RESUMEN
Complement C5a, a potent anaphylatoxin, is a candidate target molecule for the treatment of inflammatory diseases, such as myocardial ischemia/reperfusion injury, RA, and the antiphospholipid syndrome. In contrast, up until now, no specific contribution of C5a and its receptor, C5aR, was recognized in diseases of antibody-dependent type II autoimmunity. Here we identify C5a as a novel key mediator of autoimmune hemolytic anemia (AIHA) and show that mice lacking C5aR are partially resistant to this IgG autoantibody-induced disease model. Upon administration of anti-erythrocyte antibodies, upregulation of activating Fcgamma receptors (FcgammaRs) on Kupffer cells, as observed in WT mice, was absent in C5aR-deficient mice, and FcgammaR-mediated in vivo erythrophagocytosis was impaired. Surprisingly, in mice deficient in FcgammaRI and FcgammaRIII, anti-erythrocyte antibody-induced C5 and C5a production was abolished, demonstrating the existence of a previously unidentified FcgammaR-mediated C5a-generating pathway. These results show that the development of a full-blown antibody-dependent autoimmune disease requires C5a--produced by and acting on FcgammaR--and may suggest therapeutic benefits of C5 and/or C5a/C5aR blockade in AIHA and other diseases closely related to type II autoimmune injury.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Autoinmunidad/fisiología , Complemento C5a/inmunología , Inmunoglobulina G/inmunología , Receptor de Anafilatoxina C5a/inmunología , Anemia Hemolítica Autoinmune/genética , Animales , Anticuerpos Monoclonales/inmunología , Complemento C5a/genética , Eritrocitos/metabolismo , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Fagocitosis/fisiología , Receptor de Anafilatoxina C5a/genética , Receptores de IgG , Tasa de SupervivenciaRESUMEN
Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+ CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment.
Asunto(s)
Alérgenos/administración & dosificación , Alérgenos/inmunología , Asma/inmunología , Hiperreactividad Bronquial/inmunología , Receptor de Anafilatoxina C5a/fisiología , Células Th2/inmunología , Animales , Asma/genética , Bronquios/inmunología , Bronquios/metabolismo , Hiperreactividad Bronquial/genética , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Inmunidad Innata/genética , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/fisiología , Pulmón/citología , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Moco/metabolismo , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Th2/metabolismoRESUMEN
Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Inflamación/tratamiento farmacológico , Proteínas de la Membrana/genética , Receptores de Complemento/genética , Animales , Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Humanos , Inflamación/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/inmunología , Receptor de Anafilatoxina C5a , Receptores de Complemento/metabolismoRESUMEN
BACKGROUND: Mast cells (MC) are key effector cells of allergic diseases and resistance to helminthic parasites and induce or amplify diverse innate and adaptive immune responses. The signals controlling MC mobilization during inflammation are not fully understood. RESULTS: Since anaphylatoxins are attractive candidates as MC chemoattractants, we investigated expression and function of anaphylatoxin receptors in murine MC. Precursor cell-derived MC cultured with IL-3 in the presence or absence of SCF did not express significant amounts of surface C5a receptor (C5aR) or C3a receptor (C3aR). MC required approximately 4 h of stimulation with Ag (DNP-albumin, following preincubation with IgE anti-DNP), ionomycin, or PMA to enable a strong chemotactic response towards C5a, paralleled by a distinct C5aR upregulation. Likewise, C5a induced intracellular calcium fluxes solely in activated MC. In contrast, C3a proved to be a weak MC chemotaxin and unable to increase intracellular calcium. Primary peritoneal MC did not express detectable amounts of anaphylatoxin receptors, however, similar to precursor cell-derived MC, stimulation with Ag or ionomycin for 4 h induced a prominent surface expression of C5aR whereas C3aR remained undetectable. CONCLUSION: Collectively, our results suggest that Ag-dependent as well as -independent activation induces an inflammatory MC phenotype which is distinguished by neoexpression of a functional C5aR as a novel effector mechanism in MC-mediated pathogenesis.
