RESUMEN
Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.
Asunto(s)
Amidas/farmacología , Activación de Complemento/efectos de los fármacos , Complemento C3/química , Fumaratos/farmacología , Glomerulonefritis Membranoproliferativa/metabolismo , Glomerulonefritis Membranoproliferativa/terapia , Renina/química , Amidas/uso terapéutico , Quimiotaxis/efectos de los fármacos , Niño , Complemento C3/metabolismo , Complemento C3a/química , Complemento C3a/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Complemento C4/química , Complemento C5a/química , Complemento C5a/metabolismo , Complemento C5b/química , Complemento C5b/metabolismo , Factor B del Complemento/química , Factor D del Complemento/química , Femenino , Fumaratos/uso terapéutico , Membrana Basal Glomerular/patología , Glomerulonefritis Membranoproliferativa/patología , Humanos , Mastocitos/fisiología , Renina/antagonistas & inhibidores , Renina/metabolismoRESUMEN
In cystic fibrosis (CF), colonization of the airways with Pseudomonas aeruginosa is associated with disease deterioration. The mechanism behind the disease progression is not fully understood. The present work shows that the antibacterial chemokine MIG/CXCL9 is present in the airways and in sputum of CF patients. MIG/CXCL9 showed high bactericidal activity against. P. aeruginosa, including some strains from the airways of CF patients. Full-length MIG/CXCL9 was detected in sputum from healthy controls and CF patients colonized with P. aeruginosa. However, degraded MIG/CXCL9 was only found in CF sputum. In vitro, elastase of P. aeruginosa cleaved off a fragment of similar size and two additional fragments from MIG/CXCL9. The fragments showed less bactericidal activity against P. aeruginosa compared with the full-length protein. The fragments did not activate the MIG/CXCL9 receptor CXCR3 (expressed e.g. by NK cells, mast cells, and activated T cells) but instead displayed noncompetitive inhibition. In vitro, a decrease in CXCR3-bearing cells was found within and in the proximity of the bronchial epithelium of CF lung tissue compared with controls. Taken together, both bactericidal and cell-recruiting activities of MIG/CXCL9 are corrupted by P. aeruginosa through release of elastase, and this may contribute to impaired airway host defense in CF.
Asunto(s)
Proteínas Bacterianas/inmunología , Quimiocina CXCL9/inmunología , Fibrosis Quística/inmunología , Metaloendopeptidasas/inmunología , Proteolisis , Pseudomonas aeruginosa/inmunología , Receptores CXCR3/inmunología , Proteínas Bacterianas/metabolismo , Quimiocina CXCL9/metabolismo , Fibrosis Quística/enzimología , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Femenino , Humanos , Masculino , Metaloendopeptidasas/metabolismo , Pseudomonas aeruginosa/enzimología , Receptores CXCR3/metabolismo , Esputo/inmunología , Esputo/microbiologíaRESUMEN
Finegoldia magna is a Gram-positive anaerobic commensal of the human skin microbiota, but also known to act as an opportunistic pathogen. Two primary virulence factors of F. magna are the subtilisin-like extracellular serine protease SufA and the adhesive protein FAF. This study examines the molecular mechanisms F. magna uses when colonizing or establishing an infection in the skin. FAF was found to be essential in the initial adherence of F. magna to human skin biopsies. In the upper layers of the epidermis FAF mediates adhesion through binding to galectin-7 - a keratinocyte cell marker. Once the bacteria moved deeper into the skin to the basement membrane layer, SufA was found to degrade collagen IV which forms the backbone structure of the basement membrane. It also degraded collagen V, whereby F. magna could reach deeper dermal tissue sites. In the dermis, FAF interacts with collagen V and fibrillin, which presumably helps the bacteria to establish infection in this area. The findings of this study paint a clear picture of how F. magna interacts with human skin and explain how it is such a successful opportunistic pathogen in chronic wounds and ulcers.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Bacterias Grampositivas/fisiología , Péptido Hidrolasas/metabolismo , Piel/microbiología , Adhesión Bacteriana , Portador Sano/microbiología , Colágeno/metabolismo , Fibrilinas , Bacterias Grampositivas/patogenicidad , Humanos , Proteínas de Microfilamentos/metabolismo , Enfermedades Cutáneas Bacterianas/microbiologíaRESUMEN
