ABSTRACT
Neutrophil extracellular traps (NETs) are networks of decondensed chromatin loaded with antimicrobial peptides and enzymes produced against microorganisms or biochemical stimuli. Since their discovery, numerous studies made separately have revealed multiple triggers that induce similar NET morphologies allowing to classify them as lytic or non-lytic. However, the variability in NET composition depending on the inducer agent and the local milieu under similar conditions has been scarcely studied. In this work, a comparative study was conducted to evaluate structural and enzymatic divergences in NET composition induced by biochemical (phorbol myristate acetate [PMA] and hypochlorous acid [HOCl]) and microbiologic (Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa) stimuli, along with the presence of plasma from healthy donors or patients with systemic lupus erythematosus (SLE). The results showed a differential composition of DNA and the antimicrobial peptide cathelicidin (LL37) and a variable enzymatic activity (neutrophil elastase, cathepsin G, myeloperoxidase) induced by the different stimuli despite showing morphologically similar NETs. Additionally, SLE plasma´s presence increased DNA and LL37 release during NET induction independently of the trigger stimulus but with no enzymatic activity differences. This work provides new evidence about NET composition variability depending on the inducer stimulus and the local milieu.
Subject(s)
Extracellular Traps/metabolism , Lupus Erythematosus, Systemic/immunology , Neutrophils/metabolism , Biomarkers/analysis , Biomarkers/metabolism , Candida albicans/immunology , Case-Control Studies , Cathelicidins/analysis , Cathelicidins/metabolism , Cathepsin G/analysis , Cathepsin G/metabolism , Cells, Cultured , Extracellular Traps/immunology , Healthy Volunteers , Humans , Hypochlorous Acid/immunology , Leukocyte Elastase/analysis , Leukocyte Elastase/metabolism , Lupus Erythematosus, Systemic/blood , Neutrophils/immunology , Peroxidase/analysis , Peroxidase/metabolism , Primary Cell Culture , Pseudomonas aeruginosa/immunology , Staphylococcus aureus/immunology , Tetradecanoylphorbol Acetate/immunologyABSTRACT
Neutrophils are predominant immune cells that protect the human body against infections by deploying sophisticated antimicrobial strategies including phagocytosis of bacteria and neutrophil extracellular trap (NET) formation. Here, we provide an overview of the mechanisms by which neutrophils kill exogenous pathogens before we focus on one particular weapon in their arsenal: the generation of the oxidizing hypohalous acids HOCl, HOBr and HOSCN during the so-called oxidative burst by the enzyme myeloperoxidase. We look at the effects of these hypohalous acids on biological systems in general and proteins in particular and turn our attention to bacterial strategies to survive HOCl stress. HOCl is a strong inducer of protein aggregation, which bacteria can counteract by chaperone-like holdases that bind unfolding proteins without the need for energy in the form of ATP. These chaperones are activated by HOCl through thiol oxidation (Hsp33) or N-chlorination of basic amino acid side-chains (RidA and CnoX) and contribute to bacterial survival during HOCl stress. However, neutrophil-generated hypohalous acids also affect the host system. Recent studies have shown that plasma proteins act not only as sinks for HOCl, but get actively transformed into modulators of the cellular immune response through N-chlorination. N-chlorinated serum albumin can prevent aggregation of proteins, stimulate immune cells, and act as a pro-survival factor for immune cells in the presence of cytotoxic antigens. Finally, we take a look at the emerging role of HOCl as a potential signaling molecule, particularly its role in neutrophil extracellular trap formation.
Subject(s)
Bacteria/immunology , Bacterial Infections/immunology , Bromates/immunology , Hypochlorous Acid/immunology , Neutrophils/immunology , Animals , Bacterial Physiological Phenomena , Extracellular Traps/immunology , Host-Pathogen Interactions , Humans , Inflammation/immunology , Phagocytosis , Thiocyanates/immunologyABSTRACT
Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.