Asunto(s)
Mastocitos/metabolismo , Receptor de Anafilatoxina C5a/biosíntesis , Receptores de Complemento/biosíntesis , Animales , Calcio/metabolismo , Diferenciación Celular/inmunología , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Complemento C5a/farmacología , Dinitrofenoles/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Inmunoglobulina E/farmacología , Inflamación/metabolismo , Mastocitos/citología , Mastocitos/efectos de los fármacos , Ratones , Receptor de Anafilatoxina C5a/inmunología , Receptores de Complemento/inmunología , Proteínas Recombinantes/farmacología , Albúmina Sérica Bovina/farmacología , Regulación hacia ArribaRESUMEN
IgG Fc receptors (FcgammaRs, especially FcgammaRIII) and complement (in particular, C5a anaphylatoxin) are critical effectors of the acute inflammatory response to immune complexes (ICs). However, it is unknown whether and how these two key components can interact with each other in vivo. We use here a mouse model of the acute pulmonary IC hypersensitivity reaction to analyze their potential interaction. FcgammaRIII and C5aR are coexpressed on alveolar macrophages (AMs), and both FcgammaRIII and C5aR mutant mice display impaired immune responses. We find that recombinant human C5a (rhC5a) can control inverse expression of various FcgammaRs, and costimulation of ICs with rhC5a results in strong enhancement of FcgammaRIII-triggered cellular activation in vitro and in vivo. Moreover, we show here that early IC-induced bioactive C5a, and its interaction with C5aR, causes induction of activating FcgammaRIII and suppression of inhibitory FcgammaRII on AMs that appears crucial for efficient cytokine production and neutrophil recruitment in lung pathology. Therefore, C5a, which is a potent chemoattractant, has a broader critical function in regulating the inhibitory/activating FcgammaRII/III receptor pair to connect complement and FcgammaR effector pathways in immune inflammation.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Complemento C5a/inmunología , Enfermedades del Complejo Inmune/inmunología , Enfermedades Pulmonares/inmunología , Receptores de IgG/inmunología , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos CD/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Separación Celular , Células Cultivadas , Quimiotaxis/fisiología , Complemento C5a/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Enfermedades del Complejo Inmune/metabolismo , Enfermedades Pulmonares/metabolismo , Macrófagos Alveolares/citología , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/metabolismo , Receptor de Anafilatoxina C5a , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
To assess published evidence of anaphylatoxin receptor expression in renal tubular epithelial cells, monoclonal antibodies (mAbs) against human, mouse and rat receptors for C5a and C3a (C5aR, C3aR) were raised using receptor-expressing transfectants as immunogens. Applying these reagents in immunohistochemistry, we observed that mAbs with reactivities against three distinct epitopes of human C5aR N-terminus recognized tissue macrophages but not at all renal tubular epithelial cells. These findings were surprising, as strong tubular staining had been previously demonstrated by mAbs raised against a synthetic N-terminal C5aR peptide. To extend our study to mammalian kidneys, renal specimens from normal rats as well as LPS-treated Balb/c and MRL/lpr mice, which suffered from lupus-type nephritis, were examined. Similar to humans, mAbs against murine or rat C5aR strongly recognized infiltrating leukocytes in situ whereas tubular epithelial cells remained negative. As a mAb has been previously used to document C3aR expression in renal tubular epithelial cells, kidney specimens were examined using newly established mAbs against different epitopes of human, murine and rat C3aR. In contrast to published evidence, C3aR was detectable exclusively in interstitial leukocytes but not in epithelial tubular cells of normal and diseased tissues. Taken together, our findings question a direct involvement of tubular epithelial cells in anaphylatoxin-mediated renal inflammation. Furthermore, as we demonstrate in the case of anaphylatoxin receptors, cross-reactivities of mAbs may constitute as yet underestimated pitfalls in immunohistochemical antigen detection.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales/metabolismo , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunología , Animales , Línea Celular Tumoral , Células Epiteliales/inmunología , Humanos , Túbulos Renales/citología , Túbulos Renales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratas , Receptor de Anafilatoxina C5a/biosíntesisRESUMEN
