RESUMEN
CD300a is differentially expressed among B cell subsets, although its expression in immunoglobulin (Ig)M+ B cells is not well known. We identified a B cell subset expressing CD300a and high levels of IgM (IgMhiCD300a+). The results showed that IgMhiCD300a+ B cells were CD10-CD27+CD25+IgDloCD21hiCD23-CD38loCD1chi, suggesting that they are circulating marginal zone (MZ) IgM memory B cells. Regarding the immunoglobulin repertoire, IgMhiCD300a+ B cells exhibited a higher mutation rate and usage of the IgH-VDJ genes than the IgM+CD300a- counterpart. Moreover, the shorter complementarity-determining region 3 (CDR3) amino acid (AA) length from IgMhiCD300a+ B cells together with the predicted antigen experience repertoire indicates that this B cell subset has a memory phenotype. IgM memory B cells are important in T cell-independent responses. Accordingly, we demonstrate that this particular subset secretes higher amounts of IgM after stimulation with pneumococcal polysaccharides or a toll-like receptor 9 (TLR9) agonist than IgM+CD300a- cells. Finally, the frequency of IgMhiCD300a+ B cells was lower in people living with HIV-1 (PLWH) and it was inversely correlated with the years with HIV infection. Altogether, these data help to identify a memory B cell subset that contributes to T cell-independent responses to pneumococcal infections and may explain the increase in severe pneumococcal infections and the impaired responses to pneumococcal vaccination in PLWH.
Asunto(s)
Infecciones por VIH , Infecciones Neumocócicas , Humanos , Células B de Memoria , Streptococcus pneumoniae , Adyuvantes Inmunológicos , Regiones Determinantes de Complementariedad , Inmunoglobulina MRESUMEN
The CD300 molecules constitute an evolutionarily significant family of receptors that are expressed on myeloid and lymphoid cells, but also on other cell types, such as tuft cells. Many of the CD300 receptors have been shown to recognize lipids, e.g. phosphatidylserine and phosphatidylethanolamine. Over the past couple of years, accumulating evidence has shown that this family of receptors is involved in the pathogenesis of many diseases. Specifically, CD300 molecules participate in the mechanisms that viruses employ to develop immune evasion strategies and to infect host cells. The participation of CD300 molecules in viral infection includes both lipid dependent and independent mechanisms, as for example in infections with dengue virus (DENV) and murine norovirus (MNV), respectively. CD300 receptors are also involved in viral escape mechanisms, for instance inhibiting NK cell-mediated cytotoxicity against infected cells. Moreover, it is becoming increasingly recognized that the expression of CD300 receptors is altered during viral diseases. Here, we review the involvement of human and murine CD300 molecules in viral binding and entry and in cellular responses to viruses, which highlights the potential of CD300 molecules in the search of new biomarkers for various stages of infection and therapeutic targets for the treatment of viral infections.
Asunto(s)
Antígenos CD/inmunología , Virus del Dengue/inmunología , Evasión Inmune/inmunología , Norovirus/inmunología , Fosfolípidos/inmunología , Receptores Inmunológicos/inmunología , Virosis/inmunología , Animales , Antígenos CD/metabolismo , Virus del Dengue/fisiología , Humanos , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/virología , Ratones , Norovirus/fisiología , Fosfolípidos/metabolismo , Receptores Inmunológicos/metabolismo , Virosis/metabolismo , Virosis/virologíaRESUMEN
BACKGROUND: Basophils express high-affinity IgE receptors (FcεRI), which play an essential role in allergic diseases. It is important to characterize new cell-surface receptors that modulate IgE-mediated basophil activation threshold to design promising immunomodulatory therapies. OBJECTIVES: We sought to analyze the expression of CD300 receptors on human basophils and their implication in IgE-mediated basophil activation processes. METHODS: Blood samples from healthy subjects and patients with cow's milk allergy were collected through the Basque Biobank under an institutional review board-approved protocol. PBMCs were obtained by means of density centrifugation, basophils were purified with a specific isolation kit, and phenotypic and functional studies were performed by using flow cytometry. RESULTS: We demonstrate that basophils express the activating receptor CD300c, which is specifically upregulated in response to IL-3. CD300c works as a costimulatory molecule during IgE-mediated basophil activation, as shown by a significant increase in degranulation and cytokine production when basophils are activated in the presence of CD300c cross-linking compared with activation through the IgE/FcεRI axis alone. Coligation of FcεRI and CD300c increased intracellular calcium mobilization and phosphorylation of signaling intermediates evoked only by FcεRI ligation. We show that the natural ligands of CD300c, phosphatidylserine and phosphatidylethanolamine, modulate IgE-mediated basophil activation. Furthermore, we have observed that CD300c expression in children with cow's milk allergy is increased compared with that in healthy control subjects and that the intensity of expression correlates with the severity of the hypersensitivity symptoms. CONCLUSION: CD300c could be considered a biomarker and therapeutic target in patients with IgE-mediated allergic diseases because it seems to be involved in the modulation of IgE-mediated basophil activation.
Asunto(s)
Antígenos de Superficie/metabolismo , Basófilos/inmunología , Glicoproteínas de Membrana/metabolismo , Hipersensibilidad a la Leche/inmunología , Adolescente , Alérgenos/inmunología , Animales , Antígenos de Superficie/genética , Biomarcadores/metabolismo , Bovinos , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Inmunoglobulina E/metabolismo , Lactante , Masculino , Glicoproteínas de Membrana/genética , Proteínas de la Leche/inmunología , Receptor Cross-Talk , Transducción de Señal , Regulación hacia ArribaRESUMEN
Allergy is the host immune response against non-infectious substances called allergens. The prevalence of allergic diseases is increasing worldwide. However, while some drugs counteract the symptomatology caused by allergic reactions, no completely effective treatments for allergic diseases have been developed yet. In this sense, the ability of surface activating and inhibitory receptors to modulate the function of the main effector cells of allergic responses makes these molecules potential pharmacological targets. The CD300 receptor family consists of members with activating and inhibitory capabilities mainly expressed on the surface of immune cells. Multiple studies in the last few years have highlighted the importance of CD300 molecules in several pathological conditions. This review summarizes the literature on CD300 receptor expression, regulation and function in mast cells, basophils and eosinophils, the main players of allergic responses. Moreover, we review the involvement of CD300 receptors in the pathogenesis of certain allergic diseases, as well as their prospective use as therapeutic targets for the treatment of IgE-dependent allergic responses.
Asunto(s)
Hipersensibilidad/metabolismo , Mastocitos/metabolismo , Animales , Basófilos/inmunología , Basófilos/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/metabolismo , Mastocitos/inmunologíaRESUMEN
Fc receptor-like (FCRL) 5 is a novel IgG binding protein expressed on B cells, with the capacity to regulate Ag receptor signaling. We assessed FCRL5 expression on circulating B cells from healthy donors and found that FCRL5(+) cells are most enriched among atypical CD21(-/lo)/CD27(-) tissue-like memory (TLM) B cells, which are abnormally expanded in several autoimmune and infectious diseases. Using multicolor flow cytometry, FCRL5(+) TLM cells were found to express more CD11c and several inhibitory receptors than did the FCRL5(-) TLM subset. The homing receptor profiles of the two TLM subsets shared features consistent with migration away from lymphoid tissues, but they also displayed distinct differences. Analysis of IgH V regions in single cells indicated that although both subsets are diverse, the FCRL5(+) subset accumulated significantly more somatic mutations. Furthermore, the FCRL5(+) subset had more switched isotype expression and more extensive proliferative history. Microarray analysis and quantitative RT-PCR demonstrated that the two TLM subsets possess distinct gene expression profiles, characterized by markedly different CD11c, SOX5, T-bet, and RTN4R expression, as well as differences in expression of inhibitory receptors. Functional analysis revealed that the FCRL5(+) TLM subset responds poorly to multiple stimuli compared with the FCRL5(-) subset, as reflected by reduced calcium mobilization and blunted cell proliferation. We propose that the FCRL5(+) TLM subset, but not the FCRL5(-) TLM subset, underwent Ag-driven development and is severely dysfunctional. The present study elucidates the heterogeneity of TLM B cells and provides the basis to dissect their roles in the pathogenesis of inflammatory and infectious diseases.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Células Sanguíneas/inmunología , Infecciones/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores Fc/metabolismo , Señalización del Calcio , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Análisis Mutacional de ADN , Regulación de la Expresión Génica , Humanos , Cambio de Clase de Inmunoglobulina , Memoria Inmunológica , Receptores Fc/genética , Análisis de Matrices TisularesRESUMEN
The CD300a inhibitory receptor belongs to the CD300 family of cell surface molecules that regulate a diverse array of immune cell processes. The inhibitory signal of CD300a depends on the phosphorylation of tyrosine residues embedded in ITIMs of the cytoplasmic tail. CD300a is broadly expressed on myeloid and lymphoid cells, and its expression is differentially regulated depending on the cell type. The finding that CD300a recognizes phosphatidylserine and phosphatidylethanolamine, two aminophospholipids exposed on the outer leaflet of dead and activated cells, has shed new light on its role in the modulation of immune functions and in its participation in the host response to several diseases states, such as infectious diseases, cancer, allergy, and chronic inflammatory diseases. This review summarizes the literature on CD300a expression, regulation, signaling pathways, and ligand interaction, as well as its role in fine tuning immune cell functions and its clinical relevance.
Asunto(s)
Antígenos CD/metabolismo , Motivo de Inhibición del Inmunorreceptor Basado en Tirosina/genética , Fosfatidiletanolaminas/antagonistas & inhibidores , Fosfatidilserinas/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Humanos , Ligandos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Transducción de Señal , Virosis/genética , Virosis/metabolismoRESUMEN
The CD300 family of molecules modulates a broad and diverse array of immune cell processes via their paired activating and inhibitory receptor functions. The description that CD300 molecules are able to recognize lipids, such as extracellular ceramide, phosphatidylserine, and phosphatidylethanolamine, that are exposed on the outer leaflet of the plasma membrane of dead and activated cells has opened a new field of research. Through their binding to lipids and other ligands, this family of receptors is poised to have a significant role in complex biological processes and in the host response to severe pathological conditions. Indeed, published data have demonstrated their participation in the pathogenesis of several disease states. Moreover, this family of receptors has great potential as targets for diagnosis and therapeutic purposes in infectious diseases, allergies, cancer, and other pathological situations. For instance, one member of the family, CD300a, has been studied as a possible biomarker. Here, a review is provided on the cellular distribution of the human and mouse families of receptors, the stimuli that regulate their expression, their ability to tune leukocyte function and immune responses, their signaling pathways, ligand recognition, and their clinical relevance.
Asunto(s)
Antígenos CD/fisiología , Sistema Inmunológico/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/fisiología , Ligandos , Ratones , Modelos Biológicos , Familia de Multigenes/fisiología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Transducción de Señal/inmunologíaRESUMEN
CD16 (FcγRIIIa), the low-affinity receptor for IgG, expressed by the majority of human NK cells, is a potent activating receptor that facilitates Ab-dependent cell-mediated cytotoxicity (ADCC). ADCC dysfunction has been linked to cancer progression and poor prognosis for chronic infections, such as HIV; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. Importantly, CD16 cell-surface expression is downmodulated following NK cell activation and, in particular, exposure to stimulatory cytokines (IL-2 or IL-15), likely owing to the action of matrix metalloproteinases (MMPs). In this article, we identify membrane-type 6 (MT6) MMP (also known as MMP25) as a proteinase responsible for CD16 downmodulation. IL-2-induced upregulation of MT6/MMP25 cell-surface expression correlates with CD16 downmodulation. MT6/MMP25, sequestered in intracellular compartments in unstimulated NK cells, translocates to the cell surface after stimulation; moreover, it polarizes to the effector-target cell interface of the CD16-mediated immunological synapse. siRNA-mediated disruption of MT6/MMP25 expression enhances the ADCC capacity of NK cells, emphasizing the important functional role of MT6/MMP25 in the regulation of ADCC activity. Thus, this study uncovers a previously unknown role of MT6/MMP25 in human NK cells, and suggests that inhibition of MT6/MMP25 activity could improve ADCC efficacy of therapeutically administered NK cells that require IL-2 for culture and expansion.
Asunto(s)
Sinapsis Inmunológicas , Interleucina-2/farmacología , Células Asesinas Naturales/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana/fisiología , Receptores de IgG/biosíntesis , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Comunicación Celular , Compartimento Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Polaridad Celular , Células Cultivadas , Dipéptidos/farmacología , Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-15/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/ultraestructura , Activación de Linfocitos/efectos de los fármacos , Metaloproteinasas de la Matriz Asociadas a la Membrana/biosíntesis , Metaloproteinasas de la Matriz Asociadas a la Membrana/genética , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores de IgG/genética , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
FcR specific for pentameric IgM (FCMR) is expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity, and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice that lack FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells, and responsiveness to antigenic challenge.
Asunto(s)
Antígenos/inmunología , Linfocitos B/citología , Inmunoglobulina M/inmunología , Síndromes de Inmunodeficiencia/inmunología , Linfopoyesis/inmunología , Receptores Fc/inmunología , Animales , Formación de Anticuerpos/inmunología , Apoptosis/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Biopolímeros , Médula Ósea/inmunología , Médula Ósea/patología , Centro Germinal/patología , Homeostasis/inmunología , Inmunización , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Peritoneo/inmunología , Peritoneo/patología , Células Plasmáticas/patología , ARN Mensajero/biosíntesis , Receptores de Antígenos de Linfocitos B/inmunología , Receptores Fc/biosíntesis , Receptores Fc/deficiencia , Receptores Fc/genética , Bazo/inmunología , Bazo/patología , Linfocitos T/inmunologíaRESUMEN
CD300a is an immunoreceptor tyrosine-based inhibitory motif (ITIM) containing molecule that belongs to the CD300 family of paired activating/inhibitory receptors. It has been shown that its ligation inhibits activation signals on cells of both myeloid and lymphoid lineages. The ligands for CD300a have not been identified. Here, we show that a CD300a-Ig fusion protein specifically binds to apoptotic cells that are evolutionary apart, such as human and insect cells, suggesting that the ligand has to be conserved. Using surface plasmon resonance, ultracentrifugation, ELISA, and reporter cell assays, we identified phosphatidylethanolamine (PE) and phosphatidylserine (PS), 2 phospholipids that translocate to the outer leaflet of the plasma membrane of dead cells, as the ligands for CD300a. Mutational and structural modeling studies identified residues that are involved in the binding of CD300a to PE and PS and that form a cavity where the hydrophilic heads of PE and PS, can penetrate. CD300a down-regulates the uptake of apoptotic cells by macrophages and its ectopic expression in CD300a-negative cell lines also decreased the engulfment of dead cells. Collectively, our results indicate that PE and PS are ligands for CD300a, and that this interaction plays an important role in regulating the removal of dead cells.
Asunto(s)
Antígenos CD/química , Antígenos CD/metabolismo , Fagocitosis/fisiología , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Muerte Celular , Citometría de Flujo , Células HEK293 , Humanos , Ligandos , Macrófagos/inmunología , Macrófagos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de Superficie , UltracentrifugaciónRESUMEN
BACKGROUND: Sudden death (SD) constitutes one of the principal causes of death and is an important problem in healthcare provision. Cardiovascular diseases have a high prevalence in dialysis patients and constitute the principal cause of death. We sought to analyze retrospectively the incidence of SD in patients commencing dialysis and the factors related to its presence. METHODS: We evaluated all the patients who began dialysis in our center between 1/11/2003 and 15/9/2007, and who were followed up until death, transplant, or study completion on 31/12/2012. We determined the presence of SD according to the following criteria: SD at 24 h (SD 24H): unexpected death occurring in the 24 h following the start of symptoms, or when the patient was found dead and had been seen alive 24 h earlier; SD at 1 h (SD 1H): death witnessed as occurring in the first hour following the start of symptoms. RESULTS: We evaluated 285 patients, mean age 65.67 ± 15.7 years. In a follow-up of 39.9 ± 34.2 months (947.6 patient-years of follow-up) 168 died (59%), 28 (10%) patients presented SD 24H (2.9/100 patient-years), and 16 (6%) patients presented SD 1H (1.7/100 patient-years). In the multivariate analysis, having had a myocardial infarction or having had electrocardiographic abnormalities (Q wave, negative T wave, subendocardial lesion or QRS >120 ms) were the principal independent predictors of SD 24H (OR 7.83; 95% CI 2.20-27.86; p = 0.001) and of SD 1H (OR 13.43; 95% CI 1.56-115.42; p = 0.018). CONCLUSIONS: SD on dialysis is very frequent. Two groups can be identified easily, with risk profiles clearly differentiated.
Asunto(s)
Muerte Súbita/epidemiología , Fallo Renal Crónico/mortalidad , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Incidencia , Fallo Renal Crónico/terapia , Masculino , Persona de Mediana Edad , Diálisis Renal , Estudios Retrospectivos , España/epidemiologíaRESUMEN
We find that the cell surface receptor Toso is dramatically downregulated by in vitro stimulation of human T and NK cells with IL-2 in a STAT5-dependent manner. The fact that IL-2 is known to prime NK and T cells for Fas/TNF-mediated activation-induced cell death (AICD) fits nicely with the original and recent descriptions of Toso as an inhibitor of Fas/TNF-induced apoptosis. In support of this possibility, effector memory T cells express markedly lower levels of Toso than those of naive T cells, indicating that activation in vivo correlates with the downregulation of Toso. Moreover, in vitro activation of memory T cells through TCR dramatically downregulates Toso expression compared with that of naive CD4 T cells. However, overexpression of Toso in human NK cells and Jurkat T cells does not inhibit Fas-mediated apoptosis, and, in agreement with other recent reports, Toso clearly functions as an IgM receptor. Unlike CD16, Toso expression by NK cells does not convey cytotoxic potential, but its ligation does trigger intracellular signaling in NK cells. In summary, our data indicate that Toso is a functional IgM receptor that is capable of activating signaling molecules, is regulated by IL-2, and is not inherently an antiapoptotic molecule.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Interleucina-2/fisiología , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/metabolismo , Receptores Fc/metabolismo , Subgrupos de Linfocitos T/inmunología , Apoptosis/inmunología , Células HEK293 , Humanos , Células Jurkat , Células Asesinas Naturales/metabolismo , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptor fas/antagonistas & inhibidores , Receptor fas/fisiologíaRESUMEN
Cross-linking of the collagen binding receptor leukocyte-associated Ig-like receptor-1 (LAIR-1) in vitro delivers an inhibitory signal that is able to downregulate activation-mediated signals. To study the in vivo function of LAIR-1, we generated LAIR-1(-/-) mice. They are healthy and fertile and have normal longevity; however, they show certain phenotypic characteristics distinct from wild-type mice, including increased numbers of splenic B, regulatory T, and dendritic cells. As LAIR-1(-/-) mice age, the splenic T cell population shows a higher frequency of activated and memory T cells. Because LAIR-1(+/+) and LAIR-1(-/-) T cells traffic with equal proficiency to peripheral lymphoid organs, this is not likely due to abnormal T lymphocyte trafficking. LAIR-1(-/-) mice have lower serum levels of IgG1 and, in response to T-dependent immunization with trinitrophenyl-OVA, switch less efficiently to Ag specific IgG2a and IgG2b, whereas switching to IgG1 is not affected. Several mouse disease models, including experimental autoimmune encephalitis and colitis, were used to examine the effect of LAIR-1 deficiency, and no differences in the responses of LAIR-1(-/-) and LAIR-1(+/+) mice were observed. Taken together, these observations indicate that LAIR-1 plays a role in regulating immune cells and suggest that any adverse effects of its absence may be balanced in vivo by other inhibitory receptors.
Asunto(s)
Inmunofenotipificación , Leucocitos/inmunología , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Inmunofenotipificación/métodos , Leucocitos/citología , Leucocitos/metabolismo , Longevidad/genética , Longevidad/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores Inmunológicos/fisiología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Renal injury often occurs as a complication in autoimmune diseases such as systemic lupus erythematosus (SLE). It is estimated that a minimum of 20% SLE patients develop lupus nephritis, a condition that can be fatal when the pathology progresses to end-stage renal disease. Studies in animal models showed that incidence of immune cell infiltrates in the kidney was linked to pathological injury and correlated with severe lupus nephritis. Thus, preventing immune cell infiltration into the kidney is a potential approach to impede the progression to an end-stage disease. A requirement to investigate the role of kidney-infiltrating leukocytes is the development of reproducible and efficient protocols for purification and characterization of immune cells in kidney samples. This chapter describes a detailed methodology that discriminates tissue-resident leukocytes from blood-circulating cells that are found in kidney. Our protocol was designed to maximize cell viability and to reduce variability among samples, with a combination of intravascular staining and magnetic bead separation for leukocyte enrichment. Experiments included as example were performed with FcγRIIb[KO] mice, a well-characterized murine model of SLE. We identified T cells and macrophages as the primary leukocyte subsets infiltrating into the kidney during severe nephritis, and we extensively characterized them phenotypically by flow cytometry.
Asunto(s)
Modelos Animales de Enfermedad , Riñón , Leucocitos , Nefritis Lúpica , Animales , Nefritis Lúpica/patología , Nefritis Lúpica/inmunología , Ratones , Riñón/patología , Leucocitos/inmunología , Leucocitos/patología , Separación Celular/métodos , Ratones Noqueados , Macrófagos/inmunología , Macrófagos/patología , Citometría de Flujo/métodos , Linfocitos T/inmunología , Receptores de IgG/metabolismoRESUMEN
The immunomodulatory receptor CD300a is expressed on human B cells. Naive B cells express very low levels of this receptor, whereas memory B cells and plasmablasts/cells express variable levels of CD300a. Germinal center B cells are negative for CD300a expression. Stimulation of naive B cells via B-cell receptor (BCR) and Toll-like receptor 9, along with T-cell help, failed to up-regulate CD300a cell surface expression despite the increased expression of the memory marker CD27 and the down-regulation of CD305. However, Toll-like receptor 9 stimulation alone significantly increased CD300a expression on memory B cells, whereas interleukin-4 and transforming growth factor-ß1 act as negative regulators of CD300a expression on memory B cells. Coligation of BCR and CD300a inhibits Ca(2+) mobilization and nuclear factor of activated T cell transcriptional activity evoked by BCR ligation alone. Suppression of CD300a expression in primary B cells with siRNA resulted in increased BCR-mediated proliferation, thereby confirming the inhibitory capacity of CD300a. Finally, we show that CD300a expression levels are significantly down-regulated in the circulating B cells of HIV-infected patients. Altogether, these data demonstrate a novel mechanism for suppressing the activity of B cells and suggest a potential role for CD300a in the B-cell dysfunction observed in HIV-induced immunodeficiency.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/inmunología , Infecciones por VIH/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores Inmunológicos/metabolismo , Antígenos CD/inmunología , Linfocitos B/citología , Linfocitos B/metabolismo , Western Blotting , Células Cultivadas , Progresión de la Enfermedad , Regulación hacia Abajo , Citometría de Flujo , VIH/inmunología , Infecciones por VIH/metabolismo , Humanos , Memoria Inmunológica , Luciferasas/metabolismo , Factores de Transcripción NFATC/genética , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores Inmunológicos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Natural killer (NK) cells help protect the host against viral infections and tumors. NKG2D is a vital activating receptor, also expressed on subsets of T cells, whose ligands are up-regulated by cells in stress. Ligation of NKG2D leads to phosphorylation of the associated DAP10 adaptor protein, thereby activating immune cells. Understanding how the expression of NKG2D-DAP10 is regulated has implications for immunotherapy. We show that IL-2 and TGF-ß1 oppositely regulate NKG2D-DAP10 expression by NK cells. IL-2 stimulation increases NKG2D surface expression despite a decrease in NKG2D mRNA levels. Stimulation with IL-2 results in a small increase of DAP10 mRNA and a large up-regulation of DAP10 protein synthesis, indicating that IL-2-mediated effects are mostly posttranscriptional. Newly synthesized DAP10 undergoes glycosylation that is required for DAP10 association with NKG2D and stabilization of NKG2D expression. TGF-ß1 has an opposite and dominant effect to IL-2. TGF-ß1 treatment decreases DAP10, as its presence inhibits the association of RNA polymerase II with the DAP10 promoter, but not NKG2D mRNA levels. This leads to the down-regulation of DAP10 expression and, as a consequence, NKG2D protein as well. Finally, we show that other γ(c) cytokines act similarly to IL-2 in up-regulating DAP10 expression and NKG2D-DAP10 surface expression.
Asunto(s)
Membrana Celular/metabolismo , Citocinas/fisiología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptores Inmunológicos/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Membrana Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Citocinas/farmacología , Antagonismo de Drogas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-2/farmacología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/fisiología , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores Inmunológicos/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Reportedly, CD300f negatively regulates interactions between dendritic and T cells and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines, an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, cosedimentation, and surface plasmon resonance using phospholipid-containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.
Asunto(s)
Antígenos CD/inmunología , Fagocitosis/inmunología , Fosfatidilserinas/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Animales , Apoptosis/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Natural killer (NK) cells are part of the innate immune system, the classic cytotoxic population of innate lymphoid cells (ILCs). They can directly kill virus-infected or tumor cells through different mechanisms without prior sensitization using their lytic functions in response to different signals (target cell ligands and/or inflammatory cytokines) and secreting cytokines, such as interferon gamma (IFNγ) and tumor necrosis factor (TNF). NK cells use antibody-dependent cell-mediated cytotoxicity (ADCC) to recognize and kill cells expressing target antigens when they are antibody coated. Redirected cytotoxicity is a technique used to target cells that do not per se activate NK cells. Here, we use redirected degranulation, a surrogate technique that correlates with redirected lysis. The P815 cell line (mouse mastocytoma) express fragment crystallizable gamma receptor II (FcγRII) and therefore could bind the Fc portion of mouse IgG antibodies, which through their fragment antigen-binding (Fab) may recognize NK cells activating receptors leading to target cell lysis. This technique could be used to determine the inhibitory or activating capacity of different receptors or isoforms and in immunotherapy using T cell and NK cell activators.
Asunto(s)
Inmunidad Innata , Células Asesinas Naturales , Humanos , Ratones , Animales , Células Asesinas Naturales/metabolismo , Citocinas/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Línea CelularRESUMEN
Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memory-like (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy.