RESUMEN
Dendritic cells (DCs) initiate adaptive immune responses to pathogens and tumours and maintain tolerance to self and innocuous antigens. These functions occur in organs and tissues exhibiting wide variations in nutrients, growth factors, redox and oxygen tension. Understanding how these microenvironmental factors influence DCs to affect immunological outcomes is of increasing relevance with the emerging success of DC-based cellular vaccines. In a previous study, we examined whether redox, an important environmental cue, could influence DC expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). IDO-competent DCs promote long-term immune homoeostasis by limiting exaggerated inflammatory responses and directing regulatory T-cell effector function. To alter redox, we manipulated the activity of the cystine/glutamate antiporter, which functions to maintain intracellular and extracellular redox. The results of that study showed that redox perturbation strongly induced IDO expression and activity in DCs. While this study was performed using standard cell culture techniques with DCs cultured under 5% CO2 and 20% O2, it is clear that DCs capture and present antigens in inflamed tissues and secondary lymphoid organs which exhibit low oxygen tension (1-5% O2). Therefore, here we investigated whether oxygen tension influences DC expression of IDO in the context of homoeostatic and altered redox.
Asunto(s)
Células Dendríticas/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Monocitos/inmunología , Antiportadores/metabolismo , Diferenciación Celular , Hipoxia de la Célula/inmunología , Células Cultivadas , Microambiente Celular , Cisteína/deficiencia , Regulación de la Expresión Génica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Oxidación-Reducción , Oxígeno/metabolismoAsunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Células Dendríticas/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Diferenciación Celular , Células Cultivadas , Cistina/deficiencia , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Ácido Glutámico/metabolismo , Glutatión/biosíntesis , Homocisteína/análogos & derivados , Homocisteína/farmacología , Humanos , Tolerancia Inmunológica , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/metabolismo , Lipopolisacáridos/farmacología , ARN Mensajero/biosíntesisRESUMEN
The follicle-associated epithelium (FAE) of Peyer's patches (PPs) transports antigens and microorganisms into mucosal lymphoid tissues where they are captured by subepithelial dendritic cells (DCs). Feeding of cholera toxin (CT) induced migration of subepithelial DCs to interfollicular T-cell areas within 24 h. This study investigated short-term effects of CT, Escherichia coli heat-labile toxin, and non-toxic derivatives on DC migration. CT or CTB injected into ligated intestinal loops induced significant increase in CD11c+ DCs within the FAE within 90 min. In mice fed CT intragastrically, DC numbers in the FAE increased by 1 h, were maximal by 2 h, declined between 8 and 12 h, and were reversed by 24 h. Feeding of native LT, recombinant CTB, dibutyryl cyclic AMP, and to a lesser extent mutated CT(E29H) or mutated LT(R192G) had the same effect. Thus, both A and B subunits of enterotoxins, presumably acting through distinct signaling pathways, may promote capture of incoming antigens and pathogens by PP DCs.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Toxinas Bacterianas/farmacología , Movimiento Celular/efectos de los fármacos , Toxina del Cólera/farmacología , Células Dendríticas/inmunología , Enterotoxinas/farmacología , Proteínas de Escherichia coli/farmacología , Mucosa Intestinal/inmunología , Ganglios Linfáticos Agregados/inmunología , Sustitución de Aminoácidos , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/inmunología , Antígeno CD11c/inmunología , Movimiento Celular/inmunología , Toxina del Cólera/genética , Toxina del Cólera/inmunología , Células Dendríticas/citología , Enterotoxinas/genética , Enterotoxinas/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Femenino , Mucosa Intestinal/citología , Ratones , Ratones Endogámicos BALB C , Mutación Missense , Ganglios Linfáticos Agregados/citología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Factores de TiempoRESUMEN
The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the beta(2)-microglobulin (beta(2)m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical Fc gamma Rs: Fc gamma RI, Fc gamma RII, and Fc gamma RIII.
Asunto(s)
Células Dendríticas/metabolismo , Antígenos HLA/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Receptores Fc/biosíntesis , Receptores de IgG/biosíntesis , Adulto , Especificidad de Anticuerpos/genética , Biomarcadores , Línea Celular , Células Dendríticas/inmunología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas/fisiología , Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Recién Nacido , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Células Jurkat , Monocitos/inmunología , Especificidad de Órganos/inmunología , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Receptores Fc/química , Receptores Fc/genética , Receptores Fc/metabolismo , Receptores de IgG/química , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transfección , Células Tumorales Cultivadas , Células U937 , Microglobulina beta-2/metabolismoRESUMEN
In intestinal epithelia, cholera and related toxins elicit a cAMP-dependent chloride secretory response fundamental to the pathogenesis of toxigenic diarrhea. We recently proposed that specificity of cholera toxin (CT) action in model intestinal epithelia may depend on the toxin's cell surface receptor ganglioside G(M1). Binding G(M1) enabled the toxin to elicit a response, but forcing the toxin to enter the cell by binding the closely related ganglioside G(D1a) rendered the toxin inactive. The specificity of ganglioside function correlated with the ability of G(M1) to partition CT into detergent-insoluble glycosphingolipid-rich membranes (DIGs). To test the biological plausibility of these hypotheses, we examined native human intestinal epithelia. We show that human small intestinal epithelia contain DIGs that distinguish between toxin bound to G(M1) and G(D1a), thus providing a possible mechanism for enterotoxicity associated with CT. We find direct evidence for the presence of caveolin-1 in DIGs from human intestinal epithelia but find that these membranes are heterogeneous and that caveolin-1 is not a structural component of apical membrane DIGs that contain CT.
