RESUMEN
BACKGROUND: The reported prevalences of IgG autoantibodies (AAbs) to FcεRIα and IgE in sera from patients with chronic spontaneous urticaria (CSU) have varied, and these AAbs are also often observed in healthy control subjects. Regarding the histamine release activity of purified IgG from patients with CSU, the number of examined patients has been small. Thus, we sought to determine the prevalence and FcεRI crosslinking ability of these AAbs in a large number of patients with CSU and non-atopic control (NC) subjects. METHODS: We compared the concentrations of anti-IgE and anti-FcεRIα AAbs and the abilities of these AAbs to cause FcεRI aggregation in patients with CSU (n = 134) and NC subjects (n = 55) using ELISA and an in vitro elicitation test, respectively. RESULTS: The concentration of anti-IgE AAbs was significantly different between the NC subjects and the CSU patients (P < 0.0001, cutoff value: 0.558 µg/mL), whereas the concentration of anti-FcεRIα AAbs was not. A significant difference in the duration of illness was noted between patients with lower and those with higher concentrations of anti-IgE AAbs relative to the cutoff value. The abilities of anti-IgE AAbs, but not anti-FcεRIα AAbs, to induce FcεRI crosslinking were significantly higher in CSU patients than in NC subjects (P = 0.0106). CONCLUSIONS: In the Japanese population of CSU patients studied, the ability of the anti-IgE AAbs to induce FcεRI crosslinking differed significantly between NC subjects and CSU patients, suggesting the involvement of anti-IgE AAbs in the pathogenesis of CSU in the Japanese population.
Asunto(s)
Autoanticuerpos/inmunología , Inmunoglobulina E/inmunología , Receptores de IgE/inmunología , Urticaria/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Autoanticuerpos/sangre , Basófilos/inmunología , Células Cultivadas , Enfermedad Crónica , Femenino , Liberación de Histamina , Humanos , Inmunoglobulina G/sangre , Recubrimiento Inmunológico , Masculino , Mastocitos/inmunología , Persona de Mediana Edad , Adulto JovenAsunto(s)
Mastocitos/inmunología , Mastocitos/metabolismo , Multimerización de Proteína , Receptores de IgE/metabolismo , Secuencia de Aminoácidos , Animales , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Citocinas/metabolismo , Disulfuros/química , Expresión Génica , Humanos , Mutación , Dominios y Motivos de Interacción de Proteínas , Receptores de IgE/química , Receptores de IgE/genéticaRESUMEN
BACKGROUND: Interleukin (IL)-17A plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The expression of IL-17A in synovial mast cells (MCs) in RA and osteoarthritis (OA) has been reported, but the frequencies of IL-17A expression in synovial MCs have varied. The aim of this study was to investigate whether IL-17A expression is upregulated in human synovial MCs in RA and to elucidate the mechanism of IL-17A expression in synovial MCs. METHODS: Synovial tissues were obtained from patients with RA or OA undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Synovium-derived cultured MCs were generated by culturing synovial cells with stem cell factor. IL-17A expression was investigated using immunofluorescence in synovial tissues. IL-17A mRNA expression and its production from MCs were examined using RT-PCR and ELISA, respectively. RESULTS: The number of IL-17A-positive ((+)) synovial MCs and the percentage of IL-17A(+) MCs among all the IL-17A(+) cells from RA patients were not significantly increased compared with those from OA subjects. The synovium-derived cultured MCs spontaneously released small amounts of IL-17A. Neither IgE- nor IgG-dependent stimulation increased IL-17A production from the MCs. IL-33, tumor necrosis factor-α, C5a, lipopolysaccharide or IL-23 plus IL-1ß did not affect IL-17A production in MCs. CONCLUSIONS: The synovial MCs are not a main source of IL-17A in RA.
