Degranulation and expression of cytokines were modulated by diazinon in activated mast cells.

Diazinon is an organophosphorus (OP) insecticides used in agriculture, home gardening and indoor pest control in Japan. It can activate macrophages and induce pro-inflammatory responses and has been reported to cause airway hyper-reactivity, suggesting the possibility of asthma exacerbation from exposure to OP insecticides. Despite the correlation between insecticide use and the pathogenesis of allergic diseases, there have been no reports on the effects of diazinon on mast cell function. Therefore, in this study, we investigated the effects of diazinon on mast cell function in rat basophilic leukemia (RBL)-2H3 cells. Surprisingly, we found that diazinon inhibited mast cell activation, although the degree of inhibition varied with concentration. Diazinon induced reactive oxygen species (ROS) generation and HO-1 expression at a concentration of 150 µM without affecting cell viability. Diazinon inhibited A23187-mediated degranulation and Tnf and Il4 expression in RBL-2H3 cells but did not affect calcium influx. Suppression of degranulation by diazinon was reversed when the culture supernatant was removed. As a signaling event downstream of calcium influx, diazinon inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) induced by A23187, whereas the phosphorylation of p38 had little effect. IgE cross-linking-mediated degranulation as well as the induction of Tnf and IL4 expression was significantly inhibited by diazinon, while diazinon had little effect on calcium influx. In conclusion, diazinon inhibited mast cell activation, including degranulation and cytokine expression. When evaluating the in vivo effects of diazinon, its potential to inhibit mast cell activation should be considered in the pathophysiology and development of allergic diseases in terms of basic and clinical aspects, respectively, although the effect of diazinon varies depending on the cell type.

BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells.

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line.

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called "oncogene-induced senescence (OIS)", prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called "HBEC3KT"), immortalized with hTERT ("T") and Cdk4 ("K"), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated ß-galactosidase staining (SA-ßG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Involvement of heme oxygenase-1 in suppression of T cell activation by quercetin.

AIM: Acute rejection is still a major problem in transplantation and one of the most important causes of late graft loss. Cyclosporine and tacrolimus are widely used for suppression of T cell function to avoid graft rejection, but long-term use of these compounds is associated with serious toxicities. Quercetin, a flavonoid found in fruits and vegetables, has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO) -1, an enzyme involved in heme catabolism. We hypothesized that quercetin induces HO-1 in T cells and suppresses T cell function via HO-1. In the present study, we showed that quercetin suppressed the A23187-mediated expression of interleukin (IL) -2 in T cells. METHODS: Mouse splenocytes, enriched T cells, and EL4 cells, a mouse T cell line, were treated with quercetin, and then stimulated with A23187, a calcium ionophore, concanavalin A, or anti-CD3ε and anti-CD28 antibodies. Cell proliferation, expression of IL-2, calcium mobilization, apoptosis, cell cycle, and phosphorylation of extracellular signal-regulated kinase (ERK) were investigated. RESULTS: Quercetin induced HO-1, and this induction of HO-1 was implicated in the suppression of IL-2 production. Furthermore, the induction of HO-1 by quercetin suppressed the influx of calcium ions, a known trigger of IL-2 production. Additionally, quercetin suppressed T cell proliferation through promotion of cell cycle arrest via HO-1 induction, but quercetin did not induce apoptosis. To investigate the role of the signal transduction pathway in quercetin's effect on cell proliferation, we evaluated the phosphorylation of ERK in T cells. Quercetin suppressed the A23187-mediated stimulation of ERK, an effect that was mediated through HO-1. These results suggested that HO-1 is involved in the suppressive effects of quercetin on T cell activation and proliferation. CONCLUSION: Our findings indicate that the quercetin may be a promising candidate for inducing HO-1 in T cells, thereby facilitating immunosuppressive effects.

Modulation of immunological activity on macrophages induced by diazinon.

Diazinon is an organophosphorus (OP) insecticide and is widely used not only in agriculture but also homes and garden in Japan. Diazinon has been reported to increase TNF-α production in rat serum and brain, suggesting that it can modify the proinflammatory response. In this study, we investigated the effects of diazinon on macrophage functions, such as cytokine production, reactive oxygen species (ROS) generation, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expressions, cell-surface molecule expressions, and phagocytosis in RAW264.7 cells. In RAW264.7 cells, diazinon induced the production of TNF-α and IL-6. Diazinon induced ROS generation and the expressions of COX-2, iNOS, and cell-surface molecules CD40, CD86, and MHC class II, but reduced phagocytic activity in RAW264.7 cells. ERK and p38, but not JNK and p65 were involved in diazinon-induced IL-6 expression in RAW264.7 cells. We also examined these proinflammatory responses in bone marrow-derived macrophages (BMDM) and bronchoalveolar lavage fluid (BALF) cells. These results suggested that diazinon can activate macrophages and enhance inflammatory responses.

Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin's suppressive effects. METHODS: Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells. RESULTS: Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. CONCLUSIONS: Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.

Quercetin protects against pulmonary oxidant stress via heme oxygenase-1 induction in lung epithelial cells.

The lung is a primary target for oxygen toxicity because of its constant exposure to high oxygen levels and environmental oxidants. Quercetin is one of the most commonly found dietary flavonoids, and it provides cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways. In the present study, we showed that quercetin increased the levels of heme oxygenase (HO)-1 expression and protected against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in lung epithelial cell lines. Quercetin suppressed H(2)O(2)-induced apoptotic events, including hypodiploid cells, activation of caspase 3 enzyme activity and lactate dehydrogenase release. This cytoprotective effect was attenuated by the addition of the HO inhibitor, tin protoporphyrin IX. In addition, the end products of heme metabolites catalyzed by HO-1, carbon monoxide and bilirubin, protect against H(2)O(2)-induced cytotoxicity in LA-4 cells. Quercetin may well be one of the promising substances to attenuate oxidative epithelial cell injury in lung inflammation.

CD40/CD40 ligand interactions in immune responses and pulmonary immunity.

The CD40 ligand/CD40 pathway is widely recognized for its prominent role in immune regulation and homeostasis. CD40, a member of the tumor necrosis factor receptor family, is expressed by antigen-presenting cells, as well as non-immune cells and tumors. The engagement of the CD40 and CD40 ligands, which are transiently expressed on T cells and other non-immune cells under inflammatory conditions, regulates a wide spectrum of molecular and cellular processes, including the initiation and progression of cellular and humoral adaptive immunity. Based on recent research findings, the engagement of the CD40 with a deregulated amount of CD40 ligand has been implicated in a number of inflammatory diseases. We will discuss the involvement of the CD40 ligand/CD40 interaction in the pathophysiology of inflammatory diseases, including autoimmune diseases, atherothrombosis, cancer, and respiratory diseases.

Ghrelin and obestatin promote the allergic action in rat peritoneal mast cells as basic secretagogues.

Ghrelin is an endogenous ligand of the type 1a growth hormone secretagogue receptor (GHSR1a) that regulates energy balance. Ghrelin and obestatin, derived from the post-translational processing of preproghrelin, are involved in a diverse range of biological activities, yet their effect on the immune system is not fully understood. In the present study, we investigated the roles of ghrelin and obestatin on mast cell degranulation and found that both ghrelin and obestatin induce the release of histamine from rat peritoneal mast cells. This induced histamine release was inhibited by the pertussis toxin, an inhibitor of Gα(i) protein, and extracellular Ca(2+). Rat peritoneal mast cells and rat basophilic leukemia (RBL-2H3) cells did not express the ghrelin receptor GHSR1a, suggesting that histamine release induced by ghrelin occurs via a receptor-independent mechanism. We report here that ghrelin and obestatin, belonging to the family of basic secretagogues, stimulate mast cells independent of a receptor, and this may play a crucial role at the site of allergy or inflammation.

Hemoglobin induces the expression of indoleamine 2,3-dioxygenase in dendritic cells through the activation of PI3K, PKC, and NF-kappaB and the generation of reactive oxygen species.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme-free globin (Apo Hb), induced IDO expression in bone marrow-derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of I kappaB alpha. Hb-induced IDO expression was inhibited by inhibitors of PI3-kinase (PI3K), PKC and nuclear factor (NF)-kappaB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb-induced IDO expression was inhibited by anti-oxidant N-acetyl-L-cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3-amino-1,2,4-triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O(2) (-), H(2)O(2), and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF-kappaB through the PI3K-PKC-ROS and PI3K-Akt pathways is required for the Hb-induced IDO expression in BMDCs.

Peptide array-based analysis of the specific IgE and IgG4 in cow's milk allergens and its use in allergy evaluation.

Cow's milk (CM) is one of the major causes of food allergies in children. We constructed a peptide array consisting of a linear 16-mer peptide library with an offset of 3-mer, which corresponds to the primary sequences of six major CM allergens. The immune reactivity to cow's milk proteins diminishes with age and clinical tolerance commonly occurs. Although the central role of IgE in allergy is well established, the role of other specific antibody classes in obtaining immunotolerance is not well known. The hypothesis that patients become tolerant when they develop immunological changes particularly with the IgG4 isotype has been proposed. In this study, the binding pattern of the CM protein-specific IgE and IgG4 epitopes was measured using the peptide array with sera of 12 patients with persistent CM allergy (CMA), sera of 5 children who outgrew CMA, and sera of 7 CM-sensitized children without allergy symptoms. In CMA patients the IgG4/IgE fluorescence intensity ratios varied greatly from peptide to peptide, and the scatter plots of IgE versus IgG4 signals using significant IgE-binding peptides showed different distribution patterns. When setting the boundary line based on the IgG4/IgE ratio (IgG4/IgE=2), patients with persistent CMA and CM-sensitized children can be distinguished by the plot pattern of peptides. Furthermore, the number of peptide plots in these regions was less in children who outgrew CMA. The approach employed in this study will allow for the distinction between CMA and CM-sensitization, and will enable the estimation of CMA outgrow by monitoring the time elapsed data.

Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.

OBJECTIVE AND DESIGN: We investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of quercetin against degranulation of rat basophilic leukemia (RBL-2H3) cells, rat peritoneal mast cells, and mouse bone marrow-derived mast cells. METHODS: The strength of allergic reaction was evaluated by the extent of degranulation in mast cells sensitized with various stimulants. The levels of HO-1, HO-2, and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions were determined by quantitative RT-PCR, western blotting, or immunocytochemistry. RESULTS: Heme oxygenase activity was upregulated after short exposure to quercetin, followed by the induction of HO-1 expression after long exposure to quercetin. The inhibition of degranulation by quercetin was reversed using tin protoporphyrin IX (SnPP), an HO-1 inhibitor. HO-1 metabolites, bilirubin and CO, led to inhibit degranulation, and quercetin translocated Nrf2 from cytoplasm into nucleus in RBL-2H3 cells. CONCLUSION: These results strongly suggest that quercetin exerted anti-allergic actions via activation of Nrf2-HO-1 pathway.

Development of peptide arrays for detection of IgE-binding epitopes in cow's milk allergens.

Peptide arrays have become versatile tools for high throughput screening assays in biomedical and pharmaceutical research. In this study, we constructed a peptide array that contained linear peptide fragments reported as IgE-binding epitopes for cow's milk allergy (CMA). Various peptides with different solubility in aqueous solutions were dissolved in the buffer solutions containing sodium dodecyl sulfate, and we achieved a consistent spotting of peptide solutions using a piezoelectric ceramic micropump. The IgE-binding patterns were successfully detected by observing the binding of Alexa 647-labeled anti-human IgE using sera from CMA patients. Our technique in this study will provide a potent capability for the development of a peptide array for mapping IgE-epitopes in milk proteins, and it will help researchers better understand the IgE-epitopes associated with the clinical outcome of CMA.

Involvement of heme oxygenase-1 in kaempferol-induced anti-allergic actions in RBL-2H3 cells.

Kaempferol is one of the most commonly found dietary flavonoids. The exposure to kaempferol is known to inhibit degranulation from mast cells, but the inhibitory mechanism of degranulation has not been clarified yet. In this study, we investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of kaempferol against degranulation in rat basophilic leukemia (RBL-2H3) cells. Our results demonstrate upregulation of HO enzymatic activity after short (15 min) exposure to kaempferol, followed by the induction of HO-1 expression in protein. The involvement of HO-1 in the kaempferol-induced inhibition of degranulation was confirmed using tin protoporphyrin IX (SnPP), a HO-1 inhibitor. These findings strongly suggest that kaempferol exerts anti-allergic actions via activation of the HO-1.

Platelet factor 4 fragment induces histamine release from rat peritoneal mast cells.

Platelet factor 4 (PF-4) belongs to a superfamily of low-molecular weight proteins known as chemokines. However, its function has not been fully evaluated. In the present study, we investigated the effect of PF-4 on histamine release from rat peritoneal mast cells by employing its biologically-active carboxyl-terminal fragment, PF-4 (58-70). PF-4 (58-70) stimulated histamine release from mast cells in a dose-dependent manner (10(-8) to 10(-5)M). Histamine release induced by PF-4 (58-70) occurred rapidly (<30s) and was inhibited by extracellular Ca(2+). These results suggest that PF-4 might play a crucial role at the site of inflammation and/or immune response.