The anaphylatoxins C3a and C5a are involved in the pathophysiology of microbial as well as allergic inflammation in the lungs. Besides their expression in leukocytes, receptors for C3a and C5a (C3aR and C5aR) have been noted in alveolar and bronchial epithelial cells, bronchial smooth muscle cells as well as in vascular endothelial and smooth muscle cells of normal and inflamed human and murine lungs. Recently, however, expression of anaphylatoxin receptors in parenchymal cells of the lung (and kidney) has been challenged. Using well-characterized monoclonal antibodies (mabs) against murine and rat anaphylatoxin receptors, we reexamined the pulmonary distribution of C3aR and C5aR. Immunohistochemistry was performed on frozen sections of lung tissues from normal mice and rats as well as from animals subjected to lipopolysaccharide (LPS)-induced inflammation or from MRL/lpr mice suffering from autoimmune disease. Furthermore, ovalbumin (OVA)-induced models of allergic asthma in the rat and mouse were investigated. Prominent expression of both anaphylatoxin receptors was detectable in resident as well as infiltrating leukocytes. No C3aR protein was observed in alveolar macrophages. Upon LPS- and OVA-challenge as well as in autoimmune inflammation, numbers of infiltrating leukocytes expressing prominent amounts of anaphylatoxin receptors increased. Even under these highly inflammatory conditions, however, expression of C3aR and C5aR was not inducible in parenchymal cells. Thus, our findings identify infiltrating leukocytes as a prominent source of anaphylatoxin receptors in inflamed lungs. A direct involvement of parenchymal cells in anaphylatoxin-mediated pulmonary inflammation is unlikely.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Asma/metabolismo , Pulmón/metabolismo , Proteínas de la Membrana/metabolismo , Neumonía/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Asma/inducido químicamente , Asma/patología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Leucocitos/patología , Lipopolisacáridos/toxicidad , Pulmón/efectos de los fármacos , Pulmón/patología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos MRL lpr , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Ovalbúmina/toxicidad , Neumonía/inducido químicamente , Neumonía/patología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Receptor de Anafilatoxina C5a/inmunología , Receptores de Complemento/inmunologíaRESUMEN
Dendritic cells (DC) represent the most potent antigen-presenting cells (APC) of the immune system for their unique capability of presenting antigen to T-cells. Their use as cellular vaccines after charging with antigen ex vivo has been shown to induce protective and therapeutic anti-tumor immunity with regression of tumor manifestations in animal models of experimental cancer therapy. Human monocyte-derived DC (MoDC) generated in vitro in the presence of GM-CSF and IL-4 are regarded equivalent to immature DC. They can be induced to mature under various experimental conditions. MoDC, in their immature as well as mature state have been widely used for experimental as well as for clinical purposes. However, unequivocal proof for the clinical efficiency of MoDC-based anti-tumor vaccinations is still missing. There is now increasing experimental evidence demonstrating that MoDC may be hampered in their ability to migrate in response to inflammatory as well as homeostatic chemataxins. We therefore suggest that MoDC may not represent the equivalent of migratory DC in vivo limiting their use as magic bullets in tumor immunotherapy.
Asunto(s)
Células Dendríticas/inmunología , Inmunoterapia Adoptiva , Monocitos/inmunología , Neoplasias/inmunología , Animales , Presentación de Antígeno/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Factores Quimiotácticos/inmunología , Células Dendríticas/trasplante , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Inflamación/inmunología , Interleucina-4/inmunología , Ratones , Neoplasias/terapia , Linfocitos T/inmunologíaRESUMEN
About 40% of bone marrow-derived dendritic cells (BM-DCs) generated from stem cells of C57BL/6 (B6.WT) mice differentiate in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) without further stimuli to mature DCs. These cells are characterized by high levels of major histocompatibility complex class II, CD40, and CD86 on their surface. Recent studies have revealed that tumor necrosis factor (TNF) is crucial for maturation of BM-DCs. However, once matured, the phenotype of mature TNF-negative C57BL/6 (B6.TNF-/-) and B6.WT BM-DCs is comparable. Both expressed high levels of CD40 and CD86 and were positive for mRNA of the chemokine receptor (CCR)7. To extend our studies, we generated a monoclonal antibody (mAb) specific for mouse CCR7. This mAb allowed us to analyze the surface expression of CCR7 during maturation of B6.WT and B6.TNF-/- BM-DCs in the presence of GM-CSF and stimulated with TNF or lipopolysaccharide (LPS) and to compare it with the CCR7 expression on ex vivo-isolated splenic DCs with or without additional stimulation. Our results showed that CCR7 expression on murine BM-DCs is an indication of cell maturity. Incubation with LPS induced the maturation of all BM-DCs in culture but increased the number of mature CCR7+ splenic DCs only marginally.