Asunto(s)
Caveolinas , Detergentes/farmacología , Proteínas de Escherichia coli , Gangliósido G(M1)/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Toxinas Bacterianas/metabolismo , Caveolina 1 , Polaridad Celular , Toxina del Cólera/metabolismo , Técnicas de Cultivo , Enterotoxinas/metabolismo , Glicoesfingolípidos/metabolismo , Homeostasis , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Membranas/efectos de los fármacos , Membranas/metabolismo , SolubilidadRESUMEN
The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.
Asunto(s)
Inmunoglobulina G/metabolismo , Mucosa Intestinal/fisiología , Receptores Fc/inmunología , Adulto , Animales , Transporte Biológico , Células CHO , Línea Celular , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Polaridad Celular , Cricetinae , Femenino , Antígenos de Histocompatibilidad Clase I , Humanos , Inmunidad Materno-Adquirida , Absorción Intestinal , Mucosa Intestinal/inmunología , Intercambio Materno-Fetal , Ratones , Embarazo , Ratas , Células U937RESUMEN
The antifungal antibiotic clotrimazole (CLT) blocks directly and with high potency the Ca2+-activated K+ channels of human erythrocytes, erythroleukemia cells, and ferret vascular smooth muscle cells. We recently reported that CLT inhibits Cl- secretion in human intestinal T84 cells, likely by affecting K+ transport (Rufo, P.A., L. Jiang, S.J. Moe, C. Brugnara, S.L. Alper, and W.I. Lencer. 1996. J. Clin. Invest. 98:2066-2075). To determine if CLT had direct effects on K+ conductances in T84 cells, we selectively permeabilized apical membranes of confluent T84 cell monolayers using the ionophore amphotericin B. This technique permits direct measurement of basolateral K+ transport. We found that CLT and a stable des-imidazolyl derivative inhibited directly two pharmacologically distinct basolateral membrane K+conductances, but had no effect on apical membrane Cl- conductances. The effects of CLT on Cl- secretion were also examined in intact tissue. CLT inhibited forskolin-induced Cl- secretion in rabbit colonic mucosal sheets mounted in Ussing chambers by 91%. CLT also inhibited cholera toxin-induced intestinal Cl- secretion in intact mice by 94%. These data provide direct evidence that CLT blocks Cl- secretion in intestinal T84 cells by inhibition of basolateral K+ conductances, and show that CLT inhibits salt and water secretion from intact tissue in vitro and in vivo. The results further support the suggestion that CLT and its metabolites may show clinical efficacy in the treatment of secretory diarrheas of diverse etiologies.
Asunto(s)
Antifúngicos/farmacología , Cloruros/metabolismo , Clotrimazol/farmacología , Intestinos/efectos de los fármacos , Potasio/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular , Colforsina/farmacología , Diarrea/metabolismo , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/citología , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , ConejosRESUMEN
The heat-labile enterotoxin (LT) of Escherichia coli is immunologically and physiochemically related to cholera enterotoxin. A number of studies have been performed to determine the relationship of the ADP-ribosylating enzymatic activity of these enterotoxins to toxicity and adjuvanticity. These studies have generally examined the effect of abolishing the ADP-ribosyltransferase activity of A1 by a variety of chemical or genetic manipulations. In every case, loss of enzymatic activity was associated with loss of biological activity and also with the ability of the molecules to function as oral adjuvants. Consequently, we explored an alternate approach to detoxification of LT without altering its adjuvanticity. Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A1 and A2 components of the A subunit. This mutant contains a single amino acid substitution within the disulfide subtended region joining A1 and A2. This mutant toxin, designated LT(R192G), is not sensitive to proteolytic activation, has negligible activity on mouse Y-1 adrenal tumor cells, and is devoid of ADP-ribosyltransferase activity. Nonetheless, LT(R192G) retains the ability to function as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen (OVA) beyond that achieved with administration of that antigen alone. Further, LT(R192G) prevented the induction of tolerance to coadministered antigen and did not induce tolerance against itself, as demonstrated by the presence of significant serum anti-LT IgG and mucosal anti-LT IgA antibodies in immunized mice.
Asunto(s)
Adyuvantes Inmunológicos , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli , Escherichia coli/inmunología , Poli(ADP-Ribosa) Polimerasas/inmunología , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Secuencia de Bases , Células Cultivadas , Enterotoxinas/genética , Enterotoxinas/toxicidad , Escherichia coli/enzimología , Escherichia coli/genética , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/sangre , Mucosa Intestinal/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ovalbúmina/inmunología , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
There is no more demanding application of a polymer than medical uses. Polysulfone engineering resins combine high strength with long-term resistance to repeated steam sterilization. These polymers have proven successful as alternatives to stainless steel and glass. Medical grade polysulfones are biologically inert, display unique long-life under sterilization procedures, can be transparent or opaque in any color and are resistant to most common hospital chemicals.