3 inflammasome derived IL-1ß alleviated pathophysiological features of Af-induced allergic inflammation. Furthermore, PI3K-δ blockade improved Af-induced allergic inflammation through modulation of NLRP3 inflammasome, especially in epithelial cells. This modulatory role of PI3K-δ was mediated through the regulation of mitochondrial reactive oxygen species (mtROS) generation. NLRP3 inflammasome was also implicated in Aa-induced eosinophilic allergic inflammation, which was improved by PI3K-δ blockade. CONCLUSION: These findings demonstrate that fungi-induced assembly/activation of NLRP3 inflammasome in airway epithelium may be modulated by PI3K-δ, which is mediated partly through the regulation of mtROS generation. Inhibition of PI3K-δ may have potential for treating fungi-induced severe allergic lung inflammation.

GPCR Kinase (GRK)-2 Is a Key Negative Regulator of Itch: l-Glutamine Attenuates Itch via a Rapid Induction of GRK2 in an ERK-Dependent Way.

Many itch mediators activate GPCR and trigger itch via activation of GPCR-mediated signaling pathways. GPCRs are desensitized by GPCR kinases (GRKs). The aim of this study is to explore the role of GRKs in itch response and the link between GRKs and glutamine, an amino acid previously shown to be an itch reliever. Itch responses were evoked by histamine, chloroquine, and dinitrochlorobenzene-induced contact dermatitis (CD). Phosphorylation and protein expression were detected by immunofluorescent staining and Western blotting. GRK2 knockdown using small interfering RNA enhanced itch responses evoked by histamine, chloroquine, and dinitrochlorobenzene-induced CD, whereas GRK2 overexpression using GRK2-expressing adenovirus reduced the itch responses. Glutamine reduced all itch evoked by histamine, chloroquine, and dinitrochlorobenzene-induced CD. Glutamine-mediated inhibition of itch was abolished by GRK2 knockdown. Glutamine application resulted in a rapid and strong expression of GRK2 in not only dinitrochlorobenzene-induced CD (within 10 minutes) but also cultured rat dorsal root ganglion cells, F11 (within 1 minute). ERK inhibitor abrogates glutamine-mediated GRK2 expression and inhibition of itch in dinitrochlorobenzene-induced CD. Our data indicate that GRK2 is a key negative regulator of itch and that glutamine attenuates itch via a rapid induction of GRK2 in an ERK-dependent way.

FcγR/ROS/CK2α Is the Key Inducer of NF-κB Activation in a Murine Model of Asthma.

BACKGROUND: The transcription factor nuclear factor (NF)-κB plays a pivotal role in the development of allergic airway inflammation. However, the mechanism of NF-κB activation in asthma remains to be elucidated. METHODS: CK2α activation was assessed by CK2α phosphorylation and protein expression. Airway levels of histamine and cytokines were determined by ELISA. We used 2 (active and passive) forms of allergic pulmonary inflammation models. In the active form, the animals were immunized with ovalbumin (OVA) intraperitoneally, followed by an airway challenge with OVA. In the passive form, the animals were passively sensitized by intratracheal instillation with either anti-OVA IgE or anti-OVA IgG, followed by an airway challenge with OVA. The role of NADPH oxidase (NOX) in CK2α activation was assessed using NOX2-/- and NOX4-/- mice because NOX2 and NOX4 contribute to many inflammatory diseases. RESULTS: The second airway challenge increased CK2α phosphorylation and protein expression in airway epithelial cells as well as nuclear translocation of the p50 and p65 subunits of NF-κB, all of which were inhibited by the CK2α inhibitor 4,5,6,7-tetrabromobenzotriazole and the antioxidant N-acetyl-L-cysteine. CK2α phosphorylation and protein expression were significantly impaired in NOX2-/-, but not in NOX4-/-, mice. Induction of passive sensitization using anti-OVA IgE activated neither CK2α nor NF-κB. In contrast, induction of passive sensitization using anti-OVA IgG activated both CK2α and NF-κB. CONCLUSIONS: These data suggest that Fcγ receptor/reactive oxygen species/CK2α is a key inducer of NF-κB activation in airway epithelial cells in a murine model of asthma.

Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway.

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A2 via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca2+]i) in MHS macrophage cells. Ionomycin increased [Ca2+]i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca2+→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca2+ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca2+→ ERK pathway, which plays a key role in suppression of inflammatory reactions.

Platelet-activating factor enhances tumour metastasis via the reactive oxygen species-dependent protein kinase casein kinase 2-mediated nuclear factor-κB activation.

Platelet-activating factor (PAF) promotes tumour metastasis via activation of the transcription factor nuclear factor-κB (NF-κB). We here investigated the role of the protein kinase CK2 (formerly Casein Kinase 2 or II) in PAF-induced NF-κB activation and tumour metastasis, given that PAF has been reported to increase CK2 activity, and that CK2 plays a key role in NF-κB activation. PAF increased CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. CK2 inhibitors inhibited the PAF-mediated NF-κB activation and expression of NF-κB-dependent pro-inflammatory cytokines and anti-apoptotic factors. Pre-treatment with the antioxidant N-Acetyl-L-Cysteine (NAC) resulted in a significant inhibition in PAF-induced enhancement of CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. H2 O2 and known reactive oxygen species inducers, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) enhanced CK2 activity, phosphorylation and protein expression, which was again inhibited by antioxidant. PAF, LPS and TNF-α induced increased CK2 activity, phosphorylationand protein expression, which were inhibited by p38 inhibitor. PAF, LPS or TNF-α increased pulmonary metastasis of B16F10, which was inhibited by antioxidants, CK2 inhibitor and p38 inhibitor. Our data suggest that (i) reactive oxygen species activate CK2 via p38, which, in turn, induces NF-κB activation, and (ii) PAF, LPS and TNF-α increase pulmonary tumour metastasis via the induction of the reactive oxygen species (ROS)/p38/CK2/NF-κB pathway.

Cholera toxin breakdowns oral tolerance via activation of canonical NF-κB.

The mechanisms of mucosal immunogenicity and adjuvanticity of bacterial exotoxins remains unknown. In this study, we investigated the role of the transcription factor nuclear factor-κB (NF-κB) in cholera toxin (CT)-induced alteration of oral tolerance. Feeding CT abrogated ovalbumin (OVA)-induced oral tolerance, as evaluated by OVA-specific serum antibody responses, and CD4(+) T cell proliferation. CT feeding activated canonical NF-κB (one heterodimer type, p50-p65) and mRNA expression of NF-κB-dependent proinflammatory cytokines in mesenteric lymph node (MLN) and Peyer's patch (PP) cells. CT no longer showed abrogation of oral tolerance in mice pretreated with p50 small interfering RNAs (siRNAs). ADP-ribosylation inhibitors inhibited CT-induced NF-κB activation. These data suggest that CT induces canonical NF-κB activation in intestinal lymphoid cells, which plays a key role in mucosal immunogenicity and adjuvanticity.

Biphasic late airway hyperresponsiveness in a murine model of asthma.

