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1.
FEBS Open Bio ; 7(9): 1267-1280, 2017 09.
Article in English | MEDLINE | ID: mdl-28904857

ABSTRACT

The bodies and faecal pellets of the house dust mite (HDM), Dermatophagoides pteronyssinus, are the source of many allergenic and nonallergenic proteins. One of these, the 14-kDa bacteriolytic enzyme LytFM, originally isolated from the spent HDM growth medium, may contribute to bacteriolytic activity previously detected by zymography at 14 kDa in the culture supernatants of some bacterial species isolated from surface-sterilised HDM. Based on previously reported findings of lateral gene transfer between microbes and their eukaryotic hosts, we investigated the presence of lytFM in the genomes of nine Gram-positive bacteria from surface-sterilised HDM, and the expression by these isolates of LytFM and its variants LytFM1/LytFM2. The lytFM gene was detected by PCR in the genomes of three of the isolates: Bacillus licheniformis strain 1, B. licheniformis strain 2 and Staphylococcus aureus. Expression of the variant LytFM1 was detected in culture supernatants of these bacteria by mass spectrometry (MS) and ELISA, and the bacterial LytFM proteins were shown by zymography to be able to hydrolyse peptidoglycan. Our previous reports of LytFM homologues in other mite species and their phylogenetic analysis had suggested that they originated from a common mite ancestor. The phylogenetic analysis reported herein and the detection of other D. pteronyssinus proteins by MS in the culture supernatants of the three isolates that secreted LytFM1 further support the hypothesis of lateral gene transfer to the bacterial endosymbionts from their HDM host. The complete sequence homology observed between the genes amplified from the microbes and those in their eukaryotic host indicated that the lateral gene transfer was an event that occurred recently.

2.
FEBS Open Bio ; 5: 813-23, 2015.
Article in English | MEDLINE | ID: mdl-26566476

ABSTRACT

A 14 kDa protein homologous to the γ-d-glutamyl-l-diamino acid endopeptidase members of the NlpC/P60 Superfamily has been described in Dermatophagoides pteronyssinus and Dermatophagoides farinae but it is not clear whether other species produce homologues. Bioinformatics revealed homologous genes in other Sarcopteformes mite species (Psoroptes ovis and Blomia tropicalis) but not in Tetranychus urticae and Metaseiulus occidentalis. The degrees of identity (similarity) between the D. pteronyssinus mature protein and those from D. farinae, P. ovis and B. tropicalis were 82% (96%), 77% (93%) and 61% (82%), respectively. Phylogenetic studies showed the mite proteins were monophyletic and shared a common ancestor with both actinomycetes and ascomycetes. The gene encoding the D. pteronyssinus protein was polymorphic and intronless in contrast to that reported for D. farinae. Homology studies suggest that the mite, ascomycete and actinomycete proteins are involved in the catalysis of stem peptide attached to peptidoglycan. The finding of a gene encoding a P60 family member in the D. pteronyssinus genome together with the presence of a bacterial promotor suggests an evolutionary link to one or more prokaryotic endosymbionts.

4.
Physiol Rep ; 3(2)2015 Feb 01.
Article in English | MEDLINE | ID: mdl-25663523

ABSTRACT

Previous studies have shown that protease-activated receptors (PARs) play an important role in various physiological processes. In the present investigation, we determined the expression of PARs on human lung fibroblasts (HLF-1) and whether they were involved in cellular differentiation and pro-inflammatory cytokine and prostaglandin (PGE2) secretion. PAR-1, PAR-2, PAR-3, and PAR-4 were detected in fibroblasts using RT-PCR, immunocytochemistry, and flow cytometry. Increased expression of PAR-4, but not other PARs, was observed in fibroblasts stimulated with phorbol myristate acetate. The archetypical activators of PARs, namely, thrombin and trypsin, as well as PAR-1 and PAR-2 agonist peptides, stimulated transient increases in intracellular Ca(2+), and promoted increased α-smooth muscle actin expression. The proteolytic and peptidic PAR activators also stimulated the release of IL-6 and IL-8, as well as PGE2, with a rank order of potency of PAR-1 > PAR-2. The combined stimulation of PAR-1 and PAR-2 resulted in an additive release of both IL-6 and IL-8. In contrast, PAR-3 and PAR-4 agonist peptides, as well as all the PAR control peptides examined, were inactive. These results suggest an important role for PARs associated with fibroblasts in the modulation of inflammation and remodeling in the airway.