Asunto(s)
Células Dendríticas/metabolismo , Receptores de Quimiocina/genética , Animales , Anticuerpos/inmunología , Médula Ósea/metabolismo , Antígeno CD11c/metabolismo , Técnicas de Cultivo de Célula , Ratones , Pruebas de Precipitina , Receptores CCR7 , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/inmunología , Bazo/metabolismoRESUMEN
The anaphylatoxin C5a is a potent proinflammatory stimulus with immunomodulatory activities. Expression of its receptor C5aR (CD88) has been detected on cells of myeloid origin such as granulocytes and monocytes/macrophages. However, controversial results exist on the expression of C5aR on T and B lymphocytes as well as on mature dendritic cells (DC). The aim of the present study was to characterize expression of C5aR protein on myeloid and lymphoid cells in the mouse. For this purpose, rat monoclonal antibodies with specificity against the murine C5aR were generated. Using these reagents a distinct amount of C5aR antigen was observed on neutrophils and macrophages. In contrast, C5aR protein was not detectable on resting or stimulated murine T or B lymphocytes. Furthermore, no C5aR protein could be observed on splenic CD11c positive DC which have been classified in the literature as relatively mature. Taken together, our results suggest that in the mouse expression of C5aR protein may be restricted to leukocytes of myeloid origin whereas previous evidence for C5aR expression on lymphoid cells may be reevaluated.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos/inmunología , Células Mieloides/inmunología , Receptor de Anafilatoxina C5a/análisis , Animales , Células Cultivadas , Complemento C5a/metabolismo , Células Dendríticas/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos MRL lpr , Ratas , Ratas Endogámicas , Receptor de Anafilatoxina C5a/inmunologíaRESUMEN
TNF-negative C57BL/6 (B6.TNF(-/-)) mice are highly susceptible to Leishmania (L.) major infection and succumb rapidly to fatal leishmaniasis. A T helper type 1 (Th1) cell-mediated immune response is central for protective anti-leishmanial immunity. Therefore, the observed susceptibility of B6.TNF(-/-) mice to L. major parasites could be caused by a deficiency in mounting a Th1 response. Analysis of infected footpads revealed, that B6.TNF(-/-) mice exhibited a substantially diminished formation of DCs at the site of infection. Furthermore, Th1 cytokines such as IFN-gamma were reduced in footpads of infected B6.TNF(-/-) mice. Cutaneous reconstitution of B6.TNF(-/-) mice with either bone marrow derived DCs (BM-DCs) or recombinant TNF simultaneous to infection resulted in an increased expression of cytokines such as IFN-gamma and in an enhanced presence of Leishmania-antigen in skin draining lymph nodes. In addition, the individual time of survival was doubled. In conclusion we demonstrate that the expression of dermal TNF is necessary to provide an environment that initiates a local inflammatory response, but is not sufficient to induce protective immunity.
