RESUMEN
Human macrophage hybridoma cells were used to study HLA-DR expression after HIV-1 infection. HLA-DR surface expression was lost 2 wk after infection that was associated with decreased mRNA transcription. Transfecting HLA-DR-alpha and HLA-DR-beta cDNA driven by a nonphysiological CMV promoter restored expression, suggesting that regulatory DNA-binding proteins may be affected by HIV-1 infection. There was no protein binding to conserved class II DNA elements (W/Z/S box, X-1 and X-2 boxes, and Y box) in a HIV-1-infected human macrophage hybridoma cell line, 43(HIV), and in primary monocytes that lost HLA-DR expression after HIV-1(BaL) infection. PCR analysis of the HIV-1-infected cells that lost HLA-DR expression revealed mRNA for W/Z/S (RFX-5), X-1 (RFX-5), X-2 (hX-2BP), and one Y box DNA-binding protein (NF-YB), and CIITA, a non-DNA-binding protein necessary for class II transcription. There was no mRNA for the Y box-binding protein, NF-YA. However, HLA-DR expression could be restored by transfection with NF-YA driven by a CMV promoter, although HLA-DR failed to localize in either the late endosomes, lysosomes, or acidic compartments. This was associated with a loss of class II-associated invariant chain peptide and leupeptin-induced protein in the 43(HIV) cells. To address this further, non-HIV-1-infected 43 cells were infected with vaccinia virus containing HIV-1 gag, nef, pol, and env proteins. HLA-DR failed to localize in neither the late endosomes, lysosomes, or acidic compartments in the vaccinia-infected cells containing HIV-1 env protein. HIV-1 appears to have multiple effects on class II expression in monocytic cells that may contribute to the immune defects seen in HIV-1-infected patients.
Asunto(s)
VIH-1/inmunología , Antígenos HLA-DR/biosíntesis , Monocitos/metabolismo , Monocitos/virología , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Antígenos de Diferenciación de Linfocitos B/genética , Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Compartimento Celular/genética , Compartimento Celular/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/inmunología , Genes MHC Clase II , Antígenos VIH/genética , Antígenos VIH/fisiología , VIH-1/genética , Antígenos HLA-D/biosíntesis , Antígenos HLA-D/genética , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Hibridomas , Monocitos/inmunología , Factores de Transcripción NFI , Proteínas Nucleares , Unión Proteica/genética , Unión Proteica/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Células U937 , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/fisiología , Proteína 1 de Unión a la Caja YRESUMEN
Primary intestinal epithelial cells, human colonic adenocarcinoma cell lines (DLD-1, Caco-2, and HT-29), and monocytes were used as model systems to study antigen uptake, antigen-presenting cell properties, as well as the kinetics of antigen uptake in intestinal epithelial cells (IEC). Intracellular staining of fluoresceinated tetanus toxoid was not evident in the IEC until after 30 min of incubation at 37 degrees C, whereas in monocytes intracellular punctate staining of fluoresceinated tetanus toxoid was evident after 5 mins. In polarized Caco-2 cells antigen could be internalized at both the apical and basolateral surfaces with polarized transport. When analyzed by electron microscopy, gold-labeled tetanus toxoid was internalized and found within endosomes and multivesicular bodies, but not within the lysosomal compartments by 60 min. By 2 hrs, gold-labeled tetanus toxoid was evident in the secondary lysosomes. These results demonstrate that tetanus toxoid follows an endocytic pathway in intestinal epithelial cells and that the kinetics of antigen uptake is slower than that of conventional antigen-presenting cells.
Asunto(s)
Antígenos/metabolismo , Mucosa Intestinal/metabolismo , Células CACO-2 , Células Cultivadas , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/fisiopatología , Endocitosis , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Membranas Intracelulares/metabolismo , Cinética , Microscopía Confocal , Monocitos/metabolismo , Distribución TisularRESUMEN
We assessed the role of size, solubility, and prophagocytic cytokines interferon-gamma (IFN-gamma), and granulocyte-macrophage colony stimulatory factor (GM-CSF) in antigen uptake and kinetics by intestinal epithelial cells using keyhole limpet hemocyanin and ovalbumin. Both fluoresceinated keyhole limpet hemocyanin (3000-7500 kDa) and fluoresceinated ovalbumin (45 kDa) were internalized by human colonic epithelial cell lines, with kinetics similar to those of fluoresceinated tetanus toxoid, and there was decreased uptake of insoluble immune complexes and no enhancement in the uptake of soluble immune complexes. In addition, neither IFN-gamma nor GM-CSF altered the kinetics of uptake nor enhanced antigen internalization by the intestinal epithelial cell lines. These data suggest that regardless of the size of the soluble antigen, the presence of prophagocytic cytokines, or the formation of soluble immune complexes, fluid phase endocytosis of antigen by intestinal epithelial cells appears to be a relatively stable process.
Asunto(s)
Antígenos/metabolismo , Mucosa Intestinal/metabolismo , Complejo Antígeno-Anticuerpo/fisiología , Antígenos/química , Células CACO-2 , Línea Celular , Citocinas/fisiología , Endocitosis/fisiología , Células HT29 , Hemocianinas/química , Hemocianinas/farmacocinética , Humanos , Mucosa Intestinal/citología , Peso Molecular , Ovalbúmina/química , Ovalbúmina/farmacocinética , Fagocitosis/fisiología , SolubilidadRESUMEN
We investigated accessory cell function, antigen (Ag) trafficking, and uptake of immune complexes in isolated nasal epithelial cells (NEC) and airway epithelial cells (AEC), as well as in the two respiratory epithelial cell lines A549 and BEAS-2B. The NEC and AEC were capable of supporting Ag-specific as well as phytohemagglutinin-induced and anti-CD3 antibody-induced T-cell proliferation. We colocalized fluorescein isothiocyanate (FITC)-labeled Ags with human leukocyte antigen (HLA)-DR in A549 and BEAS-2B, utilizing laser confocal microscopy. Respiratory epithelial cells stimulated and unstimulated with interferon (IFN)-gamma were pulsed with FITC-labeled Ags for varying periods and evaluated for their ability to internalize Ag. In the unstimulated cells, intracellular punctate staining was evident at 60 min and persisted up to 120 min. In the IFN-gamma-stimulated cells (100 U/ml for 48 h), uptake occurred at 30 min, was maximal at 60 min, and diminished at 120 min. We conducted kinetic studies in the A549 and BEAS-2B cells, utilizing electron microscopy with colloidal gold-conjugated Ags (Au-OVA