5.
Exp Appl Acarol ; 61(4): 431-47, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23783892

ABSTRACT

Dust mites produce bacteriolytic enzymes, one of which belongs to the NlpC/P60 superfamily comprising bacterial and fungal proteins. Whether this enzyme is derived from the mite or from mite-associated microbes is unclear. To this end, the bacteriology of mites per se, and carpet and mattress dust from a group of asthmatic children and their parents was investigated. Dust from parents' and children's mattresses yielded significantly more colony forming units compared with dust from their corresponding carpets. Zymography demonstrated some dusts contained bacteriolytic enzymes, and in nine of the twelve dust samples from three of five houses examined, a prominent bacteriolytic band was obtained that corresponded to the mite band, although in one home, other lytic bands were detected. Fifty bacterial isolates were obtained from surface-sterilised, commercially obtained Dermatophagoides pteronyssinus. 16S rRNA, tuf and rpoB gene sequencing of nine Gram-positive isolates identified them as Bacillus cereus, B. licheniformis, Staphylococcus aureus, S. epidermidis, S. capitis and Micrococcus luteus, known human skin commensals. 16S rRNA sequence homologies of four of the nine isolates identified as B. licheniformis formed a distinct phylogenetic cluster. All species secreted lytic enzymes during culture although the lytic profiles obtained differed between the rods and the cocci, and none of the bands detected corresponded to those observed in dust or mites. In conclusion, mites harbour a variety of bacterial species often associated with human skin and house dusts contain bacteriolytic enzymes that may be mite-derived. The identification of a novel cluster of B. licheniformis isolates suggests an ecological adaptation to laboratory-reared D. pteronyssinus. It remains to be determined whether the previously described mite-associated 14 K lytic enzyme is derived from a microbial source.


Subject(s)
Bacillus/isolation & purification , Dermatophagoides pteronyssinus/microbiology , Pyroglyphidae/microbiology , Skin/microbiology , Animals , Humans
6.
Biol Chem ; 392(7): 633-42, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21627535

ABSTRACT

Abstract Pleural inflammation underlies the formation of most exudative pleural effusions and the plasma kallikrein-kinin system (KKS) is known to contribute. Mesothelial cells are the predominant cell type in the pleural cavity, but their potential role in plasma KKS activation and BK production has not been studied. Bradykinin concentrations were higher in pleural fluids than the corresponding serum samples in patients with a variety of diseases. Bradykinin concentrations did not correlate with disease diagnosis, but were elevated in exudative effusions. It was demonstrated, using a range of primary and transformed mesothelial and mesothelioma cell lines, that cells assembled high molecular weight kininogen and plasma prekallikrein to liberate bradykinin, a process inhibited by novobiocin, a heat shock protein 90 (HSP90) inhibitor, cysteine, bradykinin and protamine sulphate. Of the common plasma prekallikrein activators, mesothelial cells expressed HSP90, but not prolylcarboxypeptidase or Factor XII. Calcium mobilisation was induced in some mesothelium-derived cell lines by bradykinin. Des-Arg(9)-bradykinin was inactive, indicating that mesothelial cells are responsive to bradykinin, mediated via the bradykinin receptor subtype 2. In summary, pleural mesothelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and may contribute to KKS-mediated inflammation in pleural disease.