Asunto(s)
Células Dendríticas/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígeno CD11c/análisis , Movimiento Celular/inmunología , Quimiocina CCL21/inmunología , Citometría de Flujo , Pie , Inmunidad Celular , Inflamación/inmunología , Interferón gamma/inmunología , Antígenos Comunes de Leucocito/análisis , Leucocitos/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Piel/inmunologíaRESUMEN
beta-Defensins are natural peptide antibiotics whose immunomodulatory functions are poorly understood. In the present study, macrophages were found to migrate to human beta-defensins (HBD)-1 to -4 using Galpha(i) proteins as well as MAPK ERK, p38 and JNK as signal transducers. In addition, mast cells responded to HBD-1 to -4 with calcium fluxes as well as chemotaxis, which increased upon stimulation with IgE plus antigen or ionomycin. In contrast, human beta-defensins were unable to induce migration of memory lymphocytes and dendritic cells (DC). Similar to HBD, the murine beta-defensin (mBD)-8 mobilized macrophages and lacked the ability to recruit memory T cells. These findings were unexpected as CCR6 on memory T cells and DC has been previously observed to be a receptor for human beta-defensins. In support of our findings, however, RBL-2H3 as well as 300.19 cells stably expressing CCR6 proved to be unresponsive to HBD-2 and -3. Intriguingly, our observation of a PKC-independent homologous desensitization between HBD-1 to -4 suggests a common receptor for HBD. In summary, chemoattraction of macrophages and mast cells is evolutionary conserved within the beta-defensin family despite a considerable sequence variation and distinct antimicrobial activities. However, CCR6 is not a functional receptor for beta-defensins.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Mastocitos/citología , Mastocitos/efectos de los fármacos , beta-Defensinas/farmacología , Animales , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Memoria Inmunológica/inmunología , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores CCR6 , Receptores de Quimiocina/metabolismoRESUMEN
Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated alpha- and beta-defensins. Human alpha-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1-4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although alpha-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Galphai proteins and MAPK as signal transducers. Alpha-defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to beta-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and Gö6976, we observed a PKC-independent functional desensitization to occur between human alpha-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This alpha-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human beta-defensins. Conversely, alpha-defensins desensitized beta-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human alpha-defensin family and tightly regulated by beta-defensins.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Macrófagos/citología , Macrófagos/inmunología , Mastocitos/citología , Mastocitos/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , alfa-Defensinas/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Secuencia Conservada , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Evolución Molecular , Humanos , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Proteínas Quinasas Activadas por Mitógenos/fisiología , Familia de Multigenes/inmunología , Linfocitos T/metabolismo , alfa-Defensinas/químicaRESUMEN
The CC chemokine receptor 7 (CCR7) and its two ligands, CCL21 and CCL19, play an important role in migration of immune cells to lymphoid tissue. To analyze the function of CCR7 in T cell immunity to infectious agents in vivo, transgenic (tg) mice expressing CCL21 in an ubiquitous fashion were generated. These mice contained high amounts of CCL21 in the serum ( approximately 0.3 microg/ml that resulted in CCR7 down-regulation and in a strongly impaired migration of T cells toward CCL21 in vitro. Lymph nodes in CCL21-tg mice were reduced in size but with intact microanatomy and normal distribution of T and B cells. CCL21-tg mice showed a significantly decreased CD8 T cell response to lymphocytic choriomeningitis virus after footpad infection, whereas the response after systemic infection was not altered. Likewise, the CD4 T cell response to footpad infection with Leishmania major was considerably lowered and CCL21-tg mice failed to clear parasites from infected skin. Taken together, these data demonstrate the importance of CCR7 in mediating T cell immunity to viral and parasitic pathogens after local infection.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Quimiocina CCL21/inmunología , Leishmaniasis Cutánea/inmunología , Receptores CCR7/inmunología , Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Quimiocina CCL21/sangre , Quimiocina CCL21/genética , Leishmania major/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CCR7/metabolismoRESUMEN
Infection or immunization induces heterogeneous memory T cell subsets, but their origin and protective value against infection are unclear. In this study, we report the functional characterization of two memory Th subsets, defined by expression of integrin CD49b. Stable CD49b expression is induced in up to one-half of all memory Th cells. More importantly, the CD49b- and CD49b+ subsets display distinct helper activities, typified by the production of IL-10 and TNF-alpha, respectively. Although the inflammatory properties of the CD49b+ subset are protective against intracellular bacterial infection, they are associated with immunopathology in acute viral infection. Modulation of the CD49b-defined memory Th subsets may provide infection type-specific interventions, where either enhancement of the inflammatory response or reduction of immunopathology is essential.