Asunto(s)
Artritis Reumatoide/genética , Expresión Génica , Interleucina-17/genética , Mastocitos/metabolismo , Osteoartritis/genética , Sinoviocitos/metabolismo , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/inmunología , Artritis Reumatoide/cirugía , Biomarcadores , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Interleucina-17/metabolismo , Masculino , Mastocitos/inmunología , Osteoartritis/diagnóstico , Osteoartritis/inmunología , Osteoartritis/cirugía , Sinoviocitos/inmunologíaRESUMEN
BACKGROUND: The activation of liver X receptor (LXR) α or LXRß negatively regulates the expression of pro-inflammatory genes in mammalian cells. We recently reported that 25-hydroxycholesterol, a representative LXR-activating oxysterol, suppresses IL-6 production in mouse mast cells (MCs) following its engagement of the high-affinity IgE receptor (FcεRI). This finding suggests that murine MCs express functional LXRs; however, the mechanisms underlying the LXR-dependent repression of the MC-mediated production of pro-inflammatory cytokines, including IL-6, are poorly understood. Therefore, we employed the synthetic LXR ligand GW3965 to examine the functions of LXRα and LXRß in the production of pro-inflammatory cytokines by murine bone marrow-derived MCs (BMMCs). METHODS: We prepared BMMCs from wild-type (WT), LXRα(-/-), and LXRα/ß(-/-) mice. Each group of BMMCs was pretreated with GW3965 and then stimulated with IgE+antigen (Ag) or lipopolysaccharide (LPS). Cytokine production was then analyzed using specific ELISA kits. RESULTS: The activation of LXRs by GW3965 significantly attenuated the production of IL-1α and IL-1ß, but not of IL-6, in the WT and LXRα(-/-) BMMCs stimulated with IgE+Ag. However, GW3965 treatment decreased the production of IL-1α, IL-1ß, and IL-6 in WT and LXRα(-/-) BMMCs upon stimulation with LPS, while the GW3965-mediated suppression of cytokine production was nearly absent from the LXRα/ß(-/-) BMMCs. CONCLUSIONS: These findings demonstrate, for the first time, that the activation of LXRs by GW3965 attenuates the antigen- or LPS-induced production of pro-inflammatory cytokines, such as IL-1α and IL-1ß, in murine MCs and that LXRß plays an important role in the LXR-mediated repression of cytokine production.
Asunto(s)
Benzoatos/farmacología , Bencilaminas/farmacología , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Receptores Nucleares Huérfanos/agonistas , Animales , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Citocinas/genética , Expresión Génica , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Lipopolisacáridos/inmunología , Receptores X del Hígado , Ratones , Ratones Noqueados , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de IgE/agonistas , Receptores de IgE/metabolismoRESUMEN
The process of sensitisation by specific contact allergens is indispensable for the induction of allergic contact dermatitis. Oxazolone is a well-characterised contact allergen. Previous studies suggested that immune cells bearing the FcRγ subunit are essential for oxazolone-induced contact hypersensitivity, but the biological functions of the FcRγ subunit in the process of sensitisation to oxazolone remain unknown. In this study, we show that FcRγ deficiency decreases ear-swelling responses to oxazolone in mice. However, we found that oxazolone-sensitised FcRγ(-/-) mice and oxazolone-sensitised wild-type (WT) mice have comparable numbers of CD11c(+) MHCII(hi) dendritic cells (DCs) in their draining lymph nodes (LNs). In addition, oxazolone-sensitised LN cells from both FcRγ(-/-) and WT mice showed considerable production of interferon-gamma (IFNγ), interleukin-4 (IL-4) and IL-17A upon oxazolone-keyhole limpet haemocyanin loading. Consistent with these data, oxazolone-sensitised FcRγ(-/-) and FcRγ(+/+) LN cells conferred contact hypersensitivity to WT naïve mice challenged with the hapten. Our findings clearly indicate that, in an experimental mouse model, the FcRγ subunit positively regulates contact hypersensitivity to oxazolone without affecting the contact sensitisation process.