BACKGROUND: Nonspecific airway hyperresponsiveness (AHR) is one of the cardinal features of bronchial asthma. Early AHR is caused by chemical mediators released from pulmonary mast cells activated in an IgE-dependent way. However, the mechanism of late AHR remains unclear. METHODS: Features of airway allergic inflammation were analyzed, including antigen-induced AHR, using a murine model of asthma. The model was suitable for examining the sequential early molecular events occurring after the initial airway exposure to antigen. RESULTS: AHR increased at 10-12 h after airway challenge, followed by the second-phase response, which was larger and broader in resistance at 18-30 h. Pretreatment of sensitized animals with anti-tumor necrosis factor (TNF) before airway challenge or induction of allergic asthma in TNF(-/-) mice resulted in abrogation of the first-phase late AHR. Intratracheal instillation of TNF induced a single peak of AHR at 10 h. IgE and IgG immune complexes induced the development of the first-phase late AHR by TNF production. Pretreatment with cytosolic phospholipase inhibitor and 5-lipoxygenase inhibitors abolished the first-phase late AHR as well as the leukotriene B(4) levels in the airway. CpG-oligodeoxynucleotide (ODN) pretreatment reduced airway levels of Th2 cytokines, eosinophil infiltration and second-phase late AHR. However, CpG-ODN did not reduce TNF levels or the magnitude of first-phase late AHR. CONCLUSION: Biphasic late AHR occurs in a murine model of asthma. First- and second-phase late AHR is caused by TNF and Th2 response, respectively.

PTEN/MAPK pathways play a key role in platelet-activating factor-induced experimental pulmonary tumor metastasis.

In this study, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in a platelet-activating factor (PAF)-induced experimental pulmonary tumor metastasis model. An adenovirus carrying PTEN cDNA (Ad-PTEN) reversed PAF-induced increase in phosphorylation of AKT as well as pulmonary metastasis of B16F10. PAF-induced pulmonary metastasis was inhibited by MAPK inhibitors, but not by PI3K inhibitor. Ad-PTEN abrogated PAF-induced phosphorylation of MAPKs. These data indicate PTEN/MAPK pathways play a key role in PAF-induced tumor metastasis.

Glutamine inhibits platelet-activating factor-mediated pulmonary tumour metastasis.

Inflammation has been increasingly recognised as an important component of tumourigenesis. Platelet-activating factor (PAF), a potent inflammatory mediator, has the ability to enhance tumour growth and metastasis. In this study, we have investigated (i) the role of mitogen-activated protein kinases (MAPKs) and (ii) the therapeutic efficacy of the non-essential amino acid, l-glutamine (Gln), which evidences MAPKs inhibition activity in PAF-mediated B16F10 melanoma metastasis to the lungs. Mice were given intraperitoneal injection of PAF. ERK, JNK, and p38 MAPKs were activated rapidly by PAF in the lungs, and the PAF-induced metastasis of B16F10 was inhibited in a dose-dependent manner by pretreatment with either U0126 (ERK inhibitor), SP600125 (JNK inhibitor), or SB202190 (p38 inhibitor). Intraperitoneal administration of Gln after, but not before, PAF injection deactivated ERK, JNK, and p38 by dephosphorylating them. Gln inhibited PAF-induced metastasis when Gln was administered either intraperitoneally or orally. PAF induced pronounced angiogenic activity in an in vivo mouse Matrigel implantation model. MAPK inhibitors as well as Gln significantly inhibited PAF-induced angiogenesis. These data indicate that Gln exerts a beneficial effect against inflammation-associated enhanced tumour metastasis via the deactivation of MAPKs.

IgG immune complex induces the recruitment of inflammatory cells into the airway and TNF-mediated late airway hyperresponsiveness via NF-κB activation in mice.