Subject(s)
Epithelial Cells/metabolism , Kallikrein-Kinin System , Pleura/cytology , Pleura/immunology , Pleurisy/physiopathology , Animals , Body Fluids/chemistry , Bradykinin/analysis , Bradykinin/pharmacology , Calcium/metabolism , Cell Line, Tumor , Cytokines/metabolism , Epithelial Cells/drug effects , Epithelium/pathology , Gene Expression Regulation/drug effects , Humans , Membrane Proteins/metabolism , Mice , Vasodilator Agents/pharmacology
7.
Biol Chem ; 391(9): 1067-77, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20536386

ABSTRACT

Activation of the tissue kallikrein-kinin system (KKS) plays a major inflammatory role in the lung, but the contribution of the plasma KKS remains unclear. Plasma KKS involves assembly and activation of high molecular weight kininogen (HK) and plasma prekallikrein (PPK) on cell surfaces, resulting in the liberation of the inflammatory peptide, bradykinin (BK), from HK by plasma kallikrein (PK). To this end, we determined the possible contribution of plasma KKS in BK formation using airway epithelium. The HK binding proteins, urokinase plasminogen activator receptor, cytokeratin 1 and gC1qR, were expressed on transformed A549 and BEAS-2B cell lines, as well as on normal lung tissue, but Mac-1 was absent. A549 cells bound FITC-labelled HK, which was only partially inhibited by a combination of antibodies to the HK binding proteins. HK-PPK complex activation on the transformed epithelial cell lines, as well as primary epithelial cells, resulted in PK formation and liberation of BK. HK-PPK activation was inhibited by cysteine, BK and protamine, and by novobiocin, a heat shock protein 90 (HSP90) inhibitor. In summary, lung epithelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and could contribute to KKS-mediated inflammation in lung disease.


Subject(s)
Epithelial Cells/metabolism , Kallikrein-Kinin System/physiology , Lung/cytology , Bradykinin/metabolism , Carrier Proteins/biosynthesis , Epithelial Cells/pathology , Flow Cytometry , HSP90 Heat-Shock Proteins/metabolism , Humans , Immunohistochemistry , Kininogens/antagonists & inhibitors , Kininogens/metabolism , Lung/pathology , Mitochondrial Proteins/biosynthesis , Novobiocin/pharmacology , Prekallikrein/antagonists & inhibitors , Prekallikrein/metabolism , Receptors, Urokinase Plasminogen Activator/biosynthesis , Tumor Cells, Cultured
8.
Int J Mol Med ; 17(4): 551-8, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16525709

ABSTRACT

Fibrin sealant (FS), a biological adhesive material, has been recently recommended as an adjunct in autologous chondrocyte implantation (ACI). While FS has been shown to possess osteoinductive potential, little is known about its effects on chondrogenic cells. In this study, we assessed the bioactivity of FS (Tisseel) on the migration and proliferation of human articular chondrocytes in vitro. Using a co-culture assay to mimic matrix-induced ACI (MACI), chondrocytes were found to migrate from collagen membranes towards FS within 12 h of culture, with significant migratory activity evident by 24 h. In addition, 5-bromo-2'-deoxyuridine (BrdU) incorporation experiments revealed that thrombin, the active component of the tissue glue, stimulated chondrocyte proliferation, with maximal efficacy observed at 48 h post-stimulation (1-10 U/ml). In an effort to elucidate the molecular mechanisms underlying these thrombin-induced effects, we examined the expression and activation of protease-activated receptors (PARs), established thrombin receptors. Using a combination of RT-PCR and immunohistochemistry, all four PARs were detected in human chondrocytes, with PAR-1 being the major isoform expressed. Moreover, thrombin and a PAR-1, but not other PAR-isotype-specific peptide agonists, were found to induce rapid intracellular Ca2+ responses in human chondrocytes in calcium mobilization assays. Together, these data demonstrate that FS supports both the migration and proliferation of human chondrocytes. We propose that these effects are mediated, at least in part, via thrombin-induced PAR-1 signalling in human chondrocytes.


