Effects of Bothrops atrox venom and two isolated toxins on the human complement system: Modulation of pathways and generation of anaphylatoxins

The complement system plays important biological roles, including the activation of inflammatory processes in response to the generation of proteolytic fragments of its components. Here we evaluated the effects of Bothrops atrox venom and two of its toxins (the P-I metalloprotease Batroxase and the acidic phospholipase A₂ BatroxPLA₂) on the human complement system, evaluating their effects on the classical (CP), lectin (LP) and alternative (AP) pathways, as well as on different complement components associated to the generation of anaphylatoxins. Primarily, the venom and both toxins modulated the hemolytic activity of the complement CP, with the venom and Batroxase reducing this activity and BatroxPLA₂ increasing it. ELISA deposition assays indicated that B. atrox venom and Batroxase were also capable of modulating all three activation pathways (CP, LP and AP), reducing their activity after incubation with normal human serum (NHS), while BatroxPLA₂ apparently only interfered with AP. Additionally, the venom and Batroxase, but not BatroxPLA₂, promoted significant degradation of the components C3, C4, Factor B and C1-Inhibitor, as shown by Western blot and SDS-PAGE analyses, also generating anaphylatoxins C3a, C4a and C5a. Therefore, B. atrox venom and Batroxase were able to activate the complement system by direct proteolytic action on several components, generating anaphylatoxins and affecting the activation pathways, while BatroxPLA₂ only interfered with the hemolysis induced by CP and the C3 deposition related to AP. Our results indicate that Batroxase and possibly other metalloproteases should be the main toxins in B. atrox venom to induce pronounced effects on the complement system.     

金属蛋白酶在骨关节炎表达的研究进展

目的 探讨金属蛋白酶在骨关节炎中的作用和表达机制及其影响因素.方法 由第一作者应用计算机检索PubMed、中国期刊全文数据库(CNKI)、维普数据库和万方数据库1997-05/2012-08相关文献.在标题、摘要、关键词中以“Osteoarthritis; Proteinase; Cartilage;Cartilage matrix; Metalloprotease”或“骨关节炎;蛋白酶;软骨;金属蛋白酶;软骨基质”为检索词进行检索.选择文章内容与金属蛋白酶有关者,同一领域文献则选择近期发表在权威杂志文章进行分析.结果 初检得到206篇文献,排除134篇不符合标准的文献,共纳入72篇符合标准的文献.结论 经分析得出金属蛋白酶在骨关节炎中起着重要的作用,其基因表达由多种因子参与转录调控,而其酶活性由相关抑制因子或活化因子抑制或活化其活性,此外,机械负荷也参与调控金属蛋白酶的活性.     

CD23 shedding: requirements for substrate recognition and inhibition by dipeptide hydroxamic acids

CD23 (low affinity IgE receptor, FcεRII) is expressed as a Type II extracellular protein on a variety of cells such as B cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in regulation of IgE synthesis. CD23 is released from the cell surface by a metalloprotease, analogous to the cleavage of other cell surface molecules such as TNF-α. This activity has been extensively studied with respect to biochemical characterization and ability to cleave specific mutants of CD23. Both local sequence and distal domains have been shown to affect cleavage of CD23. Selective dipeptide hydroxamic acid inhibitors of CD23 processing have been identified and demonstrated to very potently and selectively inhibit CD23 processing.     

The detection of ADAM8 protein on cells of the human immune system and the demonstration of its expression on peripheral blood B cells, dendritic cells and monocyte subsets

A disintegrin and metalloprotease (ADAM) proteins have wide ranging functions, including proteolytic cleavage of cell surface molecules, cell fusion, cell adhesion and intracellular signalling. Recent evidence suggests the involvement of ADAM8 in allergic responses. For instance, ADAM8 is amongst a number of genes up-regulated in experimentally induced asthma in animals. In order to further define the involvement of ADAM8 in allergic responses, we sought in the first instance to examine its distribution on human peripheral blood B cells, resting and activated T cells, monocyte subsets and monocyte derived dendritic cells. Here we demonstrate for the first time ADAM8 protein expression on B cells and dendritic cells, and its higher expression on CD14(2+)CD16(-) monocytes compared to CD14(+)CD16(+) cells. Immature dendritic cells expressed low levels of ADAM8 when treated with a combination of GM-CSF and IL-4, but stimulation with LPS resulted in a higher level of expression, which was TLR-4 independent. Up-regulation of ADAM8 expression on dendritic cells was also observed after stimulation with TNF-alpha, but not after stimulation with anti-CD40. The demonstration of ADAM8 expression on these cells provides an opportunity for addressing the potential role of inhaled protease allergens, such as Der p 1, in modulating ADAM8 functions, particularly with regards to innate immune responses by dendritic cells and IgE synthesis by B cells.     

Cellular Responses to Whitlockite

Whitlockite crystals have been observed in both degenerating and normal articular cartilages. To determine their potential for inducing cartilage degeneration, we studied their ability to induce mitogenesis and synthesis and secretion of metalloproteases in vitro. Whitlockite crystals were found to stimulate cell proliferation and to stimulate synthesis and secretion of stromelysin and collagenase. However, they were less stimulatory than crystals that contained calcium (Ca) and phosphate without magnesium substitution for Ca. Whitlockite crystals elicit biologic cellular responses that suggest potential pathogenicity in arthritis, but are less potent than Ca phosphate crystals without magnesium. Received: 3 November 1998 / Accepted: 10 June 1999     

Immune cells and mediators involved in the inflammatory responses induced by a P-I metalloprotease and a phospholipase A2 from Bothrops atrox venom

Bothrops envenomations can promote severe inflammatory responses by inducing edema, pain, leukocyte recruitment and release of chemical mediators by local cells. In the present study, two toxins from Bothrops atrox venom (the P-I metalloprotease Batroxase and the acidic phospholipase A₂ BatroxPLA₂) were evaluated in relation to their inflammatory effects induced in vivo and in vitro, mainly focusing on the participation of different immune cells and inflammatory mediators. Both toxins mainly promoted acute inflammatory responses with significant recruitment of neutrophils in the early hours (1–4 h) after administration into the peritoneal cavity of C57BL/6 mice, and increased infiltration of mononuclear cells especially after 24 h. Among the mediators induced by both toxins are IL-6, IL-10 and PGE₂, with Batroxase also inducing the release of L-1β, and BatroxPLA₂ of LTB₄ and CysLTs. These responses pointed to possible involvement of immune cells such as macrophages and mast cells, which were then evaluated in vitro. Mice peritoneal macrophages stimulated with Batroxase produced significant levels of IL-6, IL-1β, PGE₂ and LTB₄, whereas stimulus with BatroxPLA₂ induced increases of IL-6, PGE₂ and LTB₄. Furthermore, both toxins were able to stimulate degranulation of RBL-2H3 mast cells, but with distinct concentration-dependent effects. Altogether, these results indicated that Batroxase and BatroxPLA₂ promoted local and acute inflammatory responses related to macrophages and mast cells and to the production of several mediators. Our findings should contribute for better understanding the different mechanisms of toxicity induced by P-I metalloproteases and phospholipases A₂ after snakebite envenomations.     

Changes in plasma von Willebrand factor and ADAMTS13 levels associated with left atrial remodeling in atrial fibrillation

Introduction

Previous studies have shown raised plasma von Willebrand factor (VWF) levels in patients with atrial fibrillation (AF). However, little is known about changes of VWF associated with VWF-cleaving protease (ADAMTS13) in AF. The aim of this study was to examine the relationship between changes in plasma VWF and ADAMTS13 levels, and left atrial remodeling in AF patients.

Materials and Methods

We measured plasma VWF and ADAMTS13 antigen levels in 70 paroxysmal AF (PAF) patients, 56 chronic AF (CAF) patients, and 55 control subjects.

Results

Plasma VWF levels (mU/ml) were significantly higher in CAF and PAF patients compared with the controls (2103 ± 743, 1930 ± 676, 1532 ± 555, respectively, P < 0.0001 in CAF vs. controls, P = 0.001 in PAF vs. control), while ADAMTS13 levels (mU/ml) were significantly lower in CAF and PAF patients compared with the controls (795 ± 169, 860 ± 221, 932 ± 173, respectively, P = 0.0002 in CAF vs. controls, P = 0.04 in PAF vs. control). The VWF/ADAMTS13 ratio was significantly higher in patients with CAF than PAF or controls (2.81 ± 1.30, 2.34 ± 0.92, 1.73 ± 0.83, respectively; P = 0.01 in CAF vs. PAF, P < 0.0001 in CAF vs. controls). There was a significant correlation between the VWF/ADAMTS13 ratio and left atrial diameter (positive correlation; r = 0.275, P = 0.0002) and left atrial appendage flow velocity (negative correlation; r = - 0.345, P = 0.0018).

Conclusions

These findings suggest that the imbalance between plasma VWF and ADAMTS13 levels caused by left atrial remodeling might be closely associated with intra-atrial thrombus formation in AF patients.     

丹参酮ⅡA对人股骨头成骨细胞金属蛋白酶表达的影响

目的 通过观察丹参酮ⅡA对人股骨头成骨细胞金属蛋白酶及其抑制因子表达的影响,探讨其在治疗股骨头坏死方面的作用.方法 体外培养人股骨头成骨细胞,加入IL-1β促进成骨细胞表达MMP及TIMP,对照组用qPCR方法检测成骨细胞表达MMP和TIMP的类型及量,各3个样本,单个样本重复检测3次,实验组加入丹参酮ⅡA后亦行相同检测,并行比较.结果 qPCR未见MMP8,MMP12、MMP15及TIMP4扩增;qPCR检测实验组MMP1、MMP2、MMP3、MMP7、MMP9、MMP13、MMP14、MMP17、TIMP1及TIMP3表达较对照组有显著差异(P＜0.05),实验组MMP10、MMP11及TIMP2表达较对照组无显著差异(P＞0.05).结论 人股骨头成骨细胞未表达MMP8,MMP12、MMP15及TIMP4;丹参酮ⅡA能有效抑制人股骨头成骨细胞部分MMP分泌,并有效促进部分TIMP分泌,从而达到延缓股骨头坏死的作用.     

Metalloprotease MP100: a synaptic protease in rat brain

Proteases are expressed widely throughout the nervous system and perform essential functions. We have earlier characterized and cloned the metalloprotease MP100, an enzyme originally described as a beta-amyloid precursor protein (beta-APP) processing candidate. In the present study we describe the cellular and subcellular localization of MP100 in rat brain. A punctuate intracellular immunostaining in cortical, hippocampal and cerebellar neurons suggests its high abundance in vesicular intracellular structures. The MP100 staining pattern resembled that of the presynaptic protein synaptophysin. In gel filtration chromatography of isolated rat brain synaptosomal membranes, MP100 co-fractionated with synaptophysin and beta-APP. Furthermore, pre-embedding immunoelectron microscopy of the cerebellum revealed MP100 to be localized at synaptic sites. All together, these data might indicate a role for MP100 in functions such as proteolytic modification of synaptic proteins.