BACKGROUND: Many of the inflammatory proteins that are expressed in asthmatic airways are regulated, at least partially, by nuclear factor (NF)-κB. Blockade of NF-κB activity has resulted in attenuation of the cardinal features of asthma. Thus, delineating the mechanisms involved in NF-κB activation in asthma might provide an interesting approach to improving the management of asthma. However, despite its importance, the mechanism for NF-κB activation in asthma has not yet been determined. OBJECTIVE: To examine the role of IgE and IgG antibodies (Abs) in the activation of NF-κB in mouse lungs. METHODS: To examine the effect of IgE, mice underwent intratracheal (i.t.) instillation of an IgE immune complex (IgE-IC) (anti-2,4-dinitrophenyl hapten (DNP) IgE + DNP-BSA or DNP-OVA) and anaphylactogenic anti-IgE (LO-ME-2). For IgG, mice underwent i.t. instillation with a complex of anti-chicken gamma globulin (CGG) IgG1 mAb + CGG. NF-κB activation was determined by gel shift assay. Small interfering RNA was used for blockade of p50 expression. The effect of tumor necrosis factor (TNF) blockade was determined using anti-TNF Ab. A previously established murine model of asthma was used to assess airway hyperresponsiveness (AHR). RESULTS: A single i.t. instillation of either IgE-IC or LO-ME-2 failed to induce activation of NF-κB in the lungs. In contrast, single i.t. instillation of IgG-IC was capable of inducing NF-κB activation, as well as NF-κB-dependent proinflammatory molecules, such as TNF and CXC chemokines. Pretreatment of p50 small interfering RNA decreased bronchoalveolar lavage fluid levels of TNF and macrophage inflammatory protein-2 induced by IgG-IC instillation. Single i.t. instillation of IgG-IC caused the recruitment of neutrophils and macrophages into the airway and TNF-mediated late AHR, but failed to induce Th2 cell-mediated asthmatic phenotypes. CONCLUSION: IgG, but not IgE, is the major Ab that induces not only NF-κB activation and NF-κB-dependent proinflammatory molecules in the lungs but also subsequent recruitment of inflammatory cells into the airway and TNF-mediated late AHR.

Protein kinase CK2/PTEN pathway plays a key role in platelet-activating factor-mediated murine anaphylactic shock.

Platelet-activating factor (PAF) is a major mediator in the induction of fatal hypovolemic shock in murine anaphylaxis. This PAF-mediated effect has been reported to be associated with PI3K/Akt-dependent eNOS-derived NO. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is phosphatidylinositol phosphate phosphatase, which negatively controls PI3K by dephosphorylating the signaling lipid, phosphatidylinositol 3,4,5-triphosphate. In this study, we examined the possible involvement of PTEN in PAF-mediated anaphylactic shock. Induction of anaphylaxis or PAF injection resulted in a rapid decrease in PTEN activity, followed by increases in PI3K activity and phosphorylation of Akt and eNOS. Systemic administration of adenoviruses carrying PTEN cDNA (adenoviral PTEN), but not the control AdLacZ, not only attenuated anaphylactic symptoms, but also reversed anaphylaxis- or PAF-induced changes in PTEN and PI3K activities, as well as phosphorylation of Akt and eNOS. We found that the decreased PTEN activity was associated with PTEN phosphorylation, the latter effect being prevented by the protein kinase CK2 inhibitor, DMAT. DMAT also inhibited anaphylactic symptoms as well as the anaphylaxis- or PAF-mediated PTEN/PI3K/Akt/eNOS signaling cascade. CK2 activity was increased by PAF. The present data provide, as the key mechanism underlying anaphylactic shock, PAF triggers the upstream pathway CK2/PTEN, which ultimately leads to the activation of PI3K/Akt/eNOS. Therefore, CK2/PTEN may be a potent target in the control of anaphylaxis and other many PAF-mediated pathologic conditions.

Mechanisms of platelet-activating factor-induced enhancement of VEGF expression.

It has been previously reported that platelet-activating factor (PAF) induces the expression of vascular endothelial growth factor (VEGF) via the downregulation of p53 activity. In this study, we attempted to characterize the mechanism by which p53 activity negatively regulates PAF-induced VEGF expression. PAF increased luciferase activity as well as VEGF mRNA expression in human non-small cell lung cancer cell line H1299 transfected with VEGF luciferase reporter plasmid (VEGF-Luc). Cotransfection of the cells with wt p53, but not mutant p53, effected a blockage of PAF-induced VEGF mRNA expression. The ChIP assay revealed that p53 did not bind to the VEGF promoter. Transfection of Egr-1 or Sp-1 expression vector increased VEGF luciferase activity in VEGF-Luc-transfected cells, and this was inhibited by transfection with wt p53. The results of the Immunoprecipitation and immunoblot analysis showed that p53 binds to Egr-1 and Sp-1. Additionally, our electrophoretic mobility shift assay demonstrated that PAF induced the mobilization of Egr-1 and Sp-1 to the nucleus, and this activity was inhibited by transfection with wt p53. These data indicate that PAF inhibits protein complexes between p53 and Egr-1/Sp-1 via the downregulation of p53 levels, thus increasing the free form levels of Egr-1 and Sp-1, ultimately resulting in the transcriptional activation of VEGF.