Subject(s)
Cell Movement/drug effects , Cell Proliferation/drug effects , Chondrocytes/drug effects , Fibrin Tissue Adhesive/pharmacology , Calcium/metabolism , Cartilage, Articular/cytology , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Dose-Response Relationship, Drug , Humans , Immunohistochemistry , Intracellular Fluid/drug effects , Intracellular Fluid/metabolism , Microscopy, Confocal , Peptide Fragments/pharmacology , Protein Isoforms/agonists , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, PAR-1/agonists , Receptor, PAR-1/genetics , Receptor, PAR-1/metabolism , Receptor, PAR-2/agonists , Receptor, PAR-2/genetics , Receptor, PAR-2/metabolism , Receptors, Proteinase-Activated/agonists , Receptors, Proteinase-Activated/genetics , Receptors, Proteinase-Activated/metabolism , Receptors, Thrombin/agonists , Receptors, Thrombin/genetics , Receptors, Thrombin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thrombin/pharmacology
9.
FEMS Immunol Med Microbiol ; 46(2): 198-208, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16487301

ABSTRACT

The outer membrane proteins of Moraxella catarrhalis, a bacterial pathogen which causes disease in both children and adults, play an important role in its phenotypic properties. However, their proinflammatory potential with regard to respiratory epithelium and macrophages is unclear. To this end, we examined the cytokine- and mediator-inducing capacity of a heat-killed wild-type M. catarrhalis strain and a nonautoagglutinating mutant as well as their outer membrane proteins and secretory/excretory products using the A549 respiratory epithelial cell line. The outer membrane proteins and secretory/excretory products from both isolates as well as the heat-killed bacteria all induced interleukin (IL)-6, IL-8 and prostaglandin E2, but not IL-1beta, from the A549 cell line in a dose- and time-dependent manner. Heat-killed bacteria and secretory/excretory products stimulated the release of IL-1beta, IL-6, IL-8 and prostaglandin E2 from human monocyte-derived macrophages. Both heat-killed isolates also stimulated nuclear translocation and transactivation of nuclear factor-kappaB. The heat-killed wild-type autoagglutinating isolate induced significantly greater amounts of IL-6 and IL-8 from A549 cells than the nonautoagglutinating mutant compared with the monocyte-derived macrophages but no significant differences in the amounts induced by the two strains were observed. These differences were also evident when the respiratory cell line was stimulated with outer membrane proteins as well as in the degree of nuclear factor-kappaB transactivation. There was little difference in the stimulatory activity of the secretory/excretory products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses revealed some differences in the outer membrane proteins and secretory excretory products between the two isolates. Combined, these data show that M. catarrhalis secretory excretory products and outer membrane proteins are associated with the induction of inflammatory responses in both respiratory epithelium and macrophages.


Subject(s)
Cytokines/metabolism , Dinoprostone/metabolism , Inflammation/immunology , Macrophages/immunology , Moraxella catarrhalis/immunology , Respiratory Mucosa/immunology , Bacterial Outer Membrane Proteins/immunology , Cell Line, Tumor , Culture Media, Conditioned , Epithelial Cells/immunology , Hot Temperature , Humans , Moraxella catarrhalis/genetics , Moraxella catarrhalis/growth & development , Respiratory Mucosa/cytology
10.
Am J Physiol Lung Cell Mol Physiol ; 286(2): L388-98, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14633513

ABSTRACT

Protease-activated receptors (PARs) are widely distributed in human airways, and recent evidence indicates a role for PARs in the pathophysiology of inflammatory airway disease. To further investigate the role of PARs in airway disease, we determined the expression and function of PARs in a murine model of respiratory tract viral infection. PAR-1, PAR-2, PAR-3, and PAR-4 mRNA and protein were expressed in murine airways, and confocal microscopy revealed colocalization of PAR-2 and cyclooxygenase (COX)-2 immunostaining in basal tracheal epithelial cells. Elevated levels of PAR immunostaining, which was particularly striking for PAR-1 and PAR-2, were observed in the airways of influenza A/PR-8/34 virus-infected mice compared with sham-infected mice. Furthermore, increased PAR-1 and PAR-2 expression was associated with significant changes in in vivo lung function responses. PAR-1 agonist peptide potentiated methacholine-induced increases in airway resistance in anesthetized sham-infected mice (and in indomethacin-treated, virus-infected mice), but no such potentiation was observed in virus-infected mice. PAR-2 agonist peptide transiently inhibited methacholine-induced bronchoconstriction in sham-infected mice, and this effect was prolonged in virus-infected mice. These findings suggest that during viral infection, the upregulation of PARs in the airways is coupled to increased activation of COX and enhanced generation of bronchodilatory prostanoids.


