Shao-ng He

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.

Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release

BackgroundIt has been recognized that dermal fibroblasts and matrix metalloproteases (MMP) play crucial roles in wound healing process in skin. Thrombin was found to stimulate IL-8 release from human dermal fibroblasts (HDFs). However, little is known of the effect of thrombin on secretion of MMPs from dermal fibroblasts. In the present study, the influence of thrombin on proMMP-2 and proMMP-9 activity release from primary cultured HDFs, and its potential signaling pathways were investigated.ResultsThe results showed that thrombin induced proMMP-9, but not proMMP-2 release from HDFs in a dose dependent manner at 6 h following incubation. Thrombin also upregulated expression of proMMP-9 mRNA in HDFs. Hirudin completely abolished the action of thrombin on HDFs. An agonist peptide of protease-activated receptor-1, SFLLR-NH2 stimulated an enhanced release of proMMP-9 from HDFs. AG490, an inhibitor of STAT3 inhibited basal and thrombin-provoked proMMP-9 release and phosphorylation of STAT3. PD98059, an inhibitor of MAPK and LY294002, an inhibitor PI3K failed to significantly inhibit thrombin induced proMMP-9 release.ConclusionThrombin is a potent stimulus of proMMP-9 release from HDFs. Thrombin induced proMMP-9 release is most likely through activation of PAR-1. JAK/STAT3 signaling pathway is involved in proMMP-9 release from HDFs.

INDUCTION OF INFLAMMATORY CYTOKINE RELEASE FROM HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS BY AGONISTS OF PROTEINASE‐ACTIVATED RECEPTOR‐2

1 Human endothelial cells express proteinase‐activated receptor‐2 (PAR‐2), inflammatory cytokines and trypsin (EC 3.4.21.4). However, little is known about the mechanism through which trypsin induces cytokine release from endothelial cells. 2 In the present study, we investigated the effect of trypsin on cytokine release from primary cultures of human umbilical vein endothelial cells (HUVEC) using an antibody based protein microarray and ELISA. 3 The results showed that 1 mg/mL trypsin induced release of 32 different inflammatory factors, whereas 100 mmol/L Ser‐Leu‐Ile‐Gly‐Lys‐Val‐NH2 (SLIGKV‐NH2) only stimulated secretion of 16 inflammatory factors from HUVEC, as assessed by an antibody based protein microarray. Because the release of interleukin (IL)‐1a, IL‐8, IL‐10 and IL‐12 was markedly increased following PAR‐2 activation, their release was investigated further using ELISA. Increases in release of up to approximately 4.8‐, 4.3‐, 4.1‐ and 1.8‐fold were observed for IL‐1a, IL‐10, IL‐12 and IL‐8, respectively, when HUVEC were challenged with trypsin for 16 h. Agonist peptides of PAR‐2, namely SLIGKV‐NH2 and trans‐cinnamoyl‐Leu‐Ile‐Gly‐Arg‐Leu‐Orn‐NH2 (tc‐LIGRLO‐NH2), also provoked significant release of IL‐8. Trypsin‐induced cytokine release was inhibited by its inhibitors soybean trypsin inhibitor, a1‐antitrypsin and the inhibitor peptide of PAR‐2 Phe‐Ser‐Leu‐Leu‐Arg‐Tyr‐NH2 (FSLLRY‐NH2). 4 These data indicate the action of trypsin on HUVEC is most likely through activation of PAR‐2, suggesting that PAR‐2‐related mechanisms are involved in the inflammatory process in humans.

Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2

AbstractAim:To investigate the effects of the agonists of proteinase activated receptor (PAR)-2, and histamine on degranulation of human mast cells.Methods:Human mast cells were enzymatically dispersed from tonsil and skin tissues. The dispersed cells were then cultured with various stimuli, and tryptase and histamine levels in cell supernatants collected from challenge tubes were measured.Results:PAR-2 agonist peptide SLIGKV provoked a dose-dependent release of histamine from skin mast cells. It also induced tryptase release from tonsil mast cells. tc-LIGRLO appeared less potent than SLIGKV in induction of release of histamine and tryptase. Trypsin was able to induce a “bell” shape increase in tryptase release from tonsil mast cells. It was also able to induce a dose-dependent release of histamine from both tonsil and skin mast cells. The actions of trypsin on mast cells were inhibited by soy bean trypsin inhibitor (SBTI) or α1-antitrypsin (α1-AT). Time course study revealed that both stimulated tryptase or histamine release initiated within 10 s and reached their peak release between 4 and 6 min. Pretreatment of cells with metabolic inhibitors or pertussis toxin reduced the ability of mast cells to release tryptase or histamine.Conclusion:It was demonstrated that the in vitro tryptase release properties of human tonsil and skin mast cells suggested a novel type of mast cell heterogeneity. The activation of mast cells by PAR-2 agonists indicated a self-amplification mechanism of mast cell degranulation.

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.

Induction of mast cell accumulation, histamine release and skin edema by N49 phospholipase A2

BackgroundIt has been recognized that phospholipase A2 (PLA2) is a crucial component of snake venom, which contributes greatly to snake venom induced inflammation in man. However, the mechanisms through which N49 PLA2 provoke inflammation remain unclear. Recently, a N49 PLA2, TM-N49 from Protobothrops mucrosquamatus crude venom was characterized in our laboratory. Since the purification procedure developed is able to supply us with relatively large quantity of highly purified TM-N49, we investigated the ability of TM-N49 in induction of inflammation.ResultsThe results showed that TM-N49 provoked a dose dependent increase in microvascular leakage in the skin of rats. The potency of TM-N49 in induction of skin edema appeared similar potency of bradykinin and histamine. Pretreatment of rats with compound 48/80 diminished TM-N49 induced skin reaction and reduced mast cell numbers in rats. Ginkgolide B and cyproheptadine, but not terfenadine and quinacrine, inhibited TM-N49 elicited microvascular leakage when they were co-injected with the stimulus to rat skin. Moreover, TM-N49 was found to induce histamine release from human colon, lung and tonsil mast cells, and both metabolic inhibitors and pertussis toxin were capable of inhibiting TM-N49 elicited histamine release. TM-N49 induced mast cell accumulation in the peritoneum of mice, which was inhibited by co-injection of ginkgolide B, cyproheptadine and terfenadine. Intravenous injection of monoclonal antibodies against CD18, ICAM-1 and CD11a also blocked TM-N49 induced mast cell accumulation.ConclusionTM-N49 is a potent stimulus for skin edema, mast cell activation and accumulation.

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.

CD14+ cell–derived IL-29 modulates proinflammatory cytokine production in patients with allergic airway inflammation

To cite this article: He S, Li T, Chen H, Ma W, Yao Q, Yang H, Wang H, Wang F, Zhao C, Yang P. CD14+ cell–derived IL‐29 modulates proinflammatory cytokine production in patients with allergic airway inflammation. Allergy 2011; 66: 238–246.