Mast cells play a key role in Th2 cytokine-dependent asthma model through production of adhesion molecules by liberation of TNF-α.

Mast cells are well recognized as key cells in allergic reactions, such as asthma and allergic airway diseases. However, the effects of mast cells and TNF-α on T-helper type 2 (Th2) cytokine-dependent asthma are not clearly understood. Therefore, an aim of this study was to investigate the role of mast cells on Th2 cytokine-dependent airway hyperresponsiveness and inflammation. We used genetically mast cell-deficient WBB6F1/J-Kitw/Kitw-v (W/Wv), congenic normal WBB6F1/J-Kit+/Kit+ (+/+), and mast cell-reconstituted W/Wv mouse models of allergic asthma to investigate the role of mast cells in Th2 cytokine-dependent asthma induced by ovalbumin (OVA). And we investigated whether the intratracheal injection of TNF-α directly induce the expression of ICAM-1 and VCAM-1 in W/Wv mice. This study, with OVA-sensitized and OVA-challenged mice, revealed the following typical histopathologic features of allergic diseases: increased inflammatory cells of the airway, airway hyperresponsiveness, and increased levels of TNF-α, intercellular adhesion molecule (ICAM)-1, and vascular cellular adhesion molecule (VCAM)-1. However, the histopathologic features and levels of ICAM-1 and VCAM-1 proteins in W/Wv mice after OVA challenges were significantly inhibited. Moreover, mast cell-reconstituted W/Wv mice showed restoration of histopathologic features and recovery of ICAM-1 and VCAM-1 protein levels that were similar to those found in +/+ mice. Intratracheal administration of TNF-α resulted in increased ICAM-1 and VCAM-1 protein levels in W/Wv mice. These results suggest that mast cells play a key role in a Th2 cytokine-dependent asthma model through production of adhesion molecules, including ICAM-1 and VCAM-1, by liberation of TNF-α.

Cytoplasmic phospholipase A2 metabolites play a critical role in pulmonary tumor metastasis in mice.

BACKGROUND: Cytoplasmic PLA(2) (cPLA2) has been shown to be the major enzyme responsible for arachidonic acid (AA) release. Because of this key role of cPLA(2) in AA production, cPLA(2) involvement in tumorigenesis has been suggested. However, contradictory data are found in the literature. Additionally, little is known regarding the role of cPLA(2) in pulmonary tumor metastasis. MATERIALS AND METHODS: Tumor metastases were detected by lung colonization and angiogenesis was assayed as growth of blood vessels from subcutaneous tissue into an implanted matrigel of basement membrane. The matrix metalloproteinases (MMP)-2, and MMP-9 were detected by PCR with sequence-specific primers. RESULTS: In this study, the effects of inhibitors of cPLA2, 5-lipoxygenase (5-LO), and cyclooxygenase (COX)-2 on pulmonary metastasis formation by B16F10 melanoma cells were investigated. All of these inhibitors reduced B16F10 pulmonary metastasis formation in a dose-dependent manner. Importantly, cPLA2, and 5-LO, and COX-2 inhibitors reduced platelet-activating factor-induced angiogenesis in an in vivo mouse model employing Matrigel injected subcutaneously, and also reduced expression of MMP-2 and MMP-9 in the lungs. CONCLUSION: This study demonstrates that cPLA(2) metabolites play critical roles in tumor metastasis via the promotion, at least in part, of angiogenesis and MMP expression.

A20 attenuates allergic airway inflammation in mice.