Subject(s)
Influenza A virus , Orthomyxoviridae Infections/physiopathology , Receptor, PAR-1/genetics , Receptor, PAR-2/genetics , Respiratory Mucosa/virology , Airway Resistance/drug effects , Airway Resistance/physiology , Animals , Bronchoconstrictor Agents/pharmacology , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Galactose/metabolism , Gene Expression , Indomethacin/pharmacology , Isoenzymes/genetics , Male , Methacholine Chloride/pharmacology , Mice , Mice, Inbred CBA , Orthomyxoviridae Infections/metabolism , Peptides/pharmacology , Prostaglandin-Endoperoxide Synthases/genetics , Receptor, PAR-1/agonists , Receptors, Proteinase-Activated/genetics , Respiratory Mucosa/physiology , Trachea/physiology , Trachea/virology
11.
FEMS Immunol Med Microbiol ; 38(3): 273-82, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-14522463

ABSTRACT

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.


Subject(s)
Burkholderia cepacia/pathogenicity , Cystic Fibrosis/microbiology , Cytokines/metabolism , Dinoprostone/metabolism , Inflammation , Respiratory Mucosa/metabolism , Respiratory Mucosa/microbiology , Burkholderia cepacia/immunology , Cell Line , Cell Line, Tumor , Cystic Fibrosis/immunology , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interleukin-1/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Mutation , NF-kappa B/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Respiratory Mucosa/immunology , Transcriptional Activation
12.
J Allergy Clin Immunol ; 112(3): 531-7, 2003 Sep.
Article in English | MEDLINE | ID: mdl-13679812

ABSTRACT

BACKGROUND: Allergens are frequently found within or attached to particulate material. For example, house dust mite fecal pellets (HDMFP) contain the major mite allergens, exposure to which have been implicated in the development of asthma. Although several studies have examined the ability of purified allergens to generate inflammatory responses, few studies have investigated whether HDMFP per se, are biologically active. OBJECTIVE: Our objective was to examine the ability of whole HDMFP to stimulate nitric oxide (NO) release from alveolar macrophages. METHODS: The rat alveolar macrophage cell line, NR8383, was exposed to HDMFP, Der p 1, or Der p 2, and nitrite levels in the culture supernatants were measured with the Griess reagent. NO synthase mRNA expression was determined by RT-PCR. RESULTS: HDMFP stimulated the production of NO in a dose-dependent and time-dependent manner, with maximum NO levels measured after 48 hours of exposure. Inclusion of polymyxin B did not influence NO production, suggesting that LPS was not responsible for NO production. HDMFP-mediated NO release was down-modulated after treatment with N(G)-nitro-l-arginine methyl ester (L-NAME), dexamethasone, EDTA, and cysteine, but not heat treatment. Inducible nitric oxide synthase mRNA was observed 3 hours after HDMFP exposure, with maximum levels after 48 hours. Both purified Der p 1 and Der p 2 induced NO production, and inhibition of the cysteine protease activity of Der p 1 had little effect on NO production. CONCLUSIONS: HDMFP, Der p 1 and Der p 2 are potent inducers of NO. Neither LPS nor the enzymatic activity of Der p 1 was responsible for NO production observed.


