Recombinant Der p 1 and Der f 1 exhibit cysteine protease activity but no serine protease activity

Although mite major group 1 allergens, Der p 1 and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p 1 exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p 1 or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group 1 allergens and mite-derived serine proteases. Recombinant mite group 1 allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p 1 and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group 1 allergens.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

Rho-kinase mediates lysophosphatidic acid-induced IL-8 and MCP-1 production via p38 and JNK pathways in human endothelial cells

Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-κB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-κB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-κB.     

尘螨变应原Der f 1核酸序列测定及其分子进化分析

目的：对尘螨主要变应原Der f 1进行核酸序列测定,探讨其系统进化信息。方法：根据Genbank公布的Der f 1基因序列设计引物,巢式PCR扩增Der f 1的cDNA,纯化、回收、克隆至pMD19-T simple后进行序列测定,序列比对后用Clustal W1.83构建分子进化树。结果：成功扩增出Der f 1的cDNA片段,测序表明该基因含ORF1个,长度966bp,与参考序列同源性达99．9％。该变应原具半胱氨酸蛋白酶活性,与果蝇进化关系最远,与梅氏嗜霉螨进化关系最近。结论：成功获得了尘螨变应原Der f 1基因片段．根据其编码的氨基酸序列构建出的系统进化树与形态学分类不一致。     

Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKA-independent pathways

Vasoactive intestinal peptide (VIP) has been shown to be a key regulator of intestinal epithelial functions such as mucus and chloride secretion, paracellular permeability, and cell proliferation. However, its regulatory role in intestinal epithelial chemokine production remains unknown. The aim of this study was (1) to determine whether VIP can modulate intestinal epithelial interleukin-8 (IL-8) production and (2) to identify intracellular mediators responsible for this effect. In the human colonic epithelial cell line HT29-Cl.16E, VIP stimulates IL-8 secretion dose-dependently and IL-8 mRNA level at 10(-9) M. The protein kinase A (PKA) inhibitor PKI did not abolish the effect of VIP. However, inhibition of the ERK1/2 and p38 MAPK pathways reduced the VIP-stimulated IL-8 secretion and mRNA level. Together, our results showed that VIP stimulates IL-8 production in intestinal epithelial cells via PKA-independent and MAPK-dependent pathways. These data suggest that VIPergic pathways can play an immunomodulatory role in intestinal epithelial cells, by regulating epithelial IL-8 secretion.     

House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

House dust mite allergens (HDM) cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1) and unknown enzymatic activity (Der p 2, Der p 5) induce biological responses in a human airway-derived epithelial cell line (A549), and if so, to elucidate the underlying mechanism(s) of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca²⁺ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR)1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca²⁺ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca²⁺ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.     

Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity

Recombinant pro-Der p 1 expressed in yeast Pichia pastoris was convertible into the prosequence-removed mature Der p 1 with full activities of cysteine protease and IgE-binding with or without N-glycosylation of the mature sequence as well as pro-Der f 1. The active recombinant variants will be the basis for various future studies. The major N-terminus of pro-Der p 1 with low proteolytic activity was the putative signal-cleavage site, while that of pro-Der f 1 contained not only the equivalent site but also 21 residues downstream, and pro-Der f 1 retained significant activity. Contribution of the N-terminal region of the Der p 1 prosequence including an N-glycosylation motif on effective inhibition of proteolytic activity of pro-Der p 1 was suggested.     

屋尘螨Ⅰ类变应原Der p1的体内定位

对过敏性疾病主要过敏原屋尘螨Dermatophagoides pteronyssinusⅠ类变应原（Der p1）进行了抗原定位研究。选用经表达和纯化的Der p1抗原蛋白,按常规方法免疫小鼠,分离血清获抗重组Der p1的抗体（Ⅰ抗）,用抗鼠IgG荧光抗体为Ⅱ抗,屋尘螨经石蜡切片,在荧光显微镜下对其特异性变应原的定位进行观察。HE染色显示屋尘螨消化系统占据大部分体腔。组织免疫荧光发现屋尘螨Ⅰ类变应原主要定位于螨的中肠组织及肠内容物,而螨的生殖系统、排泄系统等脏器以及几丁质甲壳均呈阴性反应。据此认为屋尘螨Ⅰ类变应原存在于螨的肠内容物和中肠组织中。     