Asunto(s)
Memoria Inmunológica , Integrina alfa2/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/prevención & control , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Citocinas/biosíntesis , Citocinas/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Perfilación de la Expresión Génica , Integrina alfa2/biosíntesis , Integrina alfa2/genética , Linfopenia/inmunología , Linfopenia/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplante , Linfocitos T Colaboradores-Inductores/trasplanteRESUMEN
The presence of plasmacytoid dendritic cells (pDC) was recently demonstrated in lesions of inflammatory skin diseases. Since anaphylatoxins or their precursors were also found in such lesions, we investigated a possible interaction between pDC and anaphylatoxins C3a and C5a. pDC precursors isolated from peripheral blood did not express the receptors for C3a and C5a, complement C3a receptor (C3aR) and complement C3a receptor (C5aR). If these pDC precursors were cultured with IL-3, the resultant immature pDC expressed both receptors. Expression of C3aR and C5aR could also be demonstrated on pDC in lesions of cutaneous lupus erythematosus and allergic contact dermatitis. Such pDC were immature since they lacked the expression of the maturation marker CD83. Blood-derived pDC matured with CpG oligonucleotides downregulated the receptors. Immature pDC responded to C3a and C5a (but not C3adesArg) stimulation with increased F-actin polymerization and chemotactic migration. In contrast, interferon alpha production, surface molecule expression, and T-cell stimulatory capacity were not significantly modulated by C3a or C5a. Thus, immature pDC represent another type of antigen-presenting cell that express C3aR and C5aR, and respond to anaphylatoxins with chemotaxis. This might be relevant in the direction of pDC to cutaneous lesions of inflammation, for example, in lupus erythematosus or contact dermatitis.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Complemento C3a/inmunología , Complemento C5a/inmunología , Células Dendríticas/inmunología , Dermatitis/inmunología , Proteínas de la Membrana/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Anafilatoxinas/inmunología , Anafilatoxinas/farmacología , Células Presentadoras de Antígenos/efectos de los fármacos , Calcio/metabolismo , Quimiotaxis , Complemento C3a/farmacología , Complemento C5a/farmacología , Islas de CpG/inmunología , Células Dendríticas/efectos de los fármacos , Humanos , Interferón-alfa/metabolismo , Ligandos , Proteínas de la Membrana/análisis , Oligonucleótidos/farmacología , Receptor de Anafilatoxina C5a/análisis , Receptores de Complemento/análisis , Linfocitos T/inmunología , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/inmunologíaRESUMEN
The complement fragment-3a (C3a) acts via a G protein-coupled C3aR and is of importance in allergic and inflammatory diseases. Recent studies suggest the presence of complement proteins in the epidermal compartment and synthesis of some of these proteins (C3, factor B, and factor H) by human primary keratinocytes (KCs) during inflammation. However, expression of C3aR and its role in human KCs is not elucidated thus far. In this study, we demonstrate the expression of C3aR on KCs as detected by quantitative real-time RT-PCR and flow cytometry. IFN-gamma and IFN-alpha strongly up-regulated the surface expression of C3aR on KCs among all other cytokines tested. After up-regulation of C3aR by IFN-gamma and IFN-alpha, we observed the induction of five genes (CCL2, CCL5, CXCL8, CXCL10, and C3) after stimulation of KCs with C3a in microarray analysis. We confirmed the induction of C3 and CCL2 at RNA and protein levels. Furthermore, incubation of C3 with skin mast cells tryptase resulted in the generation of C3 fragments with C3a activity. In conclusion, our data illustrate that epidermal KCs express functional C3aR. The increases of C3 and CCL2 synthesis by C3a and C3 activation by skin mast cell tryptase delineates a novel amplification loop of complement activation and inflammatory responses that may influence the pathogenesis of allergic/inflammatory skin diseases.
Asunto(s)
Quimiocina CCL2/metabolismo , Complemento C3/metabolismo , Complemento C3a/metabolismo , Inflamación/inmunología , Queratinocitos/metabolismo , Piel/inmunología , Comunicación Autocrina/inmunología , Células Cultivadas , Activación de Complemento/fisiología , Citocinas/biosíntesis , Citocinas/inmunología , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Expresión Génica , Humanos , Inmunohistoquímica , Inflamación/patología , Queratinocitos/inmunología , Antígeno de Macrófago-1/metabolismo , Mastocitos/enzimología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/metabolismo , Piel/citología , Piel/patología , TriptasasRESUMEN
Through its involvement in inflammation, opsonization, and cytolysis, the complement protects against infectious agents. Although most of the complement proteins are synthesized in the central nervous system (CNS), the role of the complement system in the normal or ischemic CNS remains unclear. Here we demonstrate for the first time that neural progenitor cells and immature neurons express receptors for complement fragments C3a and C5a (C3a receptor (C3aR) and C5a receptor). Mice that are deficient in complement factor C3 (C3(-/-)) lack C3a and are unable to generate C5a through proteolytic cleavage of C5 by C5-convertase. Intriguingly, basal neurogenesis is decreased both in C3(-/-) mice and in mice lacking C3aR or mice treated with a C3aR antagonist. The C3(-/-) mice had impaired ischemia-induced neurogenesis both in the subventricular zone, the main source of neural progenitor cells in adult brain, and in the ischemic region, despite normal proliferative response and larger infarct volumes. Thus, in the adult mammalian CNS, complement activation products promote both basal and ischemia-induced neurogenesis.