Subject(s)
Allergens/administration & dosage , Antigens, Dermatophagoides/administration & dosage , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , Nitric Oxide/biosynthesis , Animals , Arthropod Proteins , Cell Line , Cysteine Endopeptidases , Dexamethasone/pharmacology , Dust , Edetic Acid/pharmacology , Feces , Mites , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats
13.
Mol Membr Biol ; 20(1): 71-81, 2003.
Article in English | MEDLINE | ID: mdl-12745920

ABSTRACT

Loss of epithelial cell polarity, which can arise following disruption of tight junctions (TJs), is a precursor to the care-fully orchestrated removal of moribund cells from epithelia in apoptosis. Ordinarily, this cycle of events has minimally disruptive effects on the function of the epithelial barrier, but some agents have been identified that induce apoptosis and promote epithelial leakiness. The allergen Der p 1 is a cysteine peptidase that cleaves TJ adhesion proteins and induces apoptosis in epithelial cells. This suggests the possibility that, at least for some inducers of apoptosis, these events might be causally linked. We report here that Der p 1 induces epithelial apoptosis before outright cell detachment and that apoptosis occurs within the same time span as increased paracellular permeability in polarized epithelial monolayers. Whilst TJ-deficient BEAS-2B cells were resistant to Der p 1-induced apoptosis, the cell line 1HAEo-, which was also TJ deficient, was sensitive to Der p 1, providing evidence against TJ proteolysis as a cause of apoptosis. To provide direct evidence, we propagated cells that normally express TJs in low calcium medium that prevented intercellular junction assembly. These cells retained full susceptibility to Der p 1, indicating that Der p 1-induced apoptosis is independent from TJ proteolysis.


Subject(s)
Allergens/pharmacology , Antigens, Dermatophagoides/pharmacology , Apoptosis/drug effects , Epithelial Cells/drug effects , Tight Junctions/metabolism , Animals , Annexin A5/metabolism , Arthropod Proteins , Bronchi/cytology , Calcium/metabolism , Cell Adhesion/drug effects , Cell Line , Cell Membrane Permeability/drug effects , Cysteine Endopeptidases , Dogs , Epithelial Cells/pathology , Humans , Proteins/drug effects , Proteins/metabolism , Signal Transduction , Tight Junctions/drug effects , fas Receptor/genetics , fas Receptor/metabolism
14.
J Leukoc Biol ; 73(1): 91-9, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12525566

ABSTRACT

Although reports suggest that dendritic cells (DC) are involved in the allergic reaction characterized by a T helper cell type 2 (Th2) profile, the role of myeloid (M-DC) and plasmacytoid DC (P-DC), controlling the balance Th1/Th2, remains unknown. Here, we showed that in Dermatophagoides pteronyssinus (Dpt)-sensitized allergic patients and in healthy donors, M-DC displayed a higher capacity to capture Der p 1, a major allergen of Dpt, than did P-DC. However, Der p 1-pulsed M-DC from healthy subjects overexpressed CD80 and secreted interleukin (IL)-10, whereas M-DC from allergic patients did not. In contrast, with Der p 1-pulsed P-DC from both groups, no increase in human leukocyte antigen-DR, CD80, and CD86 and no IL-10 secretion were detected. When cocultured with allogeneic naive CD4(+) T cells from healthy donors, Der p 1-pulsed M-DC from allergic patients favored a Th1 profile [interferon (IFN)-gamma(high)/IL-4(low)] and Der p 1-pulsed P-DC, a Th2 profile (IFN-gamma(low)/IL-4(high)). In healthy donors, no T cell polarization (IFN-gamma(low)/IL-4(low)) was induced by Der p 1-pulsed M-DC or P-DC, but in response to Der p 1-pulsed M-DC, T cells secreted IL-10. The neutralization of IL-10 produced by Der p 1-pulsed M-DC from healthy donors led to an inhibition of IL-10 production by T cells and a polarization toward a type 1. Thus, IL-10 produced by M-DC might be an essential mediator controlling the balance between tolerance and allergic status. In addition, P-DC could contribute to the steady state in healthy donors or to the development of a Th2 response in allergic donors.


Subject(s)
Antigens, Dermatophagoides/immunology , Dendritic Cells/immunology , Dermatophagoides pteronyssinus/immunology , Hypersensitivity/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antigens, Dermatophagoides/pharmacology , Arthropod Proteins , Blood Cells , Case-Control Studies , Coculture Techniques , Cysteine Endopeptidases , Cytokines/analysis , HLA-DR Antigens/analysis , Humans , Myeloid Cells , T-Lymphocytes, Helper-Inducer/drug effects
15.
Eur Cytokine Netw ; 14(4): 219-28, 2003.
Article in English | MEDLINE | ID: mdl-14715413

ABSTRACT

Dendritic cells (DC) are potent antigen - presenting cells that can orientate the immune response towards a Th1 or a Th2 type. DC produce chemokines that are involved in the recruitment of either Th1 cells, such as IP10 (CXCL10), Th2 cells such as TARC (CCL17) and MDC (CCL22), or non-polarized T cells such as RANTES (CCL5) and MIP-lalpha (CCL3). We investigated whether monocyte-derived DC (MD-DC) generated from healthy donors or from patients sensitive to Dermatophagoides pteronyssinus (Dpt) and exposed to the cysteine-protease Der p 1(allergen of Dpt), could upregulate the expression of chemokines involved in type 1 or type 2 T cell recruitment. MD-DC were pulsed with either Der p 1 or with LPS as the control and the chemokines produced were evaluated using ELISA and chemotaxis assays. Der p 1-pulsed DC from allergic patients showed increased TARC (CCL17) and MDC (CCL22) production without modifying IP-10 (CXCL10) release. Der p 1-pulsed DC from healthy donors showed only increased IP-10 (CXCL10) secretion. RANTES (CCL5) and MIP-lalpha (CCL3) production were similarly increased when DC were from healthy or allergic donors. The selective Th2 clone recruitment activity of supernatants from Der p 1-pulsed DC of allergic patients was inhibited by anti-TARC (CCL17) and anti-MDC (CCL22) neutralizing Abs. By using anti-IP10 (CXCL10) blocking Abs, supernatants of Der p 1-pulsed DC from healthy donors were shown to be involved in the recruitment of Th1 cells. These results suggest that in allergic patients exposed to house dust mites, DC may favour the exacerbation of the Th2 response via the increase in type 2 chemokine production.


Subject(s)
Antigens, Dermatophagoides/immunology , Dendritic Cells/immunology , Th2 Cells/immunology , Arthropod Proteins , Chemokine CCL17 , Chemokine CCL22 , Chemokines, CC/metabolism , Cysteine Endopeptidases , Dose-Response Relationship, Immunologic , Humans , Hypersensitivity/immunology , Th2 Cells/metabolism
16.
J Immunol ; 169(8): 4572-8, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12370395

ABSTRACT

In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR-2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca(2+) concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR-2 agonist peptide-induced Ca(2+) release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1.


Subject(s)
Allergens/pharmacology , Antigens, Dermatophagoides/pharmacology , Dermatophagoides pteronyssinus/immunology , Receptors, Thrombin/antagonists & inhibitors , Receptors, Thrombin/metabolism , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Amino Acid Sequence , Animals , Arthropod Proteins , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Calcium/metabolism , Cysteine Endopeptidases/metabolism , DNA, Complementary/genetics , HeLa Cells/metabolism , Humans , Interleukin-6/antagonists & inhibitors , Interleukin-6/metabolism , Interleukin-8/antagonists & inhibitors , Interleukin-8/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , Peptides/agonists , Peptides/physiology , Receptor, PAR-1 , Receptor, PAR-2 , Receptors, Thrombin/agonists , Receptors, Thrombin/biosynthesis , Receptors, Thrombin/genetics , Respiratory Mucosa/cytology , Respiratory Mucosa/enzymology , Transfection , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/metabolism
17.
Pharmacol Ther ; 95(3): 239-57, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12243797

ABSTRACT

Protease-activated receptors (PARs) are G-protein-coupled, seven transmembrane domain receptors that act as cellular enzyme sensors. These receptors are activated by the proteolytic cleavage at the amino terminus, enabling interaction between the newly formed "tethered ligand" and the second extracellular loop of the receptor to confer cellular signalling. PARs can also be activated by small peptides that mimic the tethered ligand. In the respiratory tract, PARs may be regulated by endogenous proteases, such as airway trypsin and mast cell tryptase, as well as exogenous proteases, including inhaled aeroallergens such as those from house dust mite faecal pellets. Immunoreactive PARs have been identified in multiple cell types of the respiratory tract, and PAR activation has been reported to stimulate cellular mitogenesis and to promote tissue inflammation. Activation of PARs concurrently stimulates the release of bronchorelaxant and anti-inflammatory mediators, which may serve to induce cytoprotection and to minimise tissue trauma associated with severe chronic airways inflammation. Furthermore, airway inflammatory responses are associated with increased epithelial PAR expression and elevated concentrations of PAR-activating, and PAR-inactivating, proteases in the extracellular space. On this basis, PARs are likely to play a regulatory role in airway homeostasis, and may participate in respiratory inflammatory disorders, such as asthma and chronic obstructive pulmonary disease. Further studies focussing on the effects of newly developed PAR agonists and antagonists in appropriate models of airway inflammation will permit better insight into the role of PARs in respiratory pathophysiology and their potential as therapeutic targets.


Subject(s)
Receptors, Thrombin , Respiratory System , Animals , Humans , Lung/physiology , Molecular Biology , Receptors, Thrombin/drug effects , Receptors, Thrombin/genetics , Receptors, Thrombin/physiology , Respiratory System/drug effects
18.
FEMS Immunol Med Microbiol ; 33(2): 77-88, 2002 Jun 03.
Article in English | MEDLINE | ID: mdl-12052562

ABSTRACT

Bacteriolytic activity was detected in extracts of whole mite and spent growth medium (SGM) from the clinically important Dermatophagoides pteronyssinus and Dermatophagoides farinae mites and was most abundant in whole mite extract. Gram-positive organisms Micrococcus lysodeikticus, Bacillus megaterium and Listeria monocytogenes were preferentially lysed and the lytic activity was enhanced by thiols, destroyed by mite proteases, inhibited by HgCl2 and high concentrations of NaCl but was resistant to heat and acid treatment. Substrate SDS-PAGE analysis indicated the presence of several lytic enzymes, two of which were isolated from D. pteronyssinus spent growth medium extract by hydroxyapatite chromatography. The N-terminal amino acid sequence of one of them was then used in PCR-based cloning studies. The complete amino acid sequence of this protein was determined and cDNA found to encode a 130-amino acid residue mature protein with a 20-amino acid leader sequence. The deduced protein demonstrated sequence similarity with the C-terminal regions of a group of bacterial proteins belonging to the P60 superfamily. These data suggest that the enzyme is derived from bacteria within the mites rather than from mites per se.


Subject(s)
Bacteriolysis , Dust , Enzymes/isolation & purification , Gram-Positive Bacteria/physiology , Mites/enzymology , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Base Sequence , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/metabolism , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Enzymes/chemistry , Enzymes/genetics , Enzymes/metabolism , Gram-Positive Bacteria/enzymology , Housing , Mites/microbiology , Molecular Sequence Data
19.
J Immunol ; 168(7): 3577-85, 2002 Apr 01.
Article in English | MEDLINE | ID: mdl-11907122

ABSTRACT

Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE(2) production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 microM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE(2) from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca(2+) ion shutoff was approximately 2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.


Subject(s)
Dinoprostone/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Receptors, Thrombin/metabolism , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Animals , Anura , Bronchi/immunology , Bronchi/metabolism , Calcium/metabolism , Cell Line, Transformed/drug effects , Cell Line, Transformed/immunology , Cell Line, Transformed/metabolism , Drug Combinations , Humans , Intracellular Fluid/metabolism , Peptide Hydrolases/pharmacology , Peptides/agonists , Peptides/pharmacology , Receptor, PAR-1 , Receptor, PAR-2 , Receptors, Thrombin/agonists , Receptors, Thrombin/biosynthesis , Receptors, Thrombin/physiology , Respiratory Mucosa/drug effects , Thrombin/pharmacology , Trypsin/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/metabolism
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