RESUMEN
Surfactant protein A (SP-A) is a multifunctional host defense collectin that was first identified as a component of pulmonary surfactant. Although SP-A is also expressed in various tissues, including the urinary tract, its innate immune functions in nonpulmonary tissues are poorly understood. In this study, we demonstrated that adherence of uropathogenic Escherichia coli (UPEC) to the bladder was enhanced in SP-A-deficient mice, which suggests that SP-A plays an important role in innate immunity against UPEC. To understand the innate immune functions of SP-A in detail, we performed in vitro experiments. SP-A directly bound to UPEC in a Ca2+-dependent manner, but it did not agglutinate UPEC. Our results suggest that a bouquet-like arrangement seems unsuitable to agglutinate UPEC. Meanwhile, SP-A inhibited growth of UPEC in human urine. Furthermore, the binding of SP-A to UPEC decreased the adherence of bacteria to urothelial cells. These results indicate that direct action of SP-A on UPEC is important in host defense against UPEC. Additionally, adhesion of UPEC to urothelial cells was decreased when the cells were preincubated with SP-A. Adhesion of UPEC to urothelial cells is achieved via interaction between FimH, an adhesin located at bacterial pili, and uroplakin Ia, a glycoprotein expressed on the urothelium. SP-A directly bound to uroplakin Ia and competed with FimH for uroplakin Ia binding. These results lead us to conclude that SP-A plays important roles in host defense against UPEC.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Proteína A Asociada a Surfactante Pulmonar/inmunología , Infecciones Urinarias/inmunología , Animales , Proliferación Celular , Humanos , Inmunidad Innata/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/inmunologíaRESUMEN
Reported herein is the first case of a remarkably delayed occurrence of normal surfactant composition in an extremely preterm infant who required a total of 15 doses of artificial pulmonary surfactant (Surfacten®). A male infant, born at 26 weeks gestation, developed respiratory distress at birth. Chest radiography was consistent with respiratory distress syndrome. The infant required repeated doses of surfactant, each resulting in transient periods of decreased ventilator requirement and improved blood gas values. Surfactant proteins (SP)-A, SP-B, SP-C, and SP-D from tracheal aspirate samples were analyzed on the 13th day (deterioration period) and 36th day (recovery period) after birth. On the 13th day sufficient SP-A and SP-D but no SP-B no SP-C were detected on western blot analysis. SP-B and SP-C were eventually detected on the 36th day. This infant therefore required almost 3 months to achieve normal surfactant function.
Asunto(s)
Surfactantes Pulmonares/uso terapéutico , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Humanos , Recién Nacido , Recien Nacido Prematuro , Masculino , Factores de Tiempo , Resultado del TratamientoRESUMEN
Heregulin signaling is involved in various tumor proliferations and invasions; thus, receptors of heregulin are targets for the cancer therapy. In this study we examined the suppressing effects of extracellular domains of ErbB2, ErbB3, and ErbB4 (soluble ErbB (sErbB)) on heregulin ß signaling in human breast cancer cell line MCF7. It was found that sErbB3 suppresses ligand-induced activation of ErbB receptors, PI3K/Akt and Ras/Erk pathways most effectively; sErbB2 scarcely suppresses ligand-induced signaling, and sErbB4 suppresses receptor activation at â¼10% efficiency of sErbB3. It was revealed that sErbB3 does not decrease the effective ligands but decreases the effective receptors. By using small interfering RNA (siRNA) for ErbB receptors, we determined that sErbB3 suppresses the heregulin ß signaling by interfering ErbB3-containing heterodimers including ErbB2/ErbB3. By introducing the mutation of N418Q to sErbB3, the signaling-inhibitory effects were increased by 2-3-fold. Moreover, the sErbB3 N418Q mutant enhanced anticancer effects of lapatinib more effectively than the wild type. We also determined the structures of N-glycan on Asn-418. Results suggested that the N-glycan-deleted mutant of sErbB3 suppresses heregulin signaling via ErbB3-containing heterodimers more effectively than the wild type. Thus, we demonstrated that the sErbB3 N418Q mutant is a potent inhibitor for heregulin ß signaling.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Mutación Missense , Neurregulina-1/metabolismo , Multimerización de Proteína , Receptor ErbB-3/metabolismo , Sustitución de Aminoácidos , Antineoplásicos/farmacología , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Lapatinib , Neurregulina-1/genética , Estructura Terciaria de Proteína , Quinazolinas/farmacología , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-4RESUMEN
Although endogenous ligands for Toll-like receptor (TLR)4-myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4-MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4-MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4-MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4-MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4-MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.
