Haiwei Yang

Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom

L-amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55kDa in SDS-PAGE and an apparent molecular weight of 70kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against L-Tyr, L-Asp, L-Phe, L-Glu, L-Trp, L-His, L-Gln, L-Ile, L-Met, L-Leu and moderately active against L-Lys, L-Arg, L-Ala and L-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.

Modulation of mast cell proteinase-activated receptor expression and IL-4 release by IL-12.

It has been recognized that protease‐activated receptors (PARs), interleukin (IL)‐4 and IL‐6 are involved in the pathogenesis of allergic diseases, and that IL‐12 plays a role in adaptive immune response. However, little is known of the effect of IL‐12 on protease‐induced cytokine release from mast cells. In the present study, we examined potential influence of IL‐12 on mast cell PAR expression and IL‐4 and IL‐6 release. The results showed that IL‐12 downregulated the expression of PAR‐2 and upregulated expression of PAR‐4 on P815 cells. It also downregulated expression of PAR‐2 mRNA, and upregulated expression of PAR‐1, PAR‐3 and PAR‐4 mRNAs. However, IL‐12 enhanced trypsin‐ and tryptase‐induced PAR‐2 and PAR‐2 mRNA expression. It was observed that IL‐12 induced release of IL‐4, but reduced trypsin‐ and tryptase‐stimulated IL‐4 secretion from P815 cells. PD98059, U0126 and LY294002 not only abolished IL‐12‐induced IL‐4 release but also inhibited IL‐12‐induced phosphorylation of extracellular signal‐regulated kinase and Akt. In conclusion, IL‐12 may serve as a regulator in keeping the balance of Th1 and Th2 cytokine production in allergic inflammation.

TNF Increases Expression of IL-4 and PARs in Mast Cells

Tumor necrosis factor (TNF) is a proinflammatory cytokine which has been shown to be actively involved in the pathogenesis of allergic inflammation. However, little is known of the effects of TNF on cytokine secretion and protease activated receptor (PAR) expression in mast cells. In the present study, we examined potential influence of TNF on IL-4 and IL-12 release from P815 cells and PAR expression in P815 cells by using flow cytometry analysis, quantitative real time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that TNF induced up to 2.7-fold increase in IL-4, but not IL-12 release from P815 cells, and PAR-2 antagonist peptide FSLLRY-NH2 and PAR-4 antagonist peptide trans-cinnamoyl (tc)-YPGKF-NH2 did not affect TNF induced IL-4 release. Approximately up to 2.4 and 2.3 fold increases in expression of PAR-2 and PAR-4 were observed when cells were incubated with TNF. Pretreatment of cells with TNF for 60 min enhanced trypsin and tryptase induced PAR-2 expression by 2.4 and 2.3 fold; PAR-3 expression by 1.6 and 1.7 fold and PAR-4 expression by 1.6 and 1.7 fold, respectively. LY294002, an inhibitor PI3K abolished TNF induced IL-4 release and phosphorylation of Akt in P815 cells, indicating Akt cell signalling pathway is involved in the event. In conclusion, TNF can stimulate IL-4 release from mast cells through an Akt cell signalling pathway dependent, but PAR independent mechanism. TNF may serve as a regulator for IL-4 production and PAR expression, and through which participates in the mast cell related inflammation.

Expression and release of IL-29 by mast cells and modulation of mast cell behavior by IL-29

To cite this article: He S, Zhang H, Chen H, Yang H, Huang T, Chen Y, Lin J, Wang F, Chen X, Li T‐L, Yang P. Expression and release of IL‐29 by mast cells and modulation of mast cell behavior by IL‐29. Allergy 2010; 65: 1234–1241.

Upregulation of Toll-like receptor (TLR) expression and release of cytokines from P815 mast cells by GM-CSF

BackgroudRecently, mast cells have been recognized to express several Toll-like receptors (TLRs) on their membrane surfaces, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was reported to be able to alter expression of TLRs and cytokine production in neutrophils. However, whether GM-CSF modulates the expression of TLR and cytokine production in mast cells is not clear.ResultsUsing flow cytometry and real time PCR techniques, we found that GM-CSF upregulated expression of TLR3 and TLR7 in P815 cells in a concentration dependent manner. GM-CSF also provoked approximately up to 2.4 and 2.3 fold increase in IL-13 and IL-6 release from P815 cells, respectively following 16 h incubation. GM-CSF induced IL-13 secretion, TLR3 and TLR7 expression appeared to be through activation of mitogen-activated protein kinase (MAPK) and phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathways, whereas GM-CSF elicited IL-6 release seemed via Akt signaling pathway. At 10 ng/ml, GM-CSF significantly enhanced R-848-induced IL-6 release from P815 cells.ConclusionThe ability of GM-CSF in modulation of expression of TLR3 and TLR7 in P815 mast cells and in stimulation of IL-13 and IL-6 release from P815 mast cells in vitro suggests that GM-CSF might play an important role in enhancing the innate immune responses of mast cell to viral infection

Induction of T-helper type 2 cytokine release and up-regulated expression of protease-activated receptors on mast cells by recombinant American cockroach allergen Per a 7

Background The cockroach is a major source of indoor allergens, which cause perennial rhinitis and asthma. Per a 7 is one of the major allergens from the American cockroach, eliciting IgE reaction in over 50% of sera from allergic individuals. However, the actions of Per a 7 in the pathogenesis of allergic diseases remain poorly understood.