TNF receptor 1 can activate signaling pathways leading to the activation of NF-kappaB. A20, an NF-kappaB-inducible protein, negatively regulates these signaling pathways and acts as an anti-inflammatory mediator. Therefore, A20 is viewed as a potential therapeutic target for inflammatory disease. In this study, we examined the effect of A20 on an OVA-induced allergic airway inflammation model in mice. We used an adenovirus containing A20 cDNA (Ad-A20) that was delivered intratracheally before OVA challenge. Single administration of Ad-A20 reduced airway inflammatory cell recruitment and peribronchiolar inflammation and suppressed the production of various cytokines in bronchoalveolar fluid. In addition, Ad-A20 suppressed mucus production and prevented the development of airway hyperresponsiveness. The protective effect of Ad-A20 was mediated by the inhibition of the NF-kappaB signaling pathway. Taken together, our results suggest that the development of an immunoregulatory strategy based on A20 may have therapeutic potential for the treatment of allergic asthma.

CpG-ODN-based immunotherapy is effective in controlling the growth of metastasized tumor cells.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN) act as potent immune stimulators by activating innate immunity through toll-like receptor 9. These immunomodulatory effects of CpG-ODN have been reported to be associated with anti-tumor immunity. In this study, we used a murine B16F10 melanoma model and a CT26 colon cancer model to assess whether CpG-ODN-based immunotherapy was effective in inhibiting tumor cells that have already metastasized to distant organs. Systemic administration of CpG-ODN after melanoma cell injection resulted in a significant inhibition of pulmonary colonization. When CpG-ODN was administered after tumor cell injection, it also inhibited pulmonary metastasis of the tumor cells, albeit to a lesser degree in the latter case. Systemic administration of CpG-ODN after subcutaneous inoculation of CT26 colon cancer cells diminished pulmonary metastasis from the primary tumor sites. Additionally, CpG-ODN also inhibited the growth of pulmonary colonization of the colon tumor cells when CpG-ODN was administered after the primary tumors had been surgically removed. These data indicate that CpG-ODN was effective in inhibiting pulmonary metastasis of the B16F10 melanoma and CT26 colon cancer cells, as well as the growth of metastasized tumor cells. Our results suggest that CpG-ODN-based immunotherapy may be beneficial in controlling micrometastasis after surgery in clinical settings.

Tumor necrosis factor-alpha develops late anaphylactic reaction through cytosolic phospholipase A(2) activation.

BACKGROUND: We have recently reported that tumor necrosis factor (TNF)-alpha plays an important role in the development of a late anaphylactic reaction, but the downstream pathway beyond TNF-alpha remains unclear. OBJECTIVE: It was the aim of this study to examine whether TNF-alpha induces late-phase anaphylaxis via the activation of cytosolic phospholipase A(2) (cPLA(2)). METHODS: Using a murine model of active systemic anaphylaxis to penicillin V, the induction of the late phase of anaphylaxis was quantified by measuring the increase in hematocrit value as well as the plasma level of platelet-activating factor in TNF-alpha knockout mice. Phosphorylation of mitogen-activated protein kinases (MAPKs) and cPLA(2) was measured by immunoprecipitation. cPLA(2) activity was assessed by using 1-stearoyl-2-[1-(14)C] arachidonyl-sn-glycero-3-phosphocholine as the substrate. RESULTS: Phosphorylation and enzymatic activity of cPLA(2), and phosphorylation of the 3 known MAPKs, i.e. p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase, were markedly increased in a TNF-alpha-dependent way in the lungs of mice undergoing anaphylaxis. A specific cPLA(2) inhibitor significantly attenuated the late anaphylactic symptoms. Either p38 or an ERK inhibitor significantly attenuated not only cPLA(2) phosphorylation and activity, but also the late-phase anaphylaxis. CONCLUSION: TNF-alpha-induces cPLA(2) activation through the pathway involving p38 MAPK and ERK activation and appears to be the key mechanism leading to the development of late-phase anaphylaxis.

CD30 supports lung inflammation.

The physiological functions of CD30 have not been fully elucidated. Here we show that in CD30-deficient mice (CD30(-/-)), lung inflammation is significantly diminished in the ovalbumin (OVA) model of airway hyperreactivity. In CD30(-/-) mice, the recruitment of eosinophils into the airways after OVA-aerosol challenge of OVA-primed mice was significantly diminished when compared with wild-type (w.t.) mice. IL-13 levels were also significantly reduced in CD30(-/-) mice while levels of IFN-gamma, IL-4, IL-5 and IgE in bronchoalveolar lavage fluid, lung tissue and serum were comparable to w.t. mice. Peribronchial lymph node cells from CD30(-/-) mice, re-stimulated in vitro with OVA, secreted significantly lower levels of IL-13 than those from w.t. mice, but showed normal proliferative response and other cytokine production. Exogenous IL-13 reconstituted airway recruitment of leukocytes in OVA-challenged CD3O(-/-) mice. Adoptive transfer to naive w.t. mice of in vitro OVA-re-stimulated spleen cells from CD30(-/-) mice failed to induce eosinophilic pulmonary inflammation in contrast to transfer of primed cells from w.t. mice. These results indicate that CD30 is a regulator of T(h)2 responses in the effector-memory phase and a regulator of IL-13 production in memory cells in the lung.

Mast cells play a key role in the development of late airway hyperresponsiveness through TNF-alpha in a murine model of asthma.

We have investigated the role of TNF-alpha in mast cell-mediated late airway hyperresponsiveness (AHR) using mast cell-deficient WBB6F1-W/W(v) (W/W(v)) mice in a murine model of asthma, which exhibits a biphasic increase in AHR. TNF-alpha levels in the airway and magnitude of late AHR in response to airway allergen challenge were severely impaired in W/W(v) mice compared to their littermates. In addition to TNF-alpha, cytosolic phospholipase A(2) (cPLA(2)) phosphorylation and enzymatic activity in the lungs were also impaired in W/W(v) mice. Either anti-TNF-alpha antibody or an inhibitor of cPLA(2) abolished late AHR in congeneic +/+ mice. Intratracheal administration of TNF-alpha resulted in increases in late AHR, cPLA(2 )phosphorylation, cPLA(2 )activity, and phosphorylation of mitogen-activated protein kinases. Mast cell replacement restored airway TNF-alpha level, cPLA(2 )phosphorylation and enzymatic activity in the lungs as well as late AHR in W/W(v) mice. These data indicate that mast cells play a key role in the development of late AHR through liberation of TNF-alpha.

Critical role for matrix metalloproteinase-9 in platelet-activating factor-induced experimental tumor metastasis.

In this study, the roles of matrix metalloproteinase (MMP)-2 and MMP-9 in platelet-activating factor (PAF)-induced experimental pulmonary metastasis of the murine melanoma cell, B16F10, were investigated. An injection of PAF resulted in increases in mRNA expression, protein levels and the activities of both MMP-2 and MMP-9 in the lungs. The overall expression of MMP-9 was stronger than that of MMP-2. The increased MMP-9 expression was inhibited by both NF-kappaB and AP-1 inhibitors, whereas the increased MMP-2 expression was inhibited by only AP-1 inhibitors. Immunohistochemical analysis revealed that MMP-9 was expressed in bronchial epithelial cells as well as in the walls of blood vessels, whereas MMP-2 expression was observed only in bronchial epithelial cells. PAF significantly enhanced the pulmonary metastasis of B16F10, which was inhibited by both NF-kappaB and c-jun inhibitors. MMP-9 inhibitor, but not that of MMP-2, completely inhibited PAF-induced B16F10 metastasis. These data indicate that MMP-9, the expression of which was regulated by NF-kappaB and AP-1, plays a critical role in PAF-induced enhancement of pulmonary melanoma metastasis.