Asunto(s)
Globósidos/inmunología , Antígeno 96 de los Linfocitos/inmunología , Complejos Multiproteicos/inmunología , Receptor Toll-Like 4/inmunología , Animales , Galactosiltransferasas/genética , Galactosiltransferasas/inmunología , Galactosiltransferasas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Globósidos/genética , Globósidos/metabolismo , Lipopolisacáridos/toxicidad , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/metabolismo , Microdominios de Membrana/genética , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Mutantes , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , Choque Séptico/inducido químicamente , Choque Séptico/genética , Choque Séptico/inmunología , Choque Séptico/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca(2+)-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.
Asunto(s)
Adhesión Bacteriana , Infecciones por Escherichia coli/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Vejiga Urinaria/metabolismo , Infecciones Urinarias/metabolismo , Escherichia coli Uropatógena/metabolismo , Urotelio/metabolismo , Adhesinas de Escherichia coli/genética , Adhesinas de Escherichia coli/metabolismo , Animales , Infecciones por Escherichia coli/patología , Femenino , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Humanos , Masculino , Ratones , Proteína D Asociada a Surfactante Pulmonar/genética , Conejos , Tetraspaninas/biosíntesis , Tetraspaninas/genética , Vejiga Urinaria/microbiología , Vejiga Urinaria/patología , Infecciones Urinarias/patología , Uroplaquina Ia/biosíntesis , Uroplaquina Ia/genética , Urotelio/microbiología , Urotelio/patologíaRESUMEN
BACKGROUND: Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice. METHODS: Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice. RESULTS: The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver. CONCLUSIONS: AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins. GENERAL SIGNIFICANCE: AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.
Asunto(s)
Aldehído Reductasa/fisiología , Aldehído Reductasa/genética , Animales , Ácido Ascórbico/análisis , Proteínas de Unión al Calcio/análisis , Femenino , Glucuronatos/metabolismo , Ácido Glucurónico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Hígado/química , Masculino , Metabolómica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Pulmonary surfactant is a mixture of lipids and proteins that covers alveolar surfaces and keeps alveoli from collapsing. Four specific proteins have been identified in surfactant. Among them, two C-type lectins, surfactant proteins A and D (SP-A and SP-D), are known to be implicated in host defense and regulation of inflammatory responses of the lung. These host defense lectins are structurally characterized by N-terminal collagen-like domains and lectin domains and are called pulmonary collectins. They prevent dissemination of infectious microbes by their biological activities including agglutination and growth inhibition. They also promote clearance of microbes by enhancing phagocytosis in macrophages. In addition, they interact with the other pattern-recognition molecules, including Toll-like receptors (TLRs) and TLR-associated molecules, CD14 and MD-2, and regulate inflammatory responses. Furthermore, recent studies have demonstrated that these collectins modulate functions of neutrophil-derived innate immune molecules by interacting with them. These findings indicate that pulmonary collectins play critical roles in host defense of the lung.
Asunto(s)
Colectinas/inmunología , Neumonía/inmunología , Surfactantes Pulmonares/inmunología , Animales , HumanosRESUMEN
We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that ß-linked N-acetyl-D-glucosamine and ß-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-ß-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of ß-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.
Asunto(s)
Helicobacter pylori/metabolismo , Lipopolisacáridos/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Antígenos Bacterianos/metabolismo , Western Blotting , Línea Celular , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Glicósido Hidrolasas/metabolismo , Helicobacter pylori/citología , Helicobacter pylori/genética , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Lipopolisacáridos/inmunología , Unión Proteica , Proteína D Asociada a Surfactante Pulmonar/genética , Estómago/citología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Defensins are important molecules in the innate immune system that eliminate infectious microbes. They also exhibit cytotoxicity against host cells in higher concentrations. The mechanisms by which hosts protect their own cells from cytotoxicity of defensins have been poorly understood. We found that the cytotoxicity of human ß-defensin 3 (hBD3) against lung epithelial cells was dose-dependently attenuated by pulmonary surfactant protein A (SP-A), a collectin implicated in host defense and regulation of inflammatory responses in the lung. The direct interaction between SP-A and hBD3 may be an important factor in decreasing this cytotoxicity because preincubation of epithelial cells with SP-A did not affect the cytotoxicity. Consistent with in vitro analysis, intratracheal administration of hBD3 to SP-A(-/-) mice resulted in more severe tissue damage compared with that in WT mice. These data indicate that SP-A protects lung epithelium from tissue injury caused by hBD3. Furthermore, we found that the functional region of SP-A lies within Tyr(161)-Lys(201). Synthetic peptide corresponding to this region, tentatively called SP-A Y161-G200, also inhibited cytotoxicity of hBD3 in a dose-dependent manner. The SP-A Y161-G200 is a candidate as a therapeutic reagent that prevents tissue injury during inflammation.