Collateral damage caused by extracellular histones has an immediate impact on morbidity and mortality in many disease models. A significant increase in the levels of extracellular histones is seen in critically ill patients with trauma and sepsis. We showed that histones are released from necrotic cells in patients with invasive skin infections. Under in vitro conditions, endogenous p33, an endothelial surface protein also known as the gC1q receptor, interacts with histones released from damaged endothelial cells. Functional analyses have revealed that recombinantly expressed p33 completely neutralizes the harmful features of histones, i.e. hemolysis of erythrocytes, lysis of endothelial cells and platelet aggregation. We also noted that mice treated with a sublethal dose of histones developed severe signs of hemolysis, thrombocytopenia and lung tissue damage already 10 min after inoculation. These complications were fully counteracted when p33 was administered together with the histones. Moreover, application of p33 significantly improved survival in mice receiving an otherwise lethal dose of histones. Together, our data suggest that treatment with p33 is a promising therapeutic approach in severe infectious diseases.
Asunto(s)
Histonas/toxicidad , Proteínas Mitocondriales/farmacología , Choque , Animales , Plaquetas/inmunología , Plaquetas/patología , Células Endoteliales/inmunología , Células Endoteliales/patología , Femenino , Hemólisis/efectos de los fármacos , Hemólisis/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/inmunología , Proteínas Recombinantes/farmacología , Choque/inducido químicamente , Choque/inmunología , Choque/patología , Choque/prevención & controlRESUMEN
IgA nephropathy (IgAN) is characterized by mesangial cell proliferation and extracellular matrix expansion associated with immune deposits consisting of galactose-deficient polymeric IgA1 and C3. We have previously shown that IgA-binding regions of streptococcal M proteins colocalize with IgA in mesangial immune deposits in patients with IgAN. In the present study, the IgA-binding M4 protein from group A Streptococcus was found to bind to galactose-deficient polymeric IgA1 with higher affinity than to other forms of IgA1, as shown by surface plasmon resonance and solid-phase immunoassay. The M4 protein was demonstrated to bind to mesangial cells not via the IgA-binding region but rather via the C-terminal region, as demonstrated by flow cytometry. IgA1 enhanced binding of M4 to mesangial cells, but not vice versa. Costimulation of human mesangial cells with M4 and galactose-deficient polymeric IgA1 resulted in a significant increase in IL-6 secretion compared with each stimulant alone. Galactose-deficient polymeric IgA1 alone, but not M4, induced C3 secretion from the cells, and costimulation enhanced this effect. Additionally, costimulation enhanced mesangial cell proliferation compared with each stimulant alone. These results indicate that IgA-binding M4 protein binds preferentially to galactose-deficient polymeric IgA1 and that these proteins together induce excessive proinflammatory responses and proliferation of human mesangial cells. Thus, tissue deposition of streptococcal IgA-binding M proteins may contribute to the pathogenesis of IgAN.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Complemento C3/inmunología , Glomerulonefritis por IGA/inmunología , Inmunoglobulina A/inmunología , Interleucina-6/inmunología , Células Mesangiales/inmunología , Streptococcus/inmunología , Adolescente , Femenino , Glomerulonefritis por IGA/patología , Humanos , Masculino , Células Mesangiales/patología , Persona de Mediana EdadRESUMEN
The effect of 8 weeks treatment with oral iodinated activated charcoal (IAC) on lung function of patients with moderate chronic obstructive pulmonary disease (COPD) was examined in a double blind randomized placebo controlled parallel group study with 40 patients. In the IAC group, patients showed a statistically significant improvement of FEV1 baseline by 130 ml compared to placebo, corresponding to 8.2% improvement (p = 0.031*). Correlation statistics revealed that the improvement of FEV1 baseline was significantly correlated both to FEV1 post-bronchodilator (p = 0.0020**) and FEV1 post-exercise (0.033*) values. This demonstrates that the improved baseline lung function by IAC did not inhibit a further beta2-adrenoceptor relaxation, and thus that patients did not reach a limit for maximal improvement of the lung function after IAC treatment. Eight patients in the IAC group developed abnormal thyroid hormone levels transiently during the treatment. This side effect was not correlated to improvement of lung function (p = 0.82). No serious adverse effects directly related to the treatment were recorded. In summary, this study demonstrates that iodinated activated charcoal surprisingly and significantly improved lung function of patients with moderate COPD. The underlying mechanism of action is unclear, but is likely to be different from the drugs used today. The immediate conclusion is that further studies are now justified in order to determine clinical efficacy of IAC in COPD and explore possible mechanisms of action.