Subject(s)
Hypochlorous Acid/pharmacology , Oxidants/pharmacology , Pseudomonas aeruginosa/immunology , Thiocyanates/pharmacology , Bacterial Proteins/physiology , DNA Transposable Elements/genetics , DNA-Binding Proteins/physiology , Down-Regulation , Drug Resistance, Microbial , Genes, Regulator/genetics , Hypochlorous Acid/immunology , Hypochlorous Acid/metabolism , Mutation , Oxidants/immunology , Oxidants/metabolism , Plasmids , Polymerase Chain Reaction , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , RNA, Bacterial/chemistry , RNA, Bacterial/isolation & purification , RNA, Transfer/physiology , Thiocyanates/immunology , Thiocyanates/metabolism , Trans-Activators/genetics , Transcription Factors/physiology , Up-RegulationABSTRACT
Phagocytes destroy ingested microbes by producing hypochlorous acid (HOCl) from chloride ions (Cl-) and hydrogen peroxide within phagolysosomes, using the enzyme myeloperoxidase. HOCl, the active ingredient in bleach, has antibacterial/antiviral properties. As myeloperoxidase is needed for HOCl production, non-myeloid cells are considered incapable of producing HOCl. Here, we show that epithelial, fibroblast and hepatic cells have enhanced antiviral activity in the presence of increasing concentrations of sodium chloride (NaCl). Replication of enveloped/non-enveloped, DNA (herpes simplex virus-1, murine gammaherpesvirus 68) and RNA (respiratory syncytial virus, influenza A virus, human coronavirus 229E, coxsackievirus B3) viruses are inhibited in a dose-dependent manner. Whilst treatment with sodium channel inhibitors did not prevent NaCl-mediated virus inhibition, a chloride channel inhibitor reversed inhibition by NaCl, suggesting intracellular chloride is required for antiviral activity. Inhibition is also reversed in the presence of 4-aminobenzoic hydrazide, a myeloperoxidase inhibitor, suggesting epithelial cells have a peroxidase to convert Cl- to HOCl. A significant increase in intracellular HOCl production is seen early in infection. These data suggest that non-myeloid cells possess an innate antiviral mechanism dependent on the availability of Cl- to produce HOCl. Antiviral activity against a broad range of viral infections can be augmented by increasing availability of NaCl.
Subject(s)
Hydrogen Peroxide/immunology , Hypochlorous Acid/immunology , Immunity, Innate , Sodium Chloride/immunology , Viruses/immunology , A549 Cells , Aniline Compounds/pharmacology , Animals , Chloride Channels/antagonists & inhibitors , Chloride Channels/immunology , HeLa Cells , Humans , Ions , Mice , NIH 3T3 Cells , Peroxidase/antagonists & inhibitors , Peroxidase/immunologyABSTRACT
The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in â¼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.
Subject(s)
Fluorescent Dyes/chemistry , Hypochlorous Acid/analysis , Neutrophils/enzymology , Phagocytosis , Phagosomes/metabolism , Rhodamines/chemistry , Biological Assay , Fluorescent Dyes/metabolism , Humans , Hypochlorous Acid/immunology , Hypochlorous Acid/metabolism , Metabolism, Inborn Errors/enzymology , Metabolism, Inborn Errors/immunology , Metabolism, Inborn Errors/pathology , NADPH Oxidases/genetics , NADPH Oxidases/immunology , NADPH Oxidases/metabolism , Neutrophils/immunology , Neutrophils/pathology , Opsonin Proteins/chemistry , Peroxidase/deficiency , Peroxidase/genetics , Peroxidase/immunology , Phagosomes/immunology , Phagosomes/ultrastructure , Primary Cell Culture , Rhodamines/metabolism , Spectrometry, Fluorescence , Staphylococcus aureus/immunology , Zymosan/chemistryABSTRACT