Asunto(s)
Citotoxinas/farmacología , Pulmón/metabolismo , Péptidos/farmacología , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Mucosa Respiratoria/metabolismo , beta-Defensinas/farmacología , Animales , Línea Celular , Citotoxinas/efectos adversos , Citotoxinas/metabolismo , Humanos , Pulmón/patología , Ratones , Ratones Noqueados , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Neumonía/patología , Unión Proteica , Proteína A Asociada a Surfactante Pulmonar/genética , Mucosa Respiratoria/patología , beta-Defensinas/efectos adversos , beta-Defensinas/metabolismoRESUMEN
Hepatitis C virus (HCV) chronic infection is characterized by low-level or undetectable cellular immune response against HCV antigens. HCV proteins affect various intracellular events and modulate immune responses, although the mechanisms that mediate these effects are not fully understood. In this study, we examined the effect of HCV proteins on the differentiation of human peripheral blood monocytes to dendritic cells (DCs). The HCV core (HCVc) and non-structural 3 (NS3) proteins inhibited the expression of CD1a, CD1b and DC-SIGN during monocyte differentiation to DCs, while increasing some markers characteristic of macrophages (CD14 and HLA-DR) and also PD-L1 expression. Meanwhile, HCVc and NS3 could induce differentiating monocytes to secrete IL-10. However, anti-IL-10 mAb could not reverse HCVc and NS3 inhibition of monocyte differentiation into DCs. The HCVc and NS3 proteins increased IL-6 secretion both in immature and in fully differentiated DCs and also promoted CD4+ T-cell IL-17 production. Since T(h) 17 cells are active in many examples of immunopathology, these effects may contribute to HCV autoimmune responses in chronically infected patients.
Asunto(s)
Células Dendríticas/inmunología , Hepacivirus/inmunología , Antígenos de la Hepatitis C/inmunología , Hepatitis C Crónica/inmunología , Células Th17/inmunología , Proteínas del Núcleo Viral/inmunología , Proteínas no Estructurales Virales/inmunología , Antígenos CD/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Humanos , Inmunomodulación , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-6/metabolismoRESUMEN
Pulmonary collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), play important roles in the innate immunity of the lung. Mycobacterium avium is one of the well-known opportunistic pathogens that can replicate within macrophages. We examined the effects of pulmonary collectins in host defense against M. avium infection achieved via direct interaction between bacteria and collectins. Although both pulmonary collectins bound to M. avium in a Ca(2+)-dependent manner, these collectins revealed distinct ligand-binding specificity and biological activities. SP-A and SP-D bound to a methoxy group containing lipid and lipoarabinomannan, respectively. Binding of SP-D but not SP-A resulted in agglutination of M. avium. A chimeric protein with the carbohydrate recognition domain of SP-D, which chimera revealed a bouquet-like arrangement similar to SP-A, also agglutinated M. avium. The ligand specificity of the carbohydrate recognition domain of SP-D seems to be necessary for agglutination activity. The binding of SP-A strongly inhibited the growth of M. avium in culture media. Although pulmonary collectins did not increase membrane permeability of M. avium, they attenuated the metabolic rate of the bacteria. Observations under a scanning electron microscope revealed that SP-A almost completely covers bacterial surfaces, whereas SP-D binds to certain areas like scattered dots. These observations suggest that a distinct binding pattern of collectins correlates with the difference of their biological activities. Furthermore, the number of bacteria phagocytosed by macrophages was significantly increased in the presence of SP-D. These data indicate that pulmonary collectins play critical roles in host defense against M. avium.