Asunto(s)
Broncodilatadores/administración & dosificación , Carbón Orgánico/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Administración Oral , Anciano , Anciano de 80 o más Años , Carbón Orgánico/efectos adversos , Método Doble Ciego , Tolerancia al Ejercicio/efectos de los fármacos , Femenino , Volumen Espiratorio Forzado/fisiología , Halogenación/fisiología , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Calidad de Vida , Resultado del TratamientoRESUMEN
Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-ß-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Enterococcus faecalis/enzimología , Lactoferrina/química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Polisacáridos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Secuencia de Carbohidratos , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/crecimiento & desarrollo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Hidrólisis , Lactoferrina/farmacología , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/genética , Datos de Secuencia Molecular , Fosfopiruvato Hidratasa/metabolismo , Polisacáridos/análisis , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
OBJECTIVE: Apoptotic nucleosomes are structurally and immunologically involved in lupus nephritis. The purpose of this study was to examine the expression and function of laminins and their interactions with nucleosomes in the kidneys of patients with lupus nephritis, using surface plasmon resonance (SPR) analysis. METHODS: SPR interaction analysis was used to quantify the strength of laminin-nucleosome interactions. Electron microscopy techniques were used to determine in vivo colocalization of IgG, chromatin, and laminin ß1, as well as to characterize nucleosome-laminin interactions in vitro. RESULTS: Nucleosomes were found to possess high affinity for laminin ß1-containing laminins and to have the potential to form stable molecular complexes with these structures. In vivo, laminin ß1 was aberrantly expressed in the glomerular basement membrane (GMB) of lupus nephritis patients, and in situ, it acted as a nucleosome ligand, selectively colocalizing with nucleosomes within electron-dense deposits (EDDs). Normal adult laminin 11, which contains laminin ß2, did not bind nucleosomes, and it did not colocalize in vivo with the nucleosomes in the nephritic GBM. In addition, TGFß1 was expressed by the glomerular mesangium, glomerular endothelial cells, and by podocytes in patients with lupus nephritis. It was trapped in situ within EDDs by an as-yet-unknown ligand. As was recently described in a transgenic mouse model, paracrine kidney glomerular TGFß1 may thereby contribute to the development of glomerulopathy via the induction of laminin ß1 incorporation in the GBM, whereas systemic blood vessel-derived TGFß1 could be trapped during filtration. CONCLUSION: Our findings of the specific high-affinity binding of nucleosomes to aberrantly expressed laminin ß1 in the GBM and their colocalization in the GBM may explain important features of the initial steps in the pathogenesis of lupus nephritis, the planted antigen hypothesis.