Sustained generation of extracellular superoxide anions by membrane-associated NADPH oxidase-1 is a hallmark of malignant transformation. The resulting H2O2 drives the proliferation of malignant cells and is converted to HOCl by a Dual oxidase-related peroxidase domain that acts analogously to myeloperoxidase. Whereas H2O2 induces apoptosis nonselectively in nontransformed and transformed cells, HOCl selectively affects malignant cells, as the interaction between HOCl and extracellular superoxide anions allows for site-specific generation of apoptosis-inducing hydroxyl radicals. Transformed cells (early stages of tumor progression) and bona fide tumor cells (representing late stages of tumor progression) respond to exogenous HOCl or HOCl generated by professional phagocytes with induction of apoptosis. In contrast, only transformed cells have the potential to synthesize HOCl through interaction between their superoxide anions/H2O2 and Dual oxidase-related peroxidase released by themselves or neighbouring nontransformed or transformed effector cells. Tumor cells prevent HOCl synthesis through membrane-associated catalase that decomposes H2O2, the substrate for peroxidase, and thus prevents HOCl synthesis. Elimination of malignant cells through HOCl signaling is prevented by Helicobacter pylori-associated catalase and superoxide dismutase, whereas it is enhanced by low dose irradiation and by H2O2-producing lactobacilli in the presence of myeloperoxidase. Peroxidase and catalase that are involved in the control of HOCl signaling are also affecting apoptosis-inducing pathways based on reactive nitrogen species. Modification of tumor cell proteins by HOCl enhances the establishment of a T cell response and thus might be involved in immunogenic modulation. Therefore, targeting the control of HOCl signaling system should allow one to establish novel rational therapeutic approaches.
Subject(s)
Carcinogenesis/metabolism , Hypochlorous Acid/metabolism , Neoplasms/metabolism , Apoptosis/physiology , Carcinogenesis/immunology , Catalase/antagonists & inhibitors , Catalase/physiology , Humans , Hypochlorous Acid/immunology , Neoplasms/immunology , Signal Transduction/physiology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.
Subject(s)
Chloramines/metabolism , Hydrogen Peroxide/metabolism , Hypochlorous Acid/metabolism , Neutrophils/immunology , Superoxides/metabolism , Thiocyanates/metabolism , Cell Membrane/drug effects , Cells, Cultured , Chloramines/immunology , Gene Expression , Humans , Hydrogen Peroxide/immunology , Hypochlorous Acid/immunology , Membrane Glycoproteins/agonists , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/immunology , Neutrophils/cytology , Neutrophils/drug effects , Oxidation-Reduction , Peroxidase/genetics , Peroxidase/immunology , Signal Transduction , Superoxides/immunology , Tetradecanoylphorbol Acetate/pharmacology , Thiocyanates/immunology , Zymosan/pharmacologyABSTRACT
Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense.
Subject(s)
Candidiasis/immunology , Cystic Fibrosis/immunology , Hypochlorous Acid/immunology , Immunity, Innate , Sodium Chloride/immunology , Staphylococcal Infections/immunology , Candida albicans/physiology , Candidiasis/metabolism , Candidiasis/microbiology , Candidiasis/pathology , Chlorides/immunology , Chlorides/metabolism , Cystic Fibrosis/metabolism , Cystic Fibrosis/microbiology , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/immunology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Hypochlorous Acid/metabolism , Neutrophil Activation , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/microbiology , Phagocytes/immunology , Phagocytes/metabolism , Phagocytes/microbiology , Phagosomes/immunology , Phagosomes/metabolism , Phagosomes/microbiology , Sodium Chloride/metabolism , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus/physiologyABSTRACT
Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr-/-) mice and the non-inactivated control (Cftrfl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr-/- lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/immunology , Neutrophils/immunology , Phagocytosis , Phagosomes/immunology , Pneumonia, Bacterial/immunology , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/immunology , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Hypochlorous Acid/immunology , Mice , Mice, Knockout , Neutrophils/pathology , Phagosomes/genetics , Pneumonia, Bacterial/genetics , Pneumonia, Bacterial/pathology , Pseudomonas Infections/genetics , Pseudomonas Infections/pathologyABSTRACT
Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.
Subject(s)
Chlorides/immunology , Cystic Fibrosis Transmembrane Conductance Regulator/immunology , Hydrogen Peroxide/immunology , Hypochlorous Acid/immunology , Lysosomal Membrane Proteins/immunology , Neutrophils/immunology , Phagosomes/immunology , Cell Line , Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Hydrogen Peroxide/metabolism , Hypochlorous Acid/metabolism , Ion Transport/drug effects , Ion Transport/immunology , Lysosomal Membrane Proteins/metabolism , NADPH Oxidases/immunology , NADPH Oxidases/metabolism , Neutrophils/metabolism , Phagosomes/genetics , Phagosomes/metabolism , Piperazines/pharmacology , Quinazolines/pharmacologyABSTRACT
Members of the heme peroxidase family play an important role in host defense. Myeloperoxidase (MPO) is expressed in phagocytes and is the only animal heme peroxidase previously reported to be capable of using chloride ion as a substrate to form the highly microbicidal species hypochlorous acid (HOCl) at neutral pH. Despite the potent bacterial killing activity of HOCl, individuals who fail to express MPO typically show only a modest increase in some fungal infections. This may point to the existence of redundant host defense mechanisms. Vascular peroxidase 1 (VPO1) is newly discovered member of the heme peroxidase family. VPO1 is expressed in cells of the cardiovascular system and is secreted into the bloodstream. In the present study, we investigate whether VPO1 is capable of generating HOCl and its role in host defense. Like MPO, VPO1 in the presence of H2O2 and chloride generates HOCl. VPO1-dependent HOCl generation was demonstrated by chlorination of taurine and tyrosine using mass spectrometry. In addition, the VPO1/H2O2/Clâ» system can cause the chlorination of monochlorodimedone and the oxidation of 5-thio-2-nitrobenzoic acid. Purified VPO1 and VPO1 in plasma mediate bacterial killing that is dependent on chloride and H2O2; killing is inhibited by peroxidase inhibitors and by the H2O2 scavenger catalase. In the presence of erythrocytes, bacterial killing by VPO1 is slightly reduced. Thus, VPO1, in addition to MPO, is the second member of the heme peroxidase family capable of generating HOCl under physiological conditions. VPO1 is likely to participate in host defense, with bactericidal activity mediated through the generation of HOCl.
Subject(s)
Anti-Infective Agents/metabolism , Hemeproteins/metabolism , Hypochlorous Acid/metabolism , Peroxidases/metabolism , Plasma/enzymology , Animals , Anti-Infective Agents/immunology , Chlorides/metabolism , Hemeproteins/immunology , Humans , Hydrogen Peroxide/metabolism , Hypochlorous Acid/immunology , Mass Spectrometry , Microbial Viability/drug effects , Peroxidases/immunology , Taurine/metabolism , Tyrosine/metabolismABSTRACT
Modification/chlorination of high-density lipoprotein (HDL) by hypochlorous acid (HOCl), formed by the myeloperoxidase-H2O2-chloride system of activated phagocytes, converts an anti-atherogenic lipoprotein into a pro-inflammatory lipoprotein particle. Chlorinated HDL is present in human lesion material, binds to and is internalized by endothelial cells and impairs expression and activity of endothelial nitric oxide synthase. The present study aimed at clarifying whether exposure of endothelial cells to pro-inflammatory HOCl-HDL impacts on expression of heme oxygenase-1, a potential rescue pathway against endothelial dysfunction. Our findings revealed that HDL modified by HOCl, added as reagent or generated enzymatically, induced phosphorylation of p42/44 mitogen-activated protein kinase, expression of transcription factor early growth response-1 (Egr-1) and enhanced expression of heme oxygenase-1 in human endothelial cells. Upregulation of heme oxygenase-1 could be blocked by an inhibitor upstream of p42/44 mitogen-activated protein kinase and/or knockdown of Egr-1 by RNA-interference. Electrophoretic mobility shift assays demonstrated HOCl-HDL-mediated induction of the Egr-1 DNA binding activity. Immunocytochemical and immunoblotting experiments demonstrated HOCl-HDL-induced translocation of Egr-1 to the nucleus. The present study demonstrates a novel compensatory pathway against adverse effects of HOCl-HDL, providing cytoprotection in a number of pathological conditions including cardiovascular disease.
Subject(s)
Early Growth Response Protein 1/immunology , Endothelial Cells/immunology , Heme Oxygenase-1/immunology , Hypochlorous Acid/immunology , Lipoproteins, HDL/immunology , Mitogen-Activated Protein Kinase 1/immunology , Mitogen-Activated Protein Kinase 3/immunology , Cell Line , Early Growth Response Protein 1/analysis , Early Growth Response Protein 1/genetics , Electrophoretic Mobility Shift Assay , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Expression Regulation , Heme Oxygenase-1/genetics , Humans , Immunochemistry , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Protein Transport , Zinc Fingers , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/immunologyABSTRACT
Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.
Subject(s)
Carps/immunology , Cytotoxicity, Immunologic/immunology , Leukocytes/immunology , Animals , Antibodies, Monoclonal , Blood Platelets/immunology , Cell Line, Tumor , Cytotoxicity Tests, Immunologic , Flow Cytometry , Humans , Hydrogen Peroxide/immunology , Hypochlorous Acid/immunology , Leukocytes/ultrastructure , Lymphocytes/immunology , Mice , Microscopy, Electron , Monocytes/immunology , Neutrophils/immunologyABSTRACT
INTRODUCTION: Oxidative modification of proteins affects their biological properties. Previously we have shown that hypochlorite (HOCl), the product of activated neutrophils, enhances protein immunogenecity. Collagen type II, a primary component of cartilage, is commonly used in the induction of arthritis in animals (CIA). The aim of this study was to examine whether HOCl may affect immunogenic, tolerogenic, and arthritogenic properties of collagen. MATERIALS AND METHODS: DBA/J mice were injected with either native (CNAT) or chlorinated collagen (CHOCl) to induce arthritis. The effect of chlorination on collagen properties was measured by evaluation of incidence and severity of CIA. Moreover, the concentration of serum anti-collagen IgG antibodies and myeloperoxidase (MPO) activity in inflamed joints was determined. RESULTS: Mice immunized with CNAT in adjuvant developed arthritis (CIA) with an incidence of 69%. CNAT also exerted tolerogenic properties when injected intravenously either before or shortly after primary immunization, resulting in decreased incidence and severity of CIA, reduced MPO activity in inflamed joints, and lowered serum levels of anti-CNAT IgG anti-bodies. Chlorination of collagen significantly diminished its ability to induce CIA and to trigger generation of anti-CNAT IgG antibodies. Interestingly, chlorination did not affect tolerogenic properties of collagen administered prior to primary immunization with CNAT. CONCLUSIONS: These results suggest that chlorination of collagen may selectively affect functional epitopes of collagen. It is likely that in inflamed joints, neutrophil derived HOCl, in some circumstances, will destroy arthritogenic and immunogenic B cell epitopes, while regulatory T cell epitopes will be preserved.
Subject(s)
Arthritis, Experimental/immunology , Collagen Type II/chemistry , Collagen Type II/immunology , Hypochlorous Acid/immunology , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/pathology , Arthritis, Experimental/prevention & control , Epitopes , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Mice , Mice, Inbred DBA , Oxidation-Reduction , Peroxidase/metabolism , Serum Amyloid A Protein/metabolismSubject(s)
Hypochlorous Acid/immunology , Peroxidase/immunology , Phagocytes/immunology , Potassium/immunology , Superoxides/immunology , Animals , Candida albicans/immunology , Cathepsin G , Cathepsins/metabolism , Humans , Hydrogen Peroxide/immunology , Hydrogen-Ion Concentration , Leukocytes/immunology , Leukocytes/microbiology , Mice , NADPH Oxidases/immunology , Pancreatic Elastase/immunology , Phagocytes/microbiology , Serine Endopeptidases , Staphylococcus aureus/immunologyABSTRACT
In inflammatory arthritis, there is evidence indicating that the affected tissues produce large amounts of oxygen-free radicals and NO. Herein, we examine the biologic effects of exposure of IgG to hypochlorous acid (HOCl) and peroxynitrite (ONOO). The concentrations of IgG modified by chlorination and nitrosation were measured in synovial fluids from inflammatory and noninflammatory arthritis. Human IgG was exposed to increasing concentrations of HOCl and ONOO, and the resulting products were tested for complement component binding; binding to FcgammaRI; activation of polymorphonuclear neutrophils; effect on the Ab-combining site of Abs; and in vivo inflammatory activity in a rabbit model of acute arthritis. Rheumatoid synovial fluids contained significantly greater concentrations of nitrosated and chlorinated IgG compared with ostearthritic specimens. In vitro exposure of human IgG to HOCl and ONOO resulted in a concentration-dependent decrease in C3 and C1q fixation. The decrease in Fc domain-dependent biologic functions was confirmed by competitive binding studies to the FcgammaRI of U937 cells. HOCl-treated IgG monomer was 10 times less effective in competing for binding compared with native IgG, and ONOO-treated IgG was 2.5 times less effective. The modified IgGs were also ineffective in inducing synthesis of H(2)O(2) by human PMN. The Ag-binding domains of IgG also showed a concentration-dependent decrease in binding to Ag. The ability of the modified IgGs to induce acute inflammation in rabbit knees decreased 20-fold as gauged by the intensity of the inflammatory cell exudates. These studies clarify the modulating role of biological oxidants in inflammatory processes in which Ag-autoantibody reactions and immune complex pathogenesis may play an important role.
Subject(s)
Free Radicals/metabolism , Immunoglobulin G/metabolism , Immunoglobulin G/toxicity , Nitrates/metabolism , Oxygen/metabolism , Tyrosine/analogs & derivatives , Acute Disease , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Binding Sites, Antibody , Complement C1q/metabolism , Complement C3/metabolism , Female , Gout/immunology , Gout/metabolism , Humans , Hydrogen Peroxide/metabolism , Hypochlorous Acid/immunology , Hypochlorous Acid/metabolism , Male , Neutrophils/immunology , Neutrophils/metabolism , Osteoarthritis/immunology , Osteoarthritis/metabolism , Oxidation-Reduction , Rabbits , Receptors, IgG/metabolism , Serum Albumin, Bovine/immunology , Serum Albumin, Bovine/metabolism , Synovial Fluid/immunology , Synovial Fluid/metabolism , Tyrosine/immunology , Tyrosine/metabolismABSTRACT
The 'oxidation theory' of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although the precise mechanisms of in vivo oxidation are widely unknown, increasing evidence suggests that myeloperoxidase (MPO, EC 1.11.1.7), a protein secreted by activated phagocytes, generates modified/oxidized (lipo)proteins via intermediate formation of hypochlorous acid (HOCl). In vitro generation of HOCl transforms lipoproteins into high uptake forms for macrophages giving rise to cholesterol-engorged foam cells. To identify HOCl-modified-epitopes in human plaque tissues we have raised monoclonal antibodies (directed against human HOCl-modified LDL) that do not cross-react with other LDL modifications, i.e. peroxynitrite-LDL, hemin-LDL, Cu2+-oxidized LDL, 4-hydroxynonenal-LDL, malondialdehyde-LDL, glycated-LDL, and acetylated-LDL. The antibodies recognized a specific epitope present on various proteins after treatment with OCl- added as reagent or generated by the MPO/H2O2/halide system. Immunohistochemical studies revealed pronounced staining for HOCl-modified-epitopes in fibroatheroma (type V) and complicated (type VI) lesions, while no staining was observed in aortae of lesion-prone location (type I). HOCl-oxidation-specific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control segments. Staining was shown to be inside and outside monocytes/macrophages, endothelial cells, as well as in the extracellular matrix. A similar staining pattern using immunohistochemistry could be obtained for MPO. The colocalization of immunoreactive MPO and HOCl-modified-epitopes in serial sections of human atheroma (type IV), fibroatheroma (type V) and complicated (type VI) lesions provides further convincing evidence for MPO/H2O2/halide system-mediated oxidation of (lipo)proteins under in vivo conditions. We propose that MPO could act as an important link between the development of atherosclerotic plaque in the artery wall and chronic inflammatory events.
Subject(s)
Arteriosclerosis/enzymology , Arteriosclerosis/immunology , Hydrogen Peroxide/immunology , Peroxidase/immunology , Aged , Antibodies, Monoclonal/chemistry , Aorta/enzymology , Aorta/immunology , Blotting, Western , Dose-Response Relationship, Immunologic , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/enzymology , Endothelium, Vascular/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes , Female , Femoral Artery/enzymology , Femoral Artery/immunology , Humans , Hypochlorous Acid/immunology , Hypochlorous Acid/metabolism , Immunohistochemistry , Lipoproteins, LDL/immunology , Male , Middle Aged , Oxygen/metabolism , Tibial Arteries/enzymology , Tibial Arteries/immunology , UltracentrifugationABSTRACT
The recommended concentration for patch testing with sodium hypochlorite [NaOCl] (bleach) is 1%, generally obtained by diluting commercial bleach. In doing so, other active (potentially irritant) components of bleach, especially hypochlorous acid [HOCl] and sodium hydroxide [NaOH], are neglected. Magnitudes of potential irritant species other than NaOCl, such as alkalinity, are ordinarily not labeled on the product, though they may vary considerably between brands. Thus, patch testing with 1% hypochlorite obtained by diluting different brand bleaches can potentially elicit non-specific irritant responses, also depending upon the test volume applied. In this study, skin irritation induced by 24-h patch testing with 20 microliters or 100 microliters, with constant NaOCl concentration (1%) and different NaOH concentrations (0.01-1.0%), was studied in adult human volunteers, by means of visual scores and skin color reflectance measurements. No irritation was elicited by application of 20 microliters 1% OCl-, independent of the NaOH concentration. However, all solutions induced significant irritation in a volume of 100 microliters. Skin reactions did not show a straight pH dose response, a maximum reaction being seen to the solution containing 0.1% NaOH. Skin surface pH values had increased when monitored immediately after removal of the patch material. However, 24 h later, baseline values were again reached at most sites, demonstrating an efficient buffering capacity of human skin, even after challenge with alkaline solutions of pH 13.4. We suggest that a non-irritant concentration for diagnostic patch testing for allergic contact dermatitis with an aluminum chamber, using 17 microliters to 20 microliters test volume, could be as high as 1% NaOCl and 1% NaOH.
Subject(s)
Irritants , Patch Tests/methods , Skin/drug effects , Sodium Hypochlorite , Adult , Dose-Response Relationship, Drug , Humans , Hypochlorous Acid/chemistry , Hypochlorous Acid/immunology , Middle Aged , Skin/immunology , Sodium Hydroxide/immunology , Sodium Hypochlorite/chemistry , Sodium Hypochlorite/immunologyABSTRACT
Observation of an individual with immediate-type reaction following exaggerated dermal exposure to hypochlorite-containing cleaning products prompted review of similar hypersensitivity reactions attributed to hypochlorite or other highly reactive chemicals. This review confirms isolated incidences of hypochlorite sensitivity of the delayed type (allergic contact dermatitis), as well as immediate-type reactions from inhalation or topical challenge of sensitized individuals. We conclude that it is possible that excessive and prolonged exposure to hypochlorite may in some cases result in irritation and damage to the skin. This potentially gives rise to altered proteins which in rare cases may cause hypersensitivity. This reaction is common to other reactive small molecules with a strong irritant action.