Asunto(s)
Proteína A Asociada a Surfactante Pulmonar/inmunología , Proteína D Asociada a Surfactante Pulmonar/inmunología , Mucosa Respiratoria/inmunología , Tuberculosis/inmunología , Humanos , Immunoblotting , Macrófagos/inmunología , Macrófagos/microbiología , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Rastreo , Mycobacterium avium/inmunología , Fagocitosis/inmunología , Unión Proteica , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Resonancia por Plasmón de Superficie , Tuberculosis/metabolismoRESUMEN
Vpr, a HIV-1 accessory protein, was believed to be present in the plasma of HIV-1-positive patients, and our previous work demonstrated the presence of plasma Vpr in 20 out of 52 patients. Interestingly, our data revealed that patients' viral titer was correlated with the level of Vpr detected in their plasma. Here, we first show that rVpr, when incubated with human monocytes or MDMs, caused viral production from latently infected cells, and IL-6 was identified as a responsible factor. The induction of IL-6 by rVpr was dependent on signaling through TLR4 and its adaptor molecule, MyD88. We next provide evidence that rVpr induced the formation of OxPC and that a mAb against OxPC blocked rVpr-induced IL-6 production with the concomitant attenuation of MAPK activation. Moreover, the addition of NAC, a scavenger of ROS, abrogated the rVpr-induced formation of OxPC, the phosphorylation of C/EBP-beta, a substrate of MAPK, and IL-6 production. As rIL-6 reactivated viral replication in latently infected cells, our data indicate that rVpr-induced oxidative stress triggers cell-based innate immune responses and reactivates viral production in latently infected cells via IL-6 production. Our results suggest that Vpr should be monitored based on the viral titer, and they provide the rationale for the development of novel, anti-AIDS therapeutics targeting Vpr.
Asunto(s)
Interleucina-6/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/metabolismo , Activación Viral , Latencia del Virus , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Western Blotting , Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células Cultivadas , Humanos , Inmunidad Innata , Interleucina-6/genética , Sistema de Señalización de MAP Quinasas , Monocitos/citología , Monocitos/metabolismo , Factor 88 de Diferenciación Mieloide/antagonistas & inhibidores , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Oxidación-Reducción , Fosfolípidos/química , Fosfolípidos/metabolismo , Regiones Promotoras Genéticas/genética , Análisis por Matrices de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genética , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate immunity of the lung. Legionella pneumophila is a bacterial respiratory pathogen that can replicate within macrophages and causes opportunistic infections. L. pneumophila possesses cytolytic activity, resulting from insertion of pores in the macrophage membrane upon contact. We examined whether pulmonary collectins play protective roles against L. pneumophila infection. SP-A and SP-D bound to L. pneumophila and its lipopolysaccharide (LPS) and inhibited the bacterial growth in a Ca(2+)-dependent manner. The addition of LPS in the culture blocked the inhibitory effects on L. pneumophila growth by the collectins, indicating the importance of LPS-collectin interaction. When differentiated THP-1 cells were infected with L. pneumophila in the presence of SP-A and SP-D, the number of permeable cells was significantly decreased, indicating that pulmonary collectins inhibit pore-forming activity of L. pneumophila. The number of live bacteria within the macrophages on days 1-4 after infection was significantly decreased when infection was performed in the presence of pulmonary collectins. The phagocytosis experiments with the pH-sensitive dye-labeled bacteria revealed that pulmonary collectins promoted bacterial localization to an acidic compartment. In addition, SP-A and SP-D significantly increased the number of L. pneumophila co-localized with LAMP-1. These results indicate that pulmonary collectins protect macrophages against contact-dependent cytolytic activity of L. pneumophila and suppress intracellular growth of the phagocytosed bacteria. The promotion of lysosomal fusion with Legionella-containing phagosomes constitutes a likely mechanism of L. pneumophila growth suppression by the collectins.
Asunto(s)
Legionella pneumophila/inmunología , Enfermedad de los Legionarios/inmunología , Macrófagos Alveolares/microbiología , Proteína A Asociada a Surfactante Pulmonar/inmunología , Proteína D Asociada a Surfactante Pulmonar/inmunología , Calcio/metabolismo , Carbohidratos/inmunología , Línea Celular , Membrana Celular/inmunología , Humanos , Legionella pneumophila/crecimiento & desarrollo , Lipopolisacáridos/farmacología , Lisosomas/inmunología , Monocitos/citología , Fagocitosis/inmunologíaRESUMEN
BACKGROUND: We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined. METHODS: We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2. RESULTS: MBL bound to sTLR4 and MD-2. The interactions were Ca2+-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu195-Phe228 or Thr174-Gly194 of SP-A were replaced with the corresponding MBL sequences. GENERAL SIGNIFICANCE: These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.
