RESUMEN
Non-immune cells are increasingly recognized as important in regulating immunity, but the role of red blood cells (RBC) remains relatively unexplored, despite their abundance in the circulation and a cell surface rich in potential ligands. Here, we determine whether RBC influence the activation state of human B cells. Separation of RBC from peripheral blood mononuclear cells increased B-cell expression of HLA-DR/DP/DQ, whilst reconstitution reduced the levels of B-cell activation markers HLA-DR/DP/DQ, CD86, CD69 and CD40, as well as decreasing proliferative responses and IgM secretion. Inhibition of B cells required contact with RBC and was abrogated by either removal of sialic acids from RBC or blocking the corresponding lectin receptor CD22 on B cells. Chronic lymphocytic leukaemia B cells express low levels of CD22 and were less susceptible to inhibition by RBC, which may contribute to their activated phenotype. Taken together, the results identify a novel mechanism that may suppress inappropriate responsiveness of healthy B cells whilst circulating in the bloodstream.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Linfocitos B/inmunología , Eritrocitos/inmunología , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos CD40/metabolismo , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Inmunoglobulina M/metabolismo , Lectinas Tipo C/metabolismo , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Ácidos Siálicos/metabolismo , Regulación hacia ArribaRESUMEN
DISCLOSURES: Vickers: University of Aberdeen: Patents & Royalties: About to apply for patent. Barker: University of Aberdeen: Employment, Patents & Royalties: About to apply for patent. Cao: University of Aberdeen: Patents & Royalties: About to apply for patent.
RESUMEN
Platelet destruction in immune thrombocytopenia is caused by autoreactive antibody and T-cell responses, most commonly directed against platelet glycoprotein IIb/IIIa. Loss of self-tolerance in the disease is also associated with deficient activity of regulatory T cells. Having previously mapped seven major epitopes on platelet glycoprotein IIIa that are recognized by helper T cells from patients with immune thrombocytopenia, the aim was to test whether peptide therapy with any of these sequences, alone or in combination, could inhibit responses to the antigen in humanized mice expressing HLA-DR15. None of the individual peptides, delivered by a putative tolerogenic regimen, consistently suppressed the antibody response to subsequent immunization with human platelet glycoprotein IIb/IIIa. However, the combination of glycoprotein IIIa peptides aa6-20 and aa711-725, which contain the predominant helper epitopes in patients and elicited the strongest trends to suppress when used individually, did abrogate this response. The peptide combination also blunted, but did not reverse, the ongoing antibody response when given after immunization. Suppression of antibody was associated with reduced splenocyte T-cell responsiveness to the antigen, and with the induction of a regulatory T-cell population that is more responsive to the peptides than to purified platelet glycoprotein IIb/IIIa. Overall, these data demonstrate that combinations of peptides containing helper epitopes, such as platelet glycoprotein IIIa aa6-20 and aa711-725, can promote in vivo suppression of responses to the major antigen implicated in immune thrombocytopenia. The approach offers a promising therapeutic option to boost T-cell regulation, which should be taken forward to clinical trials.
Asunto(s)
Formación de Anticuerpos/inmunología , Antígenos HLA/inmunología , Inmunoterapia/métodos , Fragmentos de Péptidos/administración & dosificación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Púrpura Trombocitopénica Idiopática/terapia , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Formación de Anticuerpos/efectos de los fármacos , Epítopos/inmunología , Humanos , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Púrpura Trombocitopénica Idiopática/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Macrophages are key in orchestrating immune responses to micro-environmental stimuli, sensed by a complex set of surface receptors. The human cell line THP-1 has a monocytic phenotype, including the ability to differentiate into macrophages, providing a tractable, standardised surrogate for human monocyte-derived macrophages. Here we assessed the expression of 49 surface markers including Fc, complement, C-type lectin and scavenger receptors; TIMs; Siglecs; and co-stimulatory molecules by flow cytometry on both THP-1 monocytes and macrophages and following macrophage activation with seven standard conditioning/polarizing stimuli. Of the 34 surface markers detected on macrophages, 18 altered expression levels on activation. From these, expression of 9 surface markers were consistently altered by all conditioning regimens, while 9 were specific to individual polarizing stimuli. This study provides a resource for the study of macrophages and highlights that macrophage polarization states share much in common and the differences do not easily fit a simple classification system.