Differential regulation of IGF-II-induced IL-8 by extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases in human keratinocytes

In order to study the relationship between insulin like growth factor-II (IGF-II) and interleukin-8 (IL-8) that are upregulated in psoriasis, we monitored IL-8 expression in IGF-II-treated human keratinocytes and explored the signaling pathways of IL-8 expression by IGF-II. IGF-II increased the IL-8 mRNA and protein levels in human keratinocytes. The upregulation of IL-8 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src, PI3-kinase, and ERK, but not by p38. Furthermore, IGF-II remarkably increased the DNA binding activities of NF-kappaB and AP-1, and the IL-8 promoter activity. However, cotransfection with IkappaB mutant blocked the IGF-II-induced IL-8 promoter activity. In addition, cotransfection with dominant negative MEK1 mutant, but not with dominant negative p38 mutant, blocked the IGF-II-induced IL-8 promoter activity. These results suggest that IGF-II is involved in the pathogenesis of psoriasis by inducing IL-8 gene expression through the tyrosine kinase-Src-ERK1/2-AP-1 pathway, and the PI3-kinase and NF-kappaB pathway.     

LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium.

We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kappaB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kappaB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC.     

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.     

Rho-kinase regulates endothelin-1-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts

We have previously reported that endothelin-1 (ET-1) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the ET-1-stimulated IL-6 synthesis in MC3T3-E1 cells. ET-1 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific inhibitor of Rho-kinase, significantly suppressed the IL-6 synthesis induced by ET-1 as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, reduced the ET-1-stimulated IL-6 synthesis. Y27632 as well as fasudil attenuated the ET-1-induced phosphorylation of p38 MAP kinase but not p44/p42 MAP kinase. These results strongly suggest that Rho-kinase regulates ET-1-stimulated IL-6 synthesis through p38 MAP kinase activation in osteoblasts.     

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus

Background

The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade.

Methods

The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus.

Results

All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite.

Conclusions

Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases.

General significance

This finding suggests that Der p 1 may be valuable target against mites.     

TNF-alpha induces interleukin-8 and endothelin-1 expression in human endothelial cells with different redox pathways

We investigated the effect of TNF-alpha on interleukin-8 (IL-8) and endothelin-1 (ET-1) expression, and their different signal transduction pathways. Human dermal microvascular endothelial cells (HMECs) were treated with TNF-alpha. By Northern blot analysis, TNF-alpha at 50, 100, 200, and 400 U/ml significantly induced IL-8 mRNA expression by 206%, 252%, 211%, and 158%, respectively, as compared to controls (p < 0.05). Overexpression of human superoxide dismutase (SOD) by adenovirus-mediated gene transfer or addition of exogenous hydrogen peroxide (H(2)O(2)) significantly enhanced TNF-alpha-induced IL-8 mRNA expression. Furthermore, HMECs treated with TNF-alpha at 50, 100, and 200 U/ml significantly increased ET-1 mRNA expression by 71%, 82%, and 66%, respectively (p < 0.05). By contrast, SOD gene transfer and exogenous H(2)O(2) significantly inhibited TNF-alpha-induced ET-1 mRNA expression. Thus, TNF-alpha significantly induces both IL-8 and ET-1 gene expression in HMECs possibly through different redox signaling pathways. H(2)O(2) enhances TNF-alpha-induced IL-8 expression, but inhibits TNF-alpha-induced ET-1 expression.     

Suppression of sulfur mustard-increased IL-8 in human keratinocyte cell cultures by serine protease inhibitors: implications for toxicity and medical countermeasures