Asunto(s)
Colectinas/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Lectina de Unión a Manosa/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Humanos , Pulmón/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , SpodopteraRESUMEN
Lipopolysaccharide (LPS), derived from Gram-negative bacteria, is a major cause of acute lung injury and respiratory distress syndrome. Pulmonary surfactant is secreted as a complex mixture of lipids and proteins onto the alveolar surface of the lung. Surfactant phospholipids are essential in reducing surface tension at the air-liquid interface and preventing alveolar collapse at the end of the respiratory cycle. In the present study, we determined that palmitoyl-oleoyl-phosphatidylglycerol and phosphatidylinositol, which are minor components of pulmonary surfactant, and synthetic dimyristoylphosphatidylglycerol regulated the inflammatory response of alveolar macrophages. The anionic lipids significantly inhibited LPS-induced nitric oxide and tumor necrosis factor-alpha production from rat and human alveolar macrophages and a U937 cell line by reducing the LPS-elicited phosphorylation of multiple intracellular protein kinases. The anionic lipids were also effective at attenuating inflammation when administered intratracheally to mice challenged with LPS. Binding studies revealed high affinity interactions between the palmitoyl-oleoyl-phosphatidylglycerol and the Toll-like receptor 4-interacting proteins CD14 and MD-2. Our data clearly identify important anti-inflammatory properties of the minor surfactant phospholipids at the environmental interface of the lung.
Asunto(s)
Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/toxicidad , Antígeno 96 de los Linfocitos/metabolismo , Macrófagos Alveolares/metabolismo , Fosfolípidos/farmacología , Surfactantes Pulmonares/farmacología , Animales , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Pulmón/metabolismo , Ratones , Óxido Nítrico/metabolismo , Fosfolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Toll-Like 4/metabolismo , Células U937RESUMEN
The role of MD-2 in cell surface expression of Toll-like receptor (TLR) 4 has been controversial. The purposes of this study were to characterize the N-glycan of TLR4 and to investigate the roles of MD-2 in N-linked glycosylation and cell surface expression of TLR4. Lectin blot and cell surface biotinylation revealed that TLR4 exhibited the 110 kDa protein with high mannose type N-glycans and the 130 kDa protein with complex type N-glycans and that only the 130 kDa TLR4 with complex type N-glycans was expressed on the cell surface. The cells transfected with a mutant TLR4(C88A) alone expressed only the 110 kDa TLR4 with a high mannose type N-glycan, which did not appear on the cell surface. However, TLR4(C88A) acquired complex type N-glycans and was expressed on the cell surface when MD-2 was co-transfected. The amount of the 130 kDa TLR4(C88A) with complex type N-glycans expressed on the cell surface depended on that of MD-2 transfected. alpha-Mannosidase II inhibitor blocked the processing N-glycans to complex type, but TLR4 with high mannose type appeared on the cell surface, suggesting that TLR4 is destined to locate on the cell surface before processing N-glycans from a high mannose type to a complex type. From these results, we conclude that MD-2 is critical for cell surface expression of TLR4(C88A). This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.
Asunto(s)
Antígeno 96 de los Linfocitos/fisiología , Receptor Toll-Like 4/metabolismo , Línea Celular , Cisteína/genética , Cisteína/metabolismo , Análisis Mutacional de ADN , Glicosilación , Humanos , Mutagénesis Sitio-Dirigida , Polisacáridos/metabolismo , Receptor Toll-Like 4/genéticaAsunto(s)
Inmunidad Innata/genética , Proteína A Asociada a Surfactante Pulmonar/fisiología , Proteína D Asociada a Surfactante Pulmonar/fisiología , Animales , Calreticulina/fisiología , Humanos , Inmunidad Innata/inmunología , Macrófagos/inmunología , Proteínas Opsoninas , Fagocitosis , Proteína A Asociada a Surfactante Pulmonar/química , Proteína D Asociada a Surfactante Pulmonar/química , Surfactantes Pulmonares , Receptores Inmunológicos/fisiología , Receptores Toll-Like/fisiologíaRESUMEN