Asunto(s)
Diferenciación Celular/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Células THP-1/inmunología , Biomarcadores/sangre , Línea Celular , Proteínas del Sistema Complemento/inmunología , Humanos , Lectinas Tipo C/inmunología , Activación de Macrófagos/inmunologíaRESUMEN
Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4+ T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4+ Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.
Asunto(s)
Eosinófilos/metabolismo , Mycobacterium tuberculosis/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Presentación de Antígeno , Antígenos Dermatofagoides/inmunología , Antígenos Dermatofagoides/metabolismo , Antígenos de Plantas/inmunología , Antígenos de Plantas/metabolismo , Proteínas de Artrópodos/inmunología , Proteínas de Artrópodos/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Citocinas/metabolismo , Eosinófilos/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Activación de Linfocitos , Phleum , Pyroglyphidae , Tuberculina/inmunología , Tuberculina/metabolismoRESUMEN
Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3(+) regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3(+) Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4(+) Foxp3(+) population that correlated positively with fungal burden. Depletion from Foxp3(hCD2) reporter mice in vivo confirmed that Foxp3(+) cells exacerbated fungal burden and inflammatory renal disease. The CD4(+) Foxp3(+) population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3(-) cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt, and secreting IL-17A. The expanded Foxp3(+) T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3(+) T cells promotes Th17-cell responses that drive pathology.
Asunto(s)
Candida albicans/inmunología , Candidiasis/inmunología , Proliferación Celular , Factores de Transcripción Forkhead/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Candida albicans/fisiología , Candidiasis/microbiología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Interacciones Huésped-Patógeno/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Enfermedades Renales/inmunología , Enfermedades Renales/virología , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Bazo/virología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/microbiología , Células Th17/metabolismo , Células Th17/microbiologíaRESUMEN
Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3(hi) -expressing, but not SOCS1(hi) -expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1ß, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages.
Asunto(s)
Activación de Macrófagos/inmunología , Macrófagos/inmunología , Nefritis/inmunología , Peritonitis/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Enfermedad Aguda , Animales , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Silenciador del Gen , Humanos , Activación de Macrófagos/genética , Macrófagos/patología , Masculino , FN-kappa B/genética , FN-kappa B/inmunología , Nefritis/genética , Nefritis/patología , Óxido Nítrico/genética , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Peritonitis/genética , Peritonitis/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Ratas , Ratas Sprague-Dawley , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
CTLA-4 is a crucial immune regulator that mediates both negative costimulation signals to T cells, and regulatory T (Treg)-cell extrinsic control of effector responses. Here we present evidence supporting a novel mechanism for this extrinsic suppression, executed by the alternatively spliced soluble CTLA-4 isoform (sCTLA-4). Analyses of human T cells in vitro show that sCTLA-4 secretion can be increased during responses, and has potent inhibitory properties, since isoform-specific blockade of its activity significantly increased Ag-driven proliferation and cytokine (IFN-γ, IL-17) secretion. Treg cells were demonstrated to be a prominent source of sCTLA-4, which contributed to suppression in vitro when their numbers were limiting. The soluble isoform was also produced by, and inhibited, murine T cells responding to Ag in vitro, and blockade of its activity in vivo protected against metastatic spread of melanoma in mice. We conclude that sCTLA-4 is an important immune regulator, responsible for at least some of the inhibitory effects previously ascribed to the membrane-bound isoform. These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antígeno CTLA-4/inmunología , Melanoma Experimental/tratamiento farmacológico , Metástasis de la Neoplasia/prevención & control , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/genética , Células Cultivadas , Femenino , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Solubilidad , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
The K blood group remains an important target in hemolytic disease of the newborn (HDN), with no immune prophylaxis available. The aim was to characterize the Th response to K as a key step in designing specific immunotherapy and understanding the immunogenicity of the Ag. PBMCs from K-negative women who had anti-K Abs after incompatible pregnancy, and PBMCs from unimmunized controls, were screened for proliferative responses to peptide panels spanning the K or k single amino acid polymorphism. A dominant K peptide with the polymorphism at the C terminus elicited proliferation in 90% of alloimmunized women, and it was confirmed that responding cells expressed helper CD3(+)CD4(+) and "memory" CD45RO(+) phenotypes, and were MHC class II restricted. A relatively high prevalence of background peptide responses independent of alloimmunization may contribute to K immunogenicity. First, cross-reactive environmental Ag(s) pre-prime Kell-reactive Th cells, and, second, the K substitution disrupts an N-glycosylation motif, allowing the exposed amino acid chain to stimulate a Th repertoire that is unconstrained by self-tolerance in K-negative individuals. The dominant K peptide was effective in inducing linked suppression in HLA-transgenic mice and can now be taken forward for immunotherapy to prevent HDN because of anti-K responses.
Asunto(s)
Epítopos de Linfocito T/inmunología , Sistema del Grupo Sanguíneo de Kell/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Secuencia de Aminoácidos , Proliferación Celular , Células Cultivadas , Femenino , Glicosilación , Antígenos HLA/inmunología , Humanos , Factores Inmunológicos/inmunología , Sistema del Grupo Sanguíneo de Kell/química , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Embarazo , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.
Asunto(s)
Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Antígenos de Histocompatibilidad/genética , Fragmentos de Péptidos/inmunología , Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Adulto , Animales , Femenino , Humanos , Epítopos Inmunodominantes/administración & dosificación , Epítopos Inmunodominantes/inmunología , Inmunoterapia , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Fragmentos de Péptidos/administración & dosificación , Sistema del Grupo Sanguíneo Rh-Hr/química , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Adulto JovenRESUMEN
CD22, an inhibitory co-receptor of the BCR, has been identified as a potential candidate gene for the development of autoimmune haemolytic anaemia in mice. In this study, we have examined Cd22(tm1Msn) CD22-deficient mice and identified an increase in RBC turnover and stress erythropoiesis, which might be consistent with haemolysis. We then, however, eliminated CD22 deficiency as the cause of accelerated RBC turnover and established that enhanced RBC turnover occurs independently of B cells and anti-RBC autoanti-bodies. Accelerated RBC turnover in this particular strain of CD22-deficient mice is red cell intrinsic and appears to be the consequence of a defective allele of glucose phosphate isomerase, Gpi1(c). This form of Gpi1 was originally derived from wild mice and results in a substantial reduction in enzyme activity. We have identified the polymorphism that causes impaired catalytic activity in the Gpi1(c) allele, and biochemically confirmed an approximate 75% reduction of GPI1 activity in Cd22(-/-) RBCs. The Cd22(-/-).Gpi1(c) congenic mouse provides a novel animal model of GPI1-deficiency, which is one of the most common causes of chronic non-spherocytic haemolytic anaemia in humans.
Asunto(s)
Alelos , Anemia Hemolítica Congénita , Eritrocitos/inmunología , Glucosa-6-Fosfato Isomerasa , Polimorfismo Genético , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Anemia Hemolítica Congénita/genética , Anemia Hemolítica Congénita/inmunología , Animales , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Eritropoyesis/genética , Eritropoyesis/inmunología , Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/inmunología , Ratones , Ratones Endogámicos NZB , Ratones Mutantes , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunologíaRESUMEN
BACKGROUND: Many immune-mediated diseases are associated with low levels of vitamin D and sunlight. UV light or supplementation with vitamin D can increase regulatory T-cell activity and prevent animal models of autoimmune disease. Increasing population vitamin D levels may therefore alleviate the burden of human immune-mediated disease. OBJECTIVE: To determine the responses of circulating 25-hydroxyvitamin D [25(OH)D] levels, regulatory T-cell numbers, and immune function to UV light exposure in patients being treated for skin disease. METHODS: Twenty-four subjects with skin disease from the North of Scotland were recruited between December and March. At baseline, and after 2 and 4 weeks of narrowband UV light exposure, we measured peripheral blood 25(OH)D level, numbers of regulatory T cells (CD4(+)CD25(hi)FoxP3(+)), and T-cell proliferative and cytokine responses to anti-CD3/CD28 stimulation. RESULTS: Median (interquartile range) narrowband UV-B received during the study was 39.1 (30.9) as standard erythema dose, comparable to a quarter of the median summer sunlight exposure received locally. This increased the 25(OH)D level from a mean ± SD of 34 ± 17 nmol/L to 58 ± 16 nmol/L after 2 weeks and 78 ± 19 nmol/L after 4 weeks. The mean proportion of circulating regulatory T cells increased from 0.5% to 1.6% CD3(+) cells, which significantly correlated with the increased 25(OH)D level. UV treatment was also followed by reduced proliferative and IL-10 responses to anti-CD3/CD28 independent of the 25(OH)D level. CONCLUSION: Narrowband UV light reduces systemic immune responsiveness via the induction of regulatory T cells. Light and 25(OH)D levels may affect particular immune functions independently. The levels of serum 25(OH)D over which these effects are apparent should guide future interventions.
Asunto(s)
Inmunidad/efectos de la radiación , Rayos Ultravioleta , Vitamina D/análogos & derivados , Adulto , Anciano , Citocinas/biosíntesis , Femenino , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Estaciones del Año , Luz Solar , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Tiempo , Vitamina D/sangre , Vitamina D/efectos de la radiación , Adulto JovenRESUMEN
BACKGROUND: Interleukin-17A is the signature cytokine of the Th17 subset and drives inflammatory pathology, but its relevance to autoantibody-mediated diseases is unclear. Th1 cells secreting interferon-γ have been implicated in autoimmune hemolytic anemia, so the aim was to determine which cytokine is more closely associated with disease severity. DESIGN AND METHODS: Interferon-γ and interleukin-17A were measured in the sera of patients with autoimmune hemolytic anemia and healthy donors, and in peripheral blood mononuclear cell cultures stimulated with autologous red blood cells, or a panel of peptides spanning red blood cell autoantigen. RESULTS: Serum interleukin-17A, but not interferon-γ, was significantly raised in patients with autoimmune hemolytic anemia (P<0.001), and correlated with the degree of anemia. Interleukin-17A was also more prominent in the responses of peripheral blood mononuclear cells from patients with autoimmune hemolytic anemia to red blood cells, and, again unlike interferon-γ, significantly associated with more severe anemia (P<0.005). There were no interleukin-17A responses to red blood cells by peripheral blood mononuclear cells from healthy donors. Specific autoantigenic peptides were identified that elicit patients' interleukin-17A responses. CONCLUSIONS: Interleukin-17A makes a previously unrecognized contribution to the autoimmune response in autoimmune hemolytic anemia, challenging the model that the disease is driven primarily by Th1 cells. This raises the possibility that Th17, rather than Th1, cells should be the target for therapy.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Susceptibilidad a Enfermedades/inmunología , Interferón gamma/sangre , Interleucina-17/sangre , Adulto , Anciano , Anemia Hemolítica Autoinmune/metabolismo , Autoantígenos/inmunología , Estudios de Casos y Controles , Epítopos/inmunología , Eritrocitos/inmunología , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
In neonatal alloimmune thrombocytopenia, almost all human platelet antigen (HPA)-1b1b mothers who produce anti-HPA-1a antibody through carrying an HPA-1a fetus are human histocompatibility leukocyte antigen (HLA)-DRB3*0101 positive. It is predicted that the HPA-1a Leu(33) polymorphism forms part of an HLA-DRB3*0101-restricted T-helper epitope, and acts as an anchor residue for binding this class II molecule. However, it is not known whether any corresponding peptides are naturally processed and presented from platelet glycoprotein. In this study, peptides displayed by a homozygous HLA-DRB3*0101 antigen-presenting cell line were identified after pulsing with recombinant HPA-1a (Leu(33) plexin-semaphorin-integrin domain). The peptides were eluted from HLA-DR molecules, fractionated by high performance liquid chromatography, and analyzed by tandem mass spectrometry. A "nested set" of naturally presented HPA-1a-derived peptides, each containing the Trp(25)-Leu(33) core epitope, was identified, with the most abundant member being the 16-mer Met(22)-Arg(37). These peptides may provide the basis for novel treatments to tolerize the corresponding T-helper response in women at risk of neonatal alloimmune thrombocytopenia.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Antígenos de Plaqueta Humana/genética , Antígenos HLA-DR/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Células Presentadoras de Antígenos/citología , Antígenos/química , Antígenos de Plaqueta Humana/metabolismo , Cromatografía Líquida de Alta Presión , Epítopos/química , Glutatión Transferasa/metabolismo , Cadenas HLA-DRB3 , Homocigoto , Humanos , Integrina beta3 , Leucina/química , Péptidos/química , Polimorfismo Genético , Triptófano/químicaRESUMEN
Antiphospholipid syndrome is an important cause of recurrent thrombotic events. The pathogenesis of the thrombosis remains unclear, but it has been suggested that anti-phospholipid Abs, which are laboratory markers for the disease and include species capable of binding to vascular endothelial cells, play an important role. We hypothesized that these anti-endothelial Abs promote thrombosis through interference with clearance of dying cells. We show that healthy endothelial cell monolayers effectively remove apoptotic endothelial cells, but this clearance is markedly inhibited by serum or IgG from patients with antiphospholipid syndrome and anti-endothelial Abs. In addition, patient sera or IgG opsonize apoptotic endothelial cells and cause enhanced Fc-mediated uptake by professional phagocytes. Importantly, the delayed clearance of apoptotic cells by healthy endothelial cells and the enhanced Fc-mediated macrophage uptake each result in procoagulant consequences, as judged by increased thrombin generation. The effects on apoptotic cell clearance were reproduced by a mAb derived from a patient with antiphospholipid syndrome, which binds to endothelial cells and is thrombogenic in experimental models. Taken together, our data support a novel, dual mechanism by which anti-endothelial Abs are prothrombotic in antiphospholipid syndrome by inhibiting removal of procoagulant apoptotic cells and by diverting their clearance to provoke inflammatory and prothrombotic changes in professional phagocytes.
Asunto(s)
Anticuerpos Antifosfolípidos/efectos adversos , Síndrome Antifosfolípido/inmunología , Apoptosis/inmunología , Movimiento Celular/inmunología , Endotelio Vascular/inmunología , Trombosis/inmunología , Animales , Anticuerpos Antifosfolípidos/metabolismo , Síndrome Antifosfolípido/metabolismo , Sitios de Unión de Anticuerpos , Factores de Coagulación Sanguínea/metabolismo , Línea Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Hibridomas , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Proteínas Opsoninas/metabolismo , Fagocitosis , Trombosis/metabolismo , Trombosis/patología , Factores de TiempoRESUMEN
Background: Seasonal variations have been reported for immune markers. However, the relative contributions of sunlight and vitamin D variability on such seasonal changes are unknown. Objective: This double-blind, randomized, placebo-controlled trial tested whether daily 400 IU vitamin D3 supplementation affected short-term (12 weeks) and long-term (43 weeks) natural regulatory T cell (nTreg) populations in healthy participants. Design: 62 subjects were randomized equally to vitamin D versus placebo in March and assessed at baseline, April (4w), June (12w), September (25w) and January (43w). Circulating nTregs, ex vivo proliferation, IL-10 and IFN-γ productions were measured. Vitamin D metabolites and sunlight exposure were also assessed. Results: Mean serum 25-hydroxyvitamin D (25(OH)D) increased from 35.8(SD 3.0) to 65.3(2.6) nmol/L in April and remained above 75 nmol/L with vitamin D supplementation, whereas it increased from 36.4(3.2) to 49.8(3.5) nmol/L in June to fall back to 39.6(3.5) nmol/L in January with placebo. Immune markers varied similarly between groups according to the season, but independently of 25(OH)D. For nTregs, the mean (%CD3+CD4+CD127lo cells (SEM)) nadir observed in March (2.9(0.1)%) peaked in September at 4.0(0.2)%. Mean T cell proliferation peaked in June (33156(1813) CPM) returning to the nadir in January (17965(978) CPM), while IL-10 peaked in June and reached its nadir in September (median (IQR) of 262(283) to (121(194) pg/ml, respectively). Vitamin D attenuated the seasonal increase in IFN-γ by ~28% with mean ng/ml (SEM) for placebo vs vitamin D, respectively, for April 12.5(1.4) vs 10.0(1.2) (p=0.02); June 13.9(1.3) vs 10.2(1.7) (p=0.02) and January 7.4(1.1) vs 6.0(1.1) (p=0.04). Conclusions: Daily low dose Vitamin D intake did not affect the nTregs population. There were seasonal variation in nTregs, proliferative response and cytokines, suggesting that environmental changes influence immune response, but the mechanism seems independent of vitamin D status. Vitamin D attenuated the seasonal change in T cell-produced IFN-γ, suggesting a decrease in effector response which could be associated with inflammation. Clinical Trial Registration: https://www.isrctn.com, identifier (ISRCTN 73114576).
Asunto(s)
Proliferación Celular/efectos de los fármacos , Colecalciferol/administración & dosificación , Colecalciferol/inmunología , Interferón gamma/análisis , Estaciones del Año , Linfocitos T Reguladores/inmunología , Adulto , Colecalciferol/sangre , Colecalciferol/farmacología , Suplementos Dietéticos , Método Doble Ciego , Femenino , Humanos , Inflamación/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-10/análisis , Interleucina-10/inmunología , Masculino , Persona de Mediana Edad , Luz Solar , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/fisiologíaRESUMEN
In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Eritrocitos/metabolismo , Manosa/metabolismo , Fagocitos/metabolismo , Polisacáridos/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitología , Eritrocitos/parasitología , Citometría de Flujo/métodos , Hemólisis , Humanos , Ligandos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Glicoproteínas de Membrana/metabolismo , Fagocitosis , Plasmodium falciparum/fisiología , Unión Proteica , Receptores Inmunológicos/metabolismoRESUMEN
Regulatory T (Treg) cells contribute to immune evasion by malignancies. To investigate their importance in non-Hodgkin lymphoma (NHL), we enumerated Treg cells in peripheral blood mononuclear cells (PBMCs) and involved tissues from 30 patients. CD25(+)FoxP3(+)CD127(low)CD4(+) Treg cells were increased markedly in PBMCs (median = 20.4% CD4 T cells, n = 20) versus healthy controls (median = 3.2%, n = 13, P < .001) regardless of lymphoma subtype, and correlated with disease stage and serum lactate dehydrogenase (R(s) = 0.79, P < .001). T-cell hyporesponsiveness was reversed by depleting CD25(+) cells, or by adding anti-CTLA-4, supporting the view that Treg cells explain the systemic immunosuppression seen in NHL. A high proportion of Treg cells was also present in involved tissues (median = 38.8% CD4 T cells, n = 15) versus reactive nodes (median = 11.6%, n = 2, P = .02). When autologous CD25(-) PBMC fractions were incubated with tumor cells from patients (n = 6) in vitro, there was consistent strong induction and then expansion of cells with the CD4(+)CD25(+)FoxP3(+) phenotype of classic "natural" Treg cells. This population was confirmed to be suppressive in function. Direct cell-cell interaction of tumor cells with CD25(-) PBMCs was important in Treg induction, although there was heterogeneity in the mechanisms responsible. We conclude that NHL cells are powerful inducers of Treg cells, which may represent a new therapeutic target.
Asunto(s)
Linfoma no Hodgkin/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Escape del Tumor/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Linfocitos T Reguladores/citologíaRESUMEN
Atherosclerosis is now recognised as a chronic inflammatory disease occurring within the artery wall and ultimately responsible for myocardial infarction, stroke and peripheral vascular disease. A crucial step in atherogenesis is the infiltration of monocytes into the subendothelial space of large arteries where they differentiate into macrophages and become functionally active. Macrophage accumulation within plaques is a hallmark of all stages of atherosclerosis, indeed recent studies have shown their presence has the potential to act as a non-invasive marker of disease activity and plaque stability. Activated macrophages are major players in all stages of lesion development. They not only accumulate lipids but also express effector molecules that are pro-inflammatory, cytotoxic and chemotactic. Furthermore, they secrete enzymes that degrade extracellular matrix leading to plaque destabilisation and increased risk of rupture. However, macrophages are heterogeneous and when appropriately activated they have the potential to drive tissue remodelling and ultimately vascular repair. Pharmacological modulation of macrophage activities therefore represents an important strategy for the prevention and treatment of atherosclerosis and other inflammatory diseases. The aim of this review is to give a brief overview of our current understanding of macrophage activation, distribution and function within inflamed tissue. This will provide the basis for highlighting already available and future methods to exploit specifically activated macrophages as diagnostic and therapeutic targets for atherosclerosis.
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Aterosclerosis/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Macrófagos/efectos de los fármacos , Animales , Aterosclerosis/complicaciones , Aterosclerosis/fisiopatología , Progresión de la Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Humanos , Macrófagos/metabolismo , Monocitos/metabolismoRESUMEN
Autoreactive T cells in patients with Goodpasture's disease are specific for epitopes in the Goodpasture antigen (the NC1 domain of the alpha3 chain of type IV collagen) that are rapidly destroyed during antigen processing to a degree that diminishes their presentation to T cells. We hypothesized that patients' autoreactive T cells exist because antigen processing prevents presentation of the self-epitopes they recognize, circumventing specific tolerance mechanisms. We predicted that autoreactive T cells specific for these peptides should also exist in healthy individuals, albeit at low frequency and in an unprimed state. We obtained blood from healthy unrelated donors and, using a panel of 45 alpha3(IV)NC1 peptides, identified alpha3(IV)NC1-specific T cells in all donors. Thirty-six of 45 peptides elicited a proliferative T cell response from at least one subject, and 6 of the peptides evoked a response in >50% of the individuals. This consistency was not caused by selectivity of HLA class II molecules because the donors expressed a diversity of HLA antigens, but was largely a result of the substrate-specificity of the endosomal proteases Cathepsin D and E. There was a significant correlation between high susceptibility to Cathepsin D digestion and the capacity to stimulate primary T cell responses (P = 0.00006). In summary, healthy individuals have low frequencies of unstimulated alpha3(IV)NC1-reactive T cells with similar specificities to the autoreactive T cells found in patients with Goodpasture disease. In both cases, existence of the alpha3(IV)NC1-reactive T cells can be accounted for by destructive processing.