Asunto(s)
Cromatina/metabolismo , Membrana Basal Glomerular/metabolismo , Inmunoglobulina G/metabolismo , Laminina/metabolismo , Nefritis Lúpica/metabolismo , Nucleosomas/metabolismo , Adulto , Anciano , Biopsia , Femenino , Membrana Basal Glomerular/patología , Membrana Basal Glomerular/ultraestructura , Humanos , Técnicas In Vitro , Riñón/metabolismo , Riñón/patología , Riñón/ultraestructura , Nefritis Lúpica/patología , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Resonancia por Plasmón de Superficie , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The innate immune system is the first line of defense against invading microbes. Its specificity relies a great deal on host pattern recognition molecules that sense pathogen-associated molecular patterns of the invading pathogen. However, full protection is not always guaranteed, and some early defense mechanisms involved in bacterial killing, such as the complement system, can also exert cytolytic activity against host cells. Although these cascades are tightly regulated, the host has to take additional precautions to prevent its cell destruction. In this study, we describe that p33, a negatively charged surface protein found on endothelial cells also known as gC1q receptor, protects host cells from a cytolytic attack by antimicrobial peptides (AMPs), such as LL37 and ß-defensin 3. To this end, we characterized the interaction of p33 with AMPs by biochemical and functional means. Our data show that p33 forms a doughnut-shaped trimer that can bind up to three AMPs, and we identified a segment in p33 forming a ß-sheet that mediates the binding to all AMPs. Moreover, our results show that p33 abolishes the lytic activity of AMPs at an equimolar ratio, and it protects endothelial cells and erythrocytes from AMP-induced lysis. Taken together, our data suggest a novel protective mechanism of p33 in modulating innate immune response by neutralizing cytotoxic AMPs at the host cell surface.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Endoteliales/inmunología , Eritrocitos/inmunología , Proteínas Mitocondriales/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos , Sitios de Unión , Proteínas Portadoras/inmunología , Catelicidinas/farmacología , Células Cultivadas , Citoprotección/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Proteínas Mitocondriales/inmunología , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , beta-Defensinas/farmacologíaRESUMEN
Staphylococcus aureus is sometimes isolated from the airways during acute exacerbations of chronic obstructive pulmonary disease (COPD) but more commonly recognized as a cause of ventilator-associated pneumonia (VAP). Antimicrobial proteins, among them midkine (MK), are an important part of innate immunity in the airways. In this study, the levels and possible processing of MK in relation to S. aureus infection of the airways were investigated, comparing COPD and VAP, thus comparing a state of disease with preceding chronic inflammation and remodeling (COPD) with acute inflammation (that is, VAP). MK was detected in the small airways and alveoli of COPD lung tissue but less so in normal lung tissue. MK at below micromolar concentrations killed S. aureus in vitro. Proteolytic processing of MK by the staphylococcal metalloprotease aureolysin (AL), but not cysteine protease staphopain A (SA), resulted in impaired bactericidal activity. Degradation was seen foremost in the COOH-terminal portion of the molecule that harbors high bactericidal activity. In addition, MK was detected in sputum from patients suffering from VAP caused by S. aureus but less so in sputum from COPD exacerbations associated with the same bacterium. Recombinant MK was degraded more rapidly in sputum from the COPD patients than from the VAP patients and a greater proteolytic activity in COPD sputum was confirmed by zymography. Taken together, proteases of both bacteria and the host contribute to degradation of the antibacterial protein MK, resulting in an impaired defense of the airways, in particular, in COPD where the state of chronic inflammation could be of importance.
Asunto(s)
Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/metabolismo , Neumonía Asociada al Ventilador/microbiología , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Esputo/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Adulto , Anciano , Antibacterianos/uso terapéutico , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Femenino , Humanos , Inmunidad Innata , Masculino , Metaloendopeptidasas/metabolismo , Persona de Mediana Edad , Midkina , Modelos Moleculares , Factores de Crecimiento Nervioso/genética , Neumonía Asociada al Ventilador/inmunología , Neumonía Asociada al Ventilador/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Esputo/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismoRESUMEN
Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs). These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is part of the early immune response to the invading pathogen. As shown by negative staining electron microscopy and clotting assays, pro-coagulant MVs bind in the presence of plasma to the bacterial surface. Fibrinogen was identified as a linker that, through binding to the M1 protein of S. pyogenes, allows the opsonization of the bacteria by MVs. Surface plasmon resonance analysis revealed a strong interaction between pro-coagulant MVs and fibrinogen with a KD value in the nanomolar range. When performing a mass-spectrometry-based strategy to determine the protein quantity, a significant up-regulation of the fibrinogen-binding integrins CD18 and CD11b on pro-coagulant MVs was recorded. Finally we show that plasma clots induced by pro-coagulant MVs are able to prevent bacterial dissemination and possess antimicrobial activity. These findings were confirmed by in vivo experiments, as local treatment with pro-coagulant MVs dampens bacterial spreading to other organs and improved survival in an invasive streptococcal mouse model of infection. Taken together, our data implicate that pro-coagulant MVs play an important role in the early response of the innate immune system in infectious diseases.
Asunto(s)
Coagulación Sanguínea/inmunología , Antígeno CD11b/inmunología , Antígenos CD18/inmunología , Micropartículas Derivadas de Células/inmunología , Infecciones Estreptocócicas/metabolismo , Streptococcus pyogenes/inmunología , Animales , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/microbiología , Micropartículas Derivadas de Células/ultraestructura , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/patología , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/ultraestructuraRESUMEN
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in impaired host defense during cystic fibrosis (CF), where Pseudomonas aeruginosa becomes a key pathogen. We investigated the expression pattern of the antibacterial growth factor midkine (MK) in CF and the possible interference with its activity by the altered airway microenvironment. High MK expression was found in CF lung tissue compared with control samples, involving epithelia of the large and small airways, alveoli, and cells of the submucosa (i.e., neutrophils and mast cells). In CF sputum, MK was present at 100-fold higher levels, but was also subject to increased degradation, compared with MK in sputum from healthy control subjects. MK exerted a bactericidal effect on P. aeruginosa, but increasing salt concentrations and low pH impaired this activity. Molecular modeling suggested that the effects of salt and pH were attributable to electrostatic screening and a charge-neutralization of the membrane, respectively. Both the neutrophil elastase and elastase of P. aeruginosa cleaved MK to smaller fragments, resulting in impaired bactericidal activity. Thus, MK is highly expressed in CF, but its bactericidal properties may be impaired by the altered microenvironment, as reflected by the in vitro conditions used in this study.
Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Pulmón/metabolismo , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Adulto , Anciano , Antibacterianos/metabolismo , Estudios de Casos y Controles , Fibrosis Quística/patología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Pulmón/microbiología , Pulmón/patología , Masculino , Persona de Mediana Edad , Midkina , Elastasa Pancreática/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Sales (Química) , Esputo/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
The pattern recognition molecules C-reactive protein (CRP) and C1q are of big interest in relation to the pathogenesis of systemic lupus erythematosus (SLE). Circulating autoantibodies against CRP and C1q are frequently found in SLE patients with active disease, particularly in lupus nephritis (LN), and rising levels reportedly relate to disease activity and outcome. If CRP-, or dsDNA- and/or C1q-containing immune complexes (ICs) are pathogenic in LN, glomerular IgG-deposits would be expected to co-localize with these antigens. In search for proof of this concept, renal biospsies from patients with active LN (n = 5) were examined with high-resolution immunogold electron microscopy. Renal biopsies from patients with Henoch-Schönlein purpura, pauci-immune nephritis and renal cancer served as controls. IgG antibodies against CRP, C1q and nucleosomes were analyzed in pre-post flare sera. We could demonstrate that CRP, C1q, C3c and dsDNA were co-localized with IgG in electron dense deposits in the glomerular basement membrane/subendothelial space in all of the 5 LN patients. Deposits of IgG, CRP, complement and dsDNA were 10-fold higher in LN compared to controls. All SLE patients had circulating anti-nucleosome antibodies; 4/5 had serum antibodies against CRP, dsDNA, and C1q at biopsy/flare. Despite a limited number of cases, the results support the notion of a pathogenic role not only for anti-dsDNA antibodies, but also for anti-CRP and anti-C1q in LN. The glomerular ICs may have been generated by deposition of circulating ICs or by in situ IC formation.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Autoanticuerpos/sangre , Proteína C-Reactiva/inmunología , Complemento C1q/inmunología , Nefritis Lúpica/inmunología , Adulto , Anciano , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Anticuerpos Antinucleares/sangre , Complejo Antígeno-Anticuerpo/sangre , Proteína C-Reactiva/metabolismo , Complemento C1q/metabolismo , Complemento C3c/inmunología , ADN/inmunología , Femenino , Humanos , Vasculitis por IgA/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Riñón/patología , Neoplasias Renales/inmunología , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Masculino , Persona de Mediana Edad , Nefritis/inmunología , Nucleosomas/inmunologíaRESUMEN
When pathogenic bacteria breach the epithelial lining at mucosal surfaces, rapidly available innate immune mechanisms are critical to halt the infection. In the present study, we characterized the production of antibacterial polypeptides released by epithelial cells. IFN-γ, but neither TNF nor IL-1ß alone, induced release of antibacterial activity to a cell culture medium, causing a lytic appearance of killed bacteria as revealed by electron microscopy. Addition of the protein streptococcal inhibitor of complement, derived from Streptococcus pyogenes, known for its ability to neutralize antimicrobial polypeptides (AMPs), reduced the antibacterial activity of the medium. Characterization of the antibacterial incubation medium using mass spectrometric approaches and ELISAs, displayed presence of several classical AMPs, antibacterial chemokines, as well as complement factors and proteases that may interfere with bacterial killing. Many were constitutively produced, that is, being released by cells incubated in a medium alone. While a combination of IFN-γ and TNF did not increase bacterial killing, the presence of TNF boosted the amounts and detectable number of AMPs, including antibacterial chemokines. However, the methods applied in the study failed to single out certain AMPs as critical mediators, but rather demonstrate the broad range of molecules involved. Since many AMPs are highly amphiphatic in nature (i.e., cationic and hydrophobic), it is possible that difficulties in optimizing recovery present limitations in the context investigated. The findings demonstrate that epithelial cells have a constitutive production of AMPs and that IFN-γ is an important inducer of an antibacterial response in which is likely to be a critical part of the innate host defense against pathogenic bacteria at mucosal surfaces.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Células Epiteliales/inmunología , Interferón gamma/inmunología , Mucosa Respiratoria/inmunología , Streptococcus pyogenes/inmunología , Péptidos Catiónicos Antimicrobianos/inmunología , Proteínas Bacterianas/farmacología , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Interleucina-1beta , Infecciones Estreptocócicas/inmunología , Factor de Necrosis Tumoral alfaRESUMEN
Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects.
Asunto(s)
Proteínas Bacterianas/inmunología , Actividad Bactericida de la Sangre/inmunología , Inmunidad Innata , Streptococcus/inmunología , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Bacterianas/metabolismo , Bradiquinina/inmunología , Bradiquinina/metabolismo , Humanos , Quininógeno de Alto Peso Molecular/inmunología , Quininógeno de Alto Peso Molecular/metabolismo , Streptococcus/metabolismoRESUMEN
OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in several organ systems, related to the presence of circulating and tissue-deposited immune complexes (ICs) that stimulate leukocytes through Fcγ receptors (FcγR) with subsequent inflammation. Treatment with endoglycosidase S (EndoS), an IgG glycan-hydrolyzing bacterial enzyme from Streptococcus pyogenes, has shown beneficial effects in several experimental animal models of chronic inflammatory disease. This study was undertaken to investigate whether EndoS affects the proinflammatory properties of ICs and has the potential to be developed as a therapy for SLE. METHODS: ICs purified from SLE patients or RNA-containing ICs formed in vitro were treated with EndoS and used in several assays reflecting different important features of SLE pathogenesis, such as phagocytosis by polymorphonuclear cells (PMNs) and plasmacytoid dendritic cells (PDCs), complement activation, and interferon-α (IFNα) production by PDCs. RESULTS: EndoS treatment abolished all proinflammatory properties of the ICs investigated. This included FcγR-mediated phagocytosis by PDCs (P = 0.001) and subsequent production of IFNα (P = 0.002), IC-induced classical pathway of complement activation (P = 0.008), chemotaxis, and oxidative burst activity of PMNs (P = 0.002). EndoS treatment also had a direct effect on the molecular structure of ICs, causing decreased IC size and glycosylation. CONCLUSION: Our findings indicate that EndoS treatment has prominent effects on several pathogenetically important IC-mediated events, and suggest that EndoS has the potential to be developed as a novel therapy for SLE.
Asunto(s)
Complejo Antígeno-Anticuerpo/efectos de los fármacos , Proteínas Bacterianas/farmacología , Glicósido Hidrolasas/farmacología , Inmunoglobulina G/metabolismo , Inflamación/inmunología , Lupus Eritematoso Sistémico/inmunología , Polisacáridos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Complejo Antígeno-Anticuerpo/metabolismo , Quimiotaxis/fisiología , Células Dendríticas/metabolismo , Femenino , Humanos , Hidrólisis/efectos de los fármacos , Inflamación/metabolismo , Inflamación/patología , Interferón-alfa/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Masculino , Persona de Mediana Edad , Neutrófilos/fisiología , Fagocitosis/fisiología , Receptores de IgG/fisiología , Adulto JovenRESUMEN
Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.
Asunto(s)
Convertasas de Complemento C3-C5/biosíntesis , Complemento C3a/genética , Complemento C3a/metabolismo , Complemento C3b/genética , Complemento C3b/metabolismo , Síndrome Hemolítico-Urémico/inmunología , Animales , Síndrome Hemolítico Urémico Atípico , Células COS , Chlorocebus aethiops , Activación de Complemento , Convertasas de Complemento C3-C5/genética , Convertasas de Complemento C3-C5/metabolismo , Complemento C3a/biosíntesis , Complemento C3b/biosíntesis , Complemento C9/biosíntesis , Complemento C9/metabolismo , Factor B del Complemento/metabolismo , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento/genética , Glomerulonefritis/genética , Síndrome Hemolítico-Urémico/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Bacterial colonization of the lower respiratory tract is frequently seen in chronic obstructive pulmonary disease (COPD), and may cause exacerbations leading to disease progression. Antimicrobial peptides comprise an important part of innate lung immunity, and not least the cathelicidin human cationic antimicrobial protein-18/LL-37. Peptidylarginine deiminases (PADIs) post-translationally modify proteins by converting cationic peptidylarginine residues to neutral peptidylcitrulline. An increased presence of PADI2 and citrullinated proteins was demonstrated in the lungs of smokers. In this study, preformed PADI4, stored in granulocytes and extracellularly in the lumina of bronchi, was found in lung tissue of individuals suffering from COPD. In vitro, recombinant human PADI2 and PADI4 both caused a time- and dose-dependent citrullination of LL-37. The citrullination resulted in impaired antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and nontypable Haemophilus influenzae, but less so against Pseudomonas aeruginosa. Using artificial lipid bilayers, we observed discrete differences when comparing the disrupting activity of native and citrullinated LL-37, suggesting that differences in cell wall composition are important during interactions with whole bacteria. Furthermore, citrullinated LL-37 showed higher chemotactic activity against mononuclear leukocytes than did native LL-37, but was less efficient at neutralizing lipolysaccharide, and also in converting apoptotic neutrophils into a state of secondary necrosis. In addition, citrullinated LL-37 was more prone to degradation by proteases, whereas the V8 endopetidase of S. aureus cleaved the modified peptide at additional sites, compared with native LL-37. Together, these findings demonstrate novel mechanisms whereby the inflammation-dependent deiminases PADI2 and PADI4 can alter the activites of antibacterial polypeptides, affecting the course of inflammatory disorders such as COPD.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/fisiología , Bronquios/enzimología , Citrulina/metabolismo , Hidrolasas/metabolismo , Inflamación/enzimología , Fumar , Tráquea/enzimología , Péptidos Catiónicos Antimicrobianos/metabolismo , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Haemophilus influenzae/fisiología , Inmunohistoquímica , Espectrometría de Masas , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica , Proteolisis , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/fisiología , Streptococcus pneumoniae/fisiología , CatelicidinasRESUMEN
Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine expressed by epithelial cells and reported to be involved in allergic diseases and atopic eczema. The presence of several predicted α-helical regions in TSPL, a structure characterizing many classical antimicrobial peptides (AMPs), prompted us to investigate whether TSLP exerts antimicrobial activities. Recombinant human TSLP exerted antimicrobial activity, particularly against Gram-negative bacteria. Using synthetic overlapping peptide 20-mers of TSLP, it was demonstrated that the antimicrobial effect is primarily mediated by the C-terminal region of the protein. MKK34 (MKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLK), a peptide spanning a C-terminal α-helical region in TSLP, showed potent antimicrobial activities, in physiological salt conditions and in the presence of human plasma. Fluorescent studies of peptide-treated bacteria, electron microscopy and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of the classical AMP LL-37. Moreover, TSLP was degraded into multiple fragments by staphylococcal V8 proteinase. One major antimicrobial degradation fragment was found to encompass the C-terminal antimicrobial region defined by the MKK34 peptide. We here describe a novel antimicrobial role for TSLP. The antimicrobial activity is primarily mediated by the C-terminal part of the protein. In combination with the previously known cytokine function of TSLP, our result indicates dual functions of the molecule and a previously unknown role in host defense.
Asunto(s)
Antiinfecciosos/farmacología , Citocinas/farmacología , Secuencia de Aminoácidos , Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/metabolismo , Candida albicans/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Escherichia coli/efectos de los fármacos , Humanos , Elastasa de Leucocito/metabolismo , Liposomas/metabolismo , Metaloendopeptidasas/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Péptido Hidrolasas/metabolismo , Permeabilidad/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes/farmacología , Staphylococcus aureus/enzimología , Staphylococcus epidermidis/efectos de los fármacos , Catelicidinas , Linfopoyetina del Estroma TímicoRESUMEN
Epithelial linings serve as physical barriers and produce antimicrobial peptides (AMPs) to maintain host integrity. Examples are the bactericidal proteins midkine (MK) and BRAK/CXCL14 that are constitutively produced in the skin epidermal layer, where the anaerobic Gram-positive coccoid commensal Finegoldia magna resides. Consequently, this bacterium is likely to encounter both MK and BRAK/CXCL14, making these molecules possible threats to its habitat. In this study, we show that MK expression is upregulated during inflammation, concomitant with a strong downregulation of BRAK/CXCL14, resulting in changed antibacterial conditions. MK, BRAK/CXCL14, and the inflammation-dependent antimicrobial ß-defensins human ß-defensin (hBD)-2 and hBD-3 all showed bactericidal activity against both F. magna and the virulent pathogen Streptococcus pyogenes at similar concentrations. SufA, a released protease of F. magna, degraded MK and BRAK/CXCL14 but not hBD-2 nor hBD-3. Cleavage was seen at lysine and arginine residues, amino acids characteristic of AMPs. Intermediate SufA-degraded fragments of MK and BRAK/CXCL14 showed stronger bactericidal activity against S. pyogenes than F. magna, thus promoting survival of the latter. In contrast, the cysteine-protease SpeB of S. pyogenes rapidly degraded all AMPs investigated. The proteins FAF and SIC, released by F. magna and S. pyogenes, respectively, neutralized the antibacterial activity of MK and BRAK/CXCL14, protein FAF being the most efficient. Quantitation and colocalization by immunoelectron microscopy demonstrated significant levels and interactions of the molecules in in vivo and ex vivo samples. The findings reflect strategies used by a permanently residing commensal and a virulent pathogen, the latter operating during the limited time course of invasive disease.