Pulmonary surfactant protein D (SP-D) is a member of the collectin family and plays crucial roles in the innate immunity of the lung. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with MD-2 and alters lipopolysaccharide signaling. In this study, we examined and characterized the binding of SP-D to MD-2 using a soluble form of recombinant MD-2 (sMD-2). SP-D bound in a concentration- and Ca(2+)-dependent manner to sMD-2 coated onto microtiter wells. Excess mannose abolished the binding of SP-D to sMD-2. In solution, SP-D cosedimented with sMD-2 in the presence of Ca(2+). The direct binding of SP-D to sMD-2 was confirmed by BIAcore analysis. Anti-SP-D monoclonal antibody that recognizes the carbohydrate recognition domain (CRD) of SP-D significantly inhibited the binding of SP-D to sMD-2, indicating the involvement of the CRD for the binding to sMD-2. Ligand blot analysis revealed that SP-D bound to N-glycopeptidase F-treated sMD-2. In addition, the biotinylated SP-D pulled down the mutant sMD-2 with Asn(26) --> Ala and Asn(114) --> Ala substitutions, which lacks the consensus for N-glycosylation. Furthermore, the sMD-2 mutant cosedimented SP-D. These results demonstrate that SP-D directly interacts with MD-2 through the CRD.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Antígeno 96 de los Linfocitos/metabolismo , Proteína D Asociada a Surfactante Pulmonar/química , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Electroforesis , Humanos , Antígeno 96 de los Linfocitos/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , SolubilidadRESUMEN
Pulmonary surfactant protein D (SP-D) is a member of the collectin family that plays an important role in regulating innate immunity of the lung. We examined the mechanisms by which SP-D modulates lipopolysaccharide (LPS)-elicited inflammatory cell responses. SP-D bound to a complex of recombinant soluble forms of Toll-like receptor 4 (TLR4) and MD-2 with high affinity and down-regulated tumor necrosis factor-alpha secretion and NF-kappaB activation elicited by rough and smooth LPS, in alveolar macrophages and TLR4/MD-2-transfected HEK293 cells. Cell surface binding of both serotypes of LPS to TLR4/MD-2-expressing cells was attenuated by SP-D. In addition, SP-D significantly reduced MD-2 binding to both serotypes of LPS. A chimera containing the N-terminal region and the collagenous domain of surfactant protein A, and the coiled-coil neck and lectin domains of SP-D, was a weak inhibitor of LPS-induced cell responses and MD-2 binding to LPS, compared with native SP-D. The collagenase-resistant fragment consisting of the neck plus the carbohydrate recognition domain of SP-D also was a very weak inhibitor of LPS activation. This study demonstrates that SP-D down-regulates LPS-elicited inflammatory responses by altering LPS binding to its receptors and reveals the importance of the correct oligomeric structure of the protein in this process.
Asunto(s)
Inmunidad Innata/inmunología , Inmunidad Innata/fisiología , Lipopolisacáridos/toxicidad , Pulmón/inmunología , Antígeno 96 de los Linfocitos/inmunología , Proteína D Asociada a Surfactante Pulmonar/inmunología , Receptor Toll-Like 4/inmunología , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Antígeno 96 de los Linfocitos/genética , Unión Proteica , Proteína A Asociada a Surfactante Pulmonar/genética , Proteína A Asociada a Surfactante Pulmonar/inmunología , Proteína D Asociada a Surfactante Pulmonar/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Toll-like receptors (TLRs) play an essential role in defense responses. Immune cells express multiple TLRs which are simultaneously activated by microbial pathogens. PRotein Associated with Tlr4 A (PRAT4A) is a chaperone-like endoplasmic reticulum (ER)-resident protein required for the proper subcellular distribution of multiple TLRs. PRAT4A(-/-) mice show impaired expression of TLR2/4 on the cell surface and the lack of ligand-induced TLR9 relocation from the ER to endolysosome. Consequently, TLR responses to whole bacteria as well as to TLR2, 4 and 9 ligands are impaired. We here compare the interaction of these TLRs with PRAT4A. Association of endogenous PRAT4A was easily detected only with TLR4. The TLR4 region responsible for strong interaction with PRAT4A is very close to the site necessary for interaction with MD-2. By using transient expression, we were able to detect PRAT4A interaction with TLR2 and TLR9. The PRAT4A single-nucleotide mutant replacing methionine 145 with lysine (M145K) associates with TLR9 but does not rescue ligand-dependent TLR9 trafficking. By contrast, the M145K mutant weakly, if at all, associates with TLR2 and TLR4. The M145K mutant appreciably rescues cell-surface TLR2 expression and its responses in PRAT4A(-/-) bone marrow-derived dendritic cells, whereas little if any rescue of cell-surface TLR4/MD-2 expression and its responses occurs. These results demonstrate that PRAT4A differentially interacts with each TLR and suggest that a single-nucleotide change in the PRAT4A gene influences not only the strength of TLR responses but can also alter the relative activity of each TLR.