The toxicity of the chemical warfare blistering agent sulfur mustard (2,2'-dichlorodiethyl sulfide; SM) has been investigated for nearly a century; however, the toxicological mechanisms of SM remain obscure and no antidote exists. The similarity of dermal-epidermal separation caused by SM exposure, proteolysis, and certain bullous diseases has fostered the hypothesis that SM vesication involves proteolysis and/or inflammation. Compound screening conducted by the US Army Medical Research Institute of Chemical Defense established that topical application of three tested serine protease inhibitors could reduce SM toxicity in the mouse ear vesicant model. Although most of the drugs with efficacy for SM toxicity in rodent models are anti-inflammatory compounds, no in vitro assay is in current use for screening of potential anti-inflammatory SM antidotes. IL-8 is a potent neutrophil chemotactic cytokine that is increased in human epidermal keratinocyte (HEK) cell cultures following exposure to SM and has been proposed as a marker for SM-induced inflammation. This study was conducted to establish in vitro screening of IL-8 in SM-exposed HEK as a possible model for evaluating candidate compounds prior to in vivo testing. We chose two protease inhibitors, one from those shown as successful in the MEVM (ethyl p-guanidinobenzoate hydrochloride, ICD 1579) and a prototypic inhibitor of trypsin, N-tosyl-L-lysine chloromethyl ketone (TLCK). TLCK (62.5 to 1000 μmol/L) or ICD 1579 (31.25 to 1000 μmol/L) was added to HEK cell cultures 1 h after SM exposure (200 μmol/L) and dose-dependently suppressed SM-increased IL-8. The suppression of SM-increased IL-8 by a class of drug candidate compounds such as protease inhibitors may provide a mechanistic marker that helps predict future medical countermeasures for SM toxicity and reduces the need for testing in animal models. This revised version was published online in July 2006 with corrections to the Cover Date.     

Resveratrol treatment reduces expression of MCP-1, IL-6, IL-8 and RANTES in endometriotic stromal cells

Endometriosis is an inflammatory disease affecting reproductive-aged women. Immunologic disturbance, as well as inflammation, have crucial roles in the pathogenesis of endometriosis. In this study, we evaluated the effects of resveratrol treatment on expression of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), IL-8, and regulated upon activation, normal T cell expressed and secreted (RANTES) in endometrial stromal cells from patients with endometriosis compared with non-endometriotic controls. Thirteen eutopic (EuESCs) and nine ectopic (EESCs) endometrial stromal cells from endometriotic patients as well as eleven endometrial stromal cells from non-endometriotic controls (CESCs) were treated with resveratrol (100 μmol/L) or ethanol, and gene and/or protein expression of MCP-1, IL-6, IL-8 and RANTES was examined at 6, 24 and 48 hours following treatment in the cells from all origins. Resveratrol treatment significantly reduced gene and protein expression of MCP-1, IL-6, and IL-8 in EuESCs and EESCs compared with CESCs (P < .05-.001, P < .05-.001 and P < .05-<.01, respectively), and this reduction was more noticeable in EESCs than EuESCs (P < .05-<.001). Besides, resveratrol treatment significantly reduced RANTES protein expression in EESCs in all time intervals (P < .05). Resveratrol treatment significantly reduced the expression of MCP-1, IL-6, IL-8 and RANTES in EESCs.     

Sphingosine 1-phosphate induces MKP-1 expression via p38 MAPK- and CREB-mediated pathways in airway smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid elevated in asthmatic airways, is increasingly recognized as playing an important role in respiratory disease. S1P activates receptor-mediated signaling to modulate diverse cellular functions and promote airway inflammation. Although many of the stimulatory pathways activated by S1P have been delineated, especially mitogen-activated protein kinases (MAPK), the question of whether S1P exerts negative feedback control on its own signaling cascade via upregulation of phosphatases remains unexplored. We show that S1P rapidly and robustly upregulates mRNA and protein expression of the MAPK deactivator-MAPK phosphatase 1 (MKP-1). Utilizing the pivotal airway structural cell, airway smooth muscle (ASM), we confirm that S1P activates all members of the MAPK family and, in part, S1P upregulates MKP-1 expression in a p38 MAPK-dependent manner. MKP-1 is a cAMP response element binding (CREB) protein-responsive gene and here, we reveal for the first time that an adenylate cyclase/PKA/CREB-mediated pathway also contributes to S1P-induced MKP-1. Thus, by increasing MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways, S1P temporally regulates MAPK signaling pathways by upregulating the negative feedback controller MKP-1. This limits the extent and duration of pro-inflammatory MAPK signaling and represses cytokine secretion in ASM cells. Taken together, our results demonstrate that S1P stimulates both kinases and the phosphatase MKP-1 to control inflammation in ASM cells and may provide a greater understanding of the molecular mechanisms responsible for the pro-asthmatic functions induced by the potent bioactive sphingolipid S1P in the lung.