Asunto(s)
Adyuvantes Inmunológicos , Dermatitis Alérgica por Contacto/inmunología , Oxazolona , Receptores de IgE/inmunología , Receptores de IgG/inmunología , Animales , Células Dendríticas , Dermatitis Alérgica por Contacto/genética , Dermatitis Alérgica por Contacto/patología , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Dinitroclorobenceno , Inmunoglobulina E/sangre , Inmunoglobulina E/efectos de los fármacos , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de IgE/administración & dosificación , Receptores de IgG/genéticaRESUMEN
Engagement of the high-affinity IgE receptor (FcεRI) can be either protective or non-protective against apoptotic cell death (ACD) in bone marrow-derived murine mast cells (BMMCs) after IL-3 withdrawal, depending on the avidity between IgE and its antigen. We recently reported that protein L (PpL), a bacterial Igκ-binding soluble protein, is able to stimulate intracellular signaling to induce activation of BMMCs by interacting with the IgEκ-FcεRI complex. However, it is unclear if cross-linking of FcεRI with IgEκ and PpL prevents or enhances IL-3-dependent ACD in BMMCs. In the present study, we found that IL-3-dependent ACD of BMMCs is accelerated by loading soluble PpL in the presence of IgEκ-occupied FcεRIα. For this purpose, soluble PpL was incorporated into the BMMCs. Unlike soluble PpL, immobilized PpL failed to enhance ACD, although both forms of PpL induced IL-6 production equally in BMMCs. In addition, we observed that DNS5-BSA protected anti-DNS IgE-sensitized BMMCs from IL-3 depletion-mediated ACD by inducing the production of autocrine IL-3. In contrast, DNS5-PpL enhanced IL-3 withdrawal-induced ACD of anti-DNS IgE-sensitized BMMCs and reduced the production of autocrine IL-3. These findings suggest that PpL increases IL-3 withdrawal-induced ACD of IgEκ-sensitized BMMCs by incorporating PpL into the BMMCs and that this internalized PpL may interfere with survival signals via FcεRI.
Asunto(s)
Apoptosis/inmunología , Proteínas Bacterianas/inmunología , Inmunoglobulina E/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Interleucina-3/inmunología , Mastocitos/inmunología , Receptores de IgE/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/farmacología , Células Cultivadas , Proteínas Inmovilizadas/inmunología , Proteínas Inmovilizadas/farmacología , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BLRESUMEN
CCAAT/enhancer binding protein alpha (C/EBPα) is a transcription factor that influences immune cell fate and differentiation. However, the effect of C/EBPα on mast cells is not fully understood. In this study, we showed that C/EBPα suppressed granule formation in mast cells and increased macrophage inflammatory protein (MIP)-2 production from mast cells upon bacterial stimulation. These results indicate that C/EBPα regulates the balance between the allergic response and the innate immune response of mast cells. Furthermore, we showed that stimulation of mast cells with the Lactobacillus casei JCM1134(T) strain during late differentiation up-regulated C/EBPα expression in differentiated mast cells. This suggests that intestinal commensal bacteria modulate C/EBPα expression and thereby regulate mast cell function.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Mastocitos/citología , Mastocitos/metabolismo , Animales , Células de la Médula Ósea/citología , Proteína alfa Potenciadora de Unión a CCAAT/genética , Diferenciación Celular , Quimiocina CXCL2/biosíntesis , Femenino , Regulación de la Expresión Génica , Lacticaseibacillus casei/fisiología , Mastocitos/inmunología , Mastocitos/microbiología , RatonesRESUMEN
BACKGROUND: Wheal reactions to intradermally injected neuropeptides, such as substance P (SP) and vasoactive intestinal peptide, are significantly larger and longer lasting in patients with chronic urticaria (CU) than in nonatopic control (NC) subjects. Mas-related gene X2 (MrgX2) has been identified as a receptor for basic neuropeptides, such as SP and vasoactive intestinal peptide. Mast cell (MC) responsiveness to eosinophil mediators contributes to the late-phase reaction of allergy. OBJECTIVE: We sought to compare the frequency of MrgX2 expression in skin MCs from patients with CU and NC subjects and to identify the receptor for basic eosinophil granule proteins on human skin MCs. METHODS: MrgX2 expression was investigated by using immunofluorescence in skin tissues from NC subjects and patients with severe CU and on skin-derived cultured MCs. MrgX2 expression in human MCs was reduced by using a lentiviral small hairpin RNA silencing technique. Ca(2+) influx was measured in CHO cells transfected with MrgX2 in response to eosinophil granule proteins. Histamine and prostaglandin D2 levels were measured by using enzyme immunoassays. RESULTS: The number of MrgX2(+) skin MCs and the percentage of MrgX2(+) MCs in all MCs in patients with CU were significantly greater than those in NC subjects. Eosinophil infiltration in urticarial lesions was observed in 7 of 9 patients with CU. SP, major basic protein, and eosinophil peroxidase, but not eosinophil-derived neurotoxin, induced histamine release from human skin MCs through MrgX2. CONCLUSION: MrgX2 might be a new target molecule for the treatment of wheal reactions in patients with severe CU.
Asunto(s)
Mastocitos/inmunología , Proteínas del Tejido Nervioso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Piel/metabolismo , Urticaria/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Enfermedad Crónica , Proteínas en los Gránulos del Eosinófilo/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/genética , Unión Proteica , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropéptido/genética , Piel/patología , Pruebas Cutáneas , Sustancia P/administración & dosificación , Sustancia P/efectos adversos , Regulación hacia Arriba , Urticaria/inmunología , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/efectos adversos , Adulto JovenRESUMEN
BACKGROUND: Omalizumab, a monoclonal anti-IgE antibody, is currently indicated for the treatment of moderate-to-severe allergic asthma. To measure active IgE levels in sera from patients treated with omalizumab, the IgE subfraction in complex with omalizumab should be eliminated from total IgE, and free IgE levels can then be determined. With the aim of therapeutic monitoring for anti-IgE therapy, we developed a new ELISA for free IgE. METHODS: We used recombinant human soluble FcεRIα as a capture antigen and a biotinylated polyclonal anti-IgE antibody for detection. Using the newly developed ELISA, we measured the serum free IgE levels weekly in four asthmatic patients after their first omalizumab injection. We also measured the serum free IgE levels in 54 patients treated with omalizumab for over 4 weeks. RESULTS: This assay was technically robust, the mean recovery rate in serum was 93.16% ± 5.34%. For all patients, omalizumab treatment significantly reduced serum free IgE levels prior to the second omalizumab injection. To maintain the benefit of omalizumab, serum free IgE concentrations should be <50 ng/ml. However, in 14 of 54 patients treated with omalizumab for over 4 weeks, serum free IgE concentrations measured by our ELISA were >50 ng/ml. CONCLUSIONS: Our data suggest that the measurement of free IgE levels using our newly developed ELISA would be useful for monitoring serum free IgE levels during omalizumab therapy.
Asunto(s)
Asma/diagnóstico , Asma/inmunología , Inmunoglobulina E/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antiasmáticos/uso terapéutico , Anticuerpos Antiidiotipos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/tratamiento farmacológico , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina E/sangre , Masculino , Persona de Mediana Edad , Omalizumab , Estándares de Referencia , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
The CCAAT/enhancer-binding protein α (C/EBPα) is the member of a family of related basic leucine zipper (bZIP) transcription factors and is critical for granulopoiesis. We previously demonstrated that C/EBPα interacts with the ETS domain of widely expressed GABPα, which leads to cooperative transcriptional activation of the myeloid-specific promoter for human FCAR encoding the Fc receptor for IgA (FcαR, CD89) in part by facilitating recruitment of C/EBPα to the promoter. The C/EBPα molecule contains transactivation domains (TADs) at its N-terminus and a DNA-binding and dimerization bZIP structure at its C-terminus. We demonstrate here that GABPα interacts with the last 18 residues of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerizing region. Deletion of this C-terminus resulted in loss of GABPα interaction but not affecting its DNA binding ability, indicating that it is not required for homodimer formation. Moreover, the C-terminus confers the ability to functionally synergize with GABP on a heterologous TAD when fused to the C-terminus of the VP16 TAD. We identified a three-amino acid stretch (amino acids 341-343) that is important for both functional and protein interactions with GABP. Ectopic expression in K562 cells of C/EBPα mutant incapable of interacting with GABPα does not induce expression of granulocytic differentiation markers including CD15, CD11b, GCSF-R and C/EBPε, and does not inhibit proliferation, whereas wild type does. These results demonstrate the functional importance of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerization region, which may mediate cooperative activation by C/EBPα and GABP of myeloid-specific genes involved in C/EBPα-dependent granulopoiesis.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Diferenciación Celular/fisiología , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Secuencia de Bases , Proteína alfa Potenciadora de Unión a CCAAT/química , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Humanos , Células K562 , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγ(null) (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM-Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34⺠hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM-Tg mice developed passive cutaneous anaphylaxis reactions when administered anti-4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM-Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.
Asunto(s)
Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Hipersensibilidad/inmunología , Interleucina-3/inmunología , Animales , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunohistoquímica , Interleucina-3/genética , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
BACKGROUND: Myelitis is one of the rarest neurological complications of the varicella zoster virus (VZV) infection. Focal muscle weakness with or without sensory disturbance occurs in approximately 5% of the cases after acute VZV infection, with complete recovery in 50-70%. CASE PRESENTATION: This report describes two rare cases of elderly patients with VZV myelitis secondary to dermatomal zoster rash. Patient 1 was a 79-year-old woman who developed paraplegia, numbness and decreased sensation in the left arm and below thoracic (Th)-10 after sacral zoster. Spinal cord MRI showed a high-signal-intensity lesion at the cervical spinal nerve 2 on a T2-weighted image. Patient 2 was a 73-year-old man who developed right flaccid leg weakness and urinary retention after right dorsal Th 5-8 zoster. Spinal cord MRI showed a high-signal-intensity lesion at Th 3-4 on a T2-weighted image. In both cases, although the conventional single polymerase chain reaction (PCR) assays all showed negative results, the original nested PCR assay detected VZV DNA in the cerebrospinal fluid (CSF) specimen collected on admission. In addition, the anti-VZV IgG antibody by enzyme immunoassay and antibody index were elevated in the CSF specimens during the clinical courses of both patients. On the basis of these findings, both patients were diagnosed with VZV myelitis and were treated with high-dose acyclovir and corticosteroid. This combined treatment was appropriate and effective for the improvement of their functional outcomes. CONCLUSION: The detection of VZV DNA in CSF by nested PCR assay and the evaluation of the antibody index to VZV had significant diagnostic value.
RESUMEN
BACKGROUND: Basophils and mast cells are important initiator/effector cells capable of rapidly responding to IgE-mediated stimulation, but the precise mechanisms regulating their functions in vivo have not been fully identified. In this study, we assessed whether low levels of antigen can modulate activation of basophils and mast cells. METHODS: Human basophils and cultured mast cells were pretreated with low concentrations of anti-FcεRI α-chain mAb (CRA-1 mAb), and their cell functions were assessed. RESULTS: Basophils preincubated with CRA-1 mAb at as low as 1 ng/ml for 1 h showed significantly enhanced degranulation in response to various secretagogues such as MCP-1, FMLP, leukotriene B4 and Ca ionophore A23187. FMLP-induced leukotriene C4 production by basophils was also enhanced by CRA-1 mAb pretreatment. Degranulation was further enhanced when CRA-1 mAb-pretreated basophils were additionally treated with IL-3, IL-33 or leptin before stimulation with MCP-1. Priming by subthreshold CRA-1 mAb was a slow process, since 1 h of pretreatment was needed for maximal enhancement. Basophil priming also resulted from preincubation with subthreshold doses of an allergen, Der f 2. In parallel mAb experiments, CRA-1 mAb showed weak priming effects on human umbilical cord blood-derived cultured mast cells; a higher dose, 100 ng/ml, was necessary for this priming. CONCLUSION: These results indicate that subthreshold doses of CRA-1 mAb or allergens can prime basophils and induce exaggerated responses to various IgE-independent stimuli. This may be a potentially important mechanism that explains environmental allergen-induced exacerbation of IgE-mediated allergic diseases such as asthma.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Basófilos/inmunología , Basófilos/metabolismo , Receptores de IgE/inmunología , Alérgenos/inmunología , Basófilos/efectos de los fármacos , Degranulación de la Célula/inmunología , Células Cultivadas , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Leucotrieno C4/biosíntesis , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/metabolismoRESUMEN
BACKGROUND: Substantial evidence suggests that human synovial mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-33 is believed to play an important role in the pathogenesis of RA. We recently reported that FcγRI is responsible for producing abundant tumor necrosis factor alpha (TNF-α) from cultured synovium-derived MCs (SyMCs) in response to aggregated immunoglobulin G (IgG). However, whether or not IL-33 affects immune complex (IC)-induced synovial MC activation remains unknown. This study sought to evaluate the effect of IL-33 on IC-induced synovial MC activation. METHODS: Cultured SyMCs were generated by culturing synovial cells with stem cell factor. ST2 expression was analyzed using FACS and immunohistochemical techniques. Mediators released from the MCs were measured using EIAs or ELISAs. RESULTS: SyMCs obtained from patients with RA or osteoarthritis (OA) expressed ST2 on their surfaces. We confirmed the expression of ST2 in MCs using immunofluorescence staining in joint tissue obtained from RA patients. IC-triggered histamine release was not enhanced by IL-33. However, IL-33 synergistically enhanced IC-induced IL-8 and TNF-α production in SyMCs. CONCLUSIONS: ICs and IL-33 may exacerbate inflammation associated with RA by abundantly producing TNF-α and IL-8 from SyMCs.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Interleucina-8/biosíntesis , Interleucinas/farmacología , Mastocitos/inmunología , Mastocitos/metabolismo , Membrana Sinovial/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Células Cultivadas , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Receptores de Superficie Celular/metabolismo , Receptores de IgG/metabolismoRESUMEN
BACKGROUND: Zinc (Zn) affects many aspects of immune function, including thymic development and the activities of immune cells. Zn is also involved in many steps of high-affinity IgE receptor (FcεRI)-induced mast cell (MC) activation, which is required for degranulation and cytokine production. Intracellular Zn levels increase in mouse MCs after FcεRI stimulation. We previously reported that Zn distribution in a human MC line, LAD2, changed dramatically following FcεRI aggregation with synchrotron radiation microbeams. However, the kinetics of Zn distribution and the underlying mechanisms following FcεRI cross-linking remain unknown. METHODS: We used cord-blood-derived MCs and LAD2 cells. Degranulation was assessed by ß-hexosaminidase (ß-hex) release. Extracellular Zn levels were determined by inductively coupled plasma atomic emission spectrometry or based on the fluorescence intensity of a Zn indicator. We also used RNAi to knockdown ZnT1 expression. mRNA expression levels were determined by real-time RT-PCR. RESULTS: Zn was rapidly released from human MCs after FcεRI aggregation. The kinetics and optimal conditions for FcεRI cross-linking for Zn release were different from those for degranulation. Treating LAD2 cells with an intracellular Ca(2+) chelator significantly inhibited IgE-mediated ß-hex release but not Zn release. We investigated IgE-mediated ß-hex and Zn release with specific inhibitors of signaling pathways. Zn and ß-hex release were partly correlated with but also partly independent of IgE. Knockdown of the Zn efflux transporter, ZnT1, significantly inhibited Zn release from human MCs. CONCLUSIONS: Our results indicate that IgE-dependent Zn release from human MCs involves signaling cascades that are distinct from those of degranulation. Thus, Zn may have a unique function as a mediator of allergic inflammation.
Asunto(s)
Inmunoglobulina E/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , Receptores de IgE/metabolismo , Zinc/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/inmunología , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica , Liberación de Histamina , Humanos , Interferencia de ARNRESUMEN
BACKGROUND: Chronic urticaria (CU) appears to be of autoimmune origin in about half of all patients, since several autoreactive immunoglobulin Gs (IgGs), such as anti-FcεRIα and anti-IgE, are detected in the sera of such patients. However, whether autoreactive IgE is associated with CU remains unclear. In this study, we attempted to identify autoreactive IgE antibodies in sera from patients with CU. METHODS: Sera were collected from 67 normal subjects, 85 patients with CU and 28 patients with atopic dermatitis (AD). An autologous serum skin test (ASST) was performed on 27 of the CU patients. Autoreactive IgE and IgG levels against self-antigens were measured using enzyme-linked immunosorbent assays. The basophils were activated with dsDNA, and the CD63 expression level was examined using a fluorescence-activated cell sorter. RESULTS: The anti-dsDNA IgE levels were significantly higher in patients with CU and AD than in normal subjects, but no differences in the anti-dsDNA IgG levels were seen. The levels of thioredoxin-, peroxiredoxin- and thyroglobulin-reactive IgE and IgG were not significantly higher in the CU patients than in the other 2 groups. There was no significant difference in the levels of anti-dsDNA IgE between ASST-positive and ASST-negative patients. The basophils from 2 out of 9 CU patients exhibited degranulation in response to dsDNA. CONCLUSIONS: Our data suggest that anti-dsDNA IgE is involved in the pathogenesis of some cases of CU.
Asunto(s)
Basófilos/inmunología , Degranulación de la Célula/inmunología , ADN/inmunología , Inmunoglobulina E/inmunología , Urticaria/inmunología , Adulto , Enfermedad Crónica , Dermatitis Atópica/inmunología , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana EdadRESUMEN
Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.
Asunto(s)
Fosfolipasas A2 Grupo III/inmunología , Mastocitos/inmunología , Comunicación Paracrina/inmunología , Prostaglandina D2/inmunología , Receptores de Prostaglandina/inmunología , Animales , Western Blotting , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Fosfolipasas A2 Grupo III/genética , Fosfolipasas A2 Grupo III/metabolismo , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/inmunología , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/genética , Lipocalinas/inmunología , Lipocalinas/metabolismo , Mastocitos/metabolismo , Mastocitos/ultraestructura , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Análisis de Secuencia por Matrices de Oligonucleótidos , Comunicación Paracrina/genética , Prostaglandina D2/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly induced GFP expression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes.
Asunto(s)
Antígenos de Superficie/fisiología , Regulación de la Expresión Génica , Mastocitos/metabolismo , Glicoproteínas de Membrana/fisiología , Monocitos/metabolismo , Receptores de IgG/metabolismo , Animales , Antígenos de Superficie/metabolismo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Células HL-60 , Humanos , Sistema Inmunológico , Células Jurkat , Células K562 , Ligandos , Mastocitos/citología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Células 3T3 NIH , Fagocitosis , Ratas , Transducción de Señal , Relación Estructura-Actividad , Células U937RESUMEN
Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O2()) are thought to mediate apoptosis induced by death receptor ligands, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the role of H(2)O(2) is controversial, since some evidence suggests that H(2)O(2) acts as an anti-apoptotic factor. Here, we show that exogenously applied H(2)O(2) (30-100 µM) induces cell death in TRAIL-resistant human melanoma cells via intracellular superoxide (O(2)-) generation. H(2)O(2) induced apoptotic or necrotic cell death, depending on the concentration of the oxidant applied; low concentrations of H(2)O(2) preferentially activated the caspase-dependent apoptotic pathway, while high concentrations of H(2)O(2) induced apoptotic and necrotic cell death in a caspase-independent manner. The H(2)O(2)-induced cell death was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation and ER stress responses including caspase-12 and X-box-binding protein-1 (XBP-1) activation. H(2)O(2) induced intracellular O2- generation even within the mitochondria, while TRAIL did not. The superoxide dismutase mimetic antioxidant MnTBaP [Mn (III) tetrakis (4-benzonic acid) porphyrin chloride] inhibited the H(2)O(2)-induced O(2)- generation, apoptosis and XBP-1 and caspase-12 activation at comparable concentrations. Importantly, H(2)O(2) treatment caused minimal O(2)- generation and apoptosis in normal primary melanocytes. These data show that H(2)O(2) induces endoplasmic reticulum-associated cell death via intracellular O(2)- generation and that malignant melanoma cells are more susceptible than normal cells to this oxidative cell death. The findings suggest that H(2)O(2) has therapeutic potential in the treatment of TRAIL-resistant melanoma.