A novel allergen Tab y 1 with inhibitory activity of platelet aggregation from salivary glands of horseflies

To cite this article: An S, Ma D, Wei JF, Yang X, Yang HW, Yang H, Xu X, He S, Lai R. A novel allergen Tab y 1 with inhibitory activity of platelet aggregation from salivary glands of horseflies. Allergy 2011; 66: 1420–1427.

Upregulation of Toll-like Receptor (TLR) expression and release of cytokines from mast cells by IL-12.

Background: It has been reported that peptidoglycan (PGN) and lipopolysaccharide (LPS) can provoke mast cells to release an array of cytokines via TLR2 and TLR4, respectively. However, little is known of the regulatory mechanism of TLR2 and TLR4 mediated cytokine production in mast cells. Methods: Since IL-12 plays important roles in protection of the body from microorganism infection and mast cell is a crucial source of IL-12, we investigated effects of IL-12 on expression of TLR2 and TLR4, and cytokine production in mast cells by using quantitative real time PCR, flow cytometry analysis and cellular activation of signaling ELISA techniques. Results: The results showed that IL-12 induced significant increase in expression of TLR2 and TLR4 mRNAs and proteins, respectively. It can also synergistically enhance LPS-induced TLR4 expression in P815 cells. IL-12 not only by itself, but also synergistically enhanced LPS-induced IL-13 release from P815 cells. It appears that IL-12 induced IL-13 release and TLR4 expression is through activation of MAPK and PI3K/Akt signaling pathways, whereas IL-12 induced upregulation of TLR2 is via activation of PI3K/Akt signaling pathway, but not MAPK pathway. Conclusion: The ability of IL-12 in modulation of expression of TLR2 and TLR4 in mast cells, and in stimulation of IL-13 release from mast cells provides further evidence that this cytokine may play a role in the protective immunity against bacteria infection.

Induction of IL-13 production and upregulation of gene expression of protease activated receptors in P815 cells by IL-6

Interleukin (IL)-6 is a multifunctional cytokine which has been showed to induce up-regulated expression of Fc epsilon RI receptor and histamine production in mast cells. However, little is known of its effects on Th2 cytokine secretion and protease activated receptor (PAR) expression in mast cells. In the present study, we examined potential influence of IL-6 on IL-13, IL-4 and IL-10 release from P815 cells and PAR expression on P815 cells by using flow cytometry analysis, quantitative real-time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that IL-6 induced up to 1.8-fold increase in IL-13, but not IL-4 or IL-10 release from P815 cells, and FSLLRY-NH(2) did not affect IL-6 induced IL-13 release. Tryptase elicited 2.0-fold increase in IL-13 release from P815 cells, which can be inhibited by IL-6. IL-6 elicited the up-regulated expression of PAR-1, PAR-2, PAR-3 and PAR-4 mRNAs, but had little effects on expression of PAR proteins. U0126, PD98059 and LY204002 abolished IL-6 induced IL-13 release when they were preincubated with P815 cells, indicating ERK and Akt cell signaling pathways may be involved in the event. In conclusion, IL-6 can stimulate IL-13 release from mast cells through an ERK and Akt cell signaling pathway dependent, but PAR independent mechanism.

Cockroach Allergen per a 7 Down-Regulates Expression of Toll-Like Receptor 9 and IL-12 Release from P815 Cells Through PI3K and MAPK Signaling Pathways

Background: As a major source of indoor allergens, cockroach causes perennial rhinitis and asthma. Recently, cockroach feces were reported to contain TLR2 agonist, which could directly activate neutrophils to release cytokines. CpG oligodeoxynucleotide (ODN), a Toll-like receptor (TLR)9 activator was also found to induce proinflammatory cytokine release from mast cells. However, influence of specific cockroach allergen on Th1 cytokine release and expression of TLR9 in mast cells remains uninvestigated. Methods: To investigate effects of Per a 7 on TLR expression and cytokine release from mast cells and their cell signaling mechanisms, P815 cells were challenged by recombinant Per a 7 (rPer a 7), and expression of TLR9 mRNA and protein was assessed mainly by real time PCR and flow cytometry analysis. Detection of phosphorylation of cell signaling components was performed with Western blotting technique. Results: The results showed that rPer a 7 induced up to 72 and 46% down-regulation of expression of TLR9 mRNA and protein, respectively following 16 h incubation period. It induced also approximately 41.1% reduction of IL-12 release. When PD98059, U0126 and LY294002 were pre-incubated with the cells for 30 min they diminished rPer a 7 induced reduction of TLR9 expression and IL-12 release, indicating these events are via activation of ERK and PI3K/Akt signaling pathways. Conclusion: Reduction of IL-12 production and expression of TLR9 in P815 mastocytoma cells by Per a 7 suggests that this major cockroach allergen might contribute to the development of cockroach allergy.