Asunto(s)
Complemento C3a/fisiología , Complemento C5a/fisiología , Isquemia/patología , Neuronas/citología , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Células Madre/citología , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Activación de Complemento , Complemento C3a/genética , Complemento C5a/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Receptor de Anafilatoxina C5a/genética , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/genética , Células Madre/metabolismoRESUMEN
To analyze the role of S19 ribosomal protein (RP S19) in apoptosis, murine NIH3T3 were transfected with either hemagglutinin peptide-tagged (HA) wild-type human RP S19 or a mutant (Gln137Asn) that is resistant to transglutaminase-catalyzed cross-linked-dimerization. Transfection with the mutant HA-RP S19 inhibited manganese (II) (Mn II)-induced apoptosis whereas the wild-type HA-RP S19 augmented apoptosis and a mock transfection had no effect. Release of the wild-type HA-RP S19 dimer but not the mutant HA-RP S19 was observed during the apoptosis. The reduced rate of apoptosis of the cells transfected with the mutant HA-RP S19 was overcome by addition of extracellular wild-type RP S19 dimer. The apoptosis rates in cells transfected with either form of human HA-RP S19 and in mock transfectants were reduced to about 40% by the presence of anti-RP S19 antibody in the culture medium. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) analysis showed that the cell surface expression of the receptor for cross-linked RP S19 dimer, C5a receptor, increased during apoptosis, concomitant with phosphatidylserine exposure. The expression of the C5a receptor gene also increased twofold. Apoptosis rates in the transfected and control cell lines were also reduced by the presence of an anti-mouse C5a receptor monoclonal antibody or of a peptide C5a receptor antagonist. These results indicated the presence of an RP S19 dimer- and C5a receptor-mediated autocrine-type augmentation mechanism during Mn II-induced apoptosis in the mouse fibroblastic cell line. In contrast to the RP S19 dimer, C5a actually inhibited apoptosis, suggesting that signaling through the C5a receptor varies depending on the ligand bound.
Asunto(s)
Apoptosis/fisiología , Fibroblastos/citología , Receptor de Anafilatoxina C5a/metabolismo , Proteínas Ribosómicas/fisiología , Células 3T3 , Animales , Secuencia de Bases , Cartilla de ADN , Dimerización , Electroforesis en Gel de Poliacrilamida , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismoRESUMEN
Regulated migration and spatial localization of dendritic cells (DCs) are critical events during the initiation of physiologic immune responses and maintenance of tolerance. Here we have used cells deficient in the Wiskott-Aldrich syndrome protein (WASp) to demonstrate the importance of dynamic remodeling of the actin cytoskeleton for these trafficking processes to occur in vitro and in vivo. On fibronectin-coated surfaces, WASp-null immature murine DCs exhibited defects both of attachment and detachment, resulting in impaired net translocation compared with normal cells. The chemokinetic response to CCL21, which is critical for normal lymphatic trafficking, was also abrogated in the absence of WASp. In vivo in both fluorescein isothiocyanate (FITC) and oxazolone contact hypersensitivity models, WASp-null Langerhans cell (LC) migration was compromised, as judged by exit from the skin as well as by homing to the draining lymph node (LN). Furthermore, following systemic challenge with lipopolysaccharide (LPS) or toxoplasma-derived antigen, WASp-null DCs showed incomplete redistribution to T-cell areas in the spleen. Instead, they were retained ectopically in the marginal zone. DC trafficking in vivo is therefore dependent on a normally regulated actin cytoskeleton, which performs an essential function during maintenance of physiologic immunity and when disturbed may contribute significantly to the immunopathology of Wiskott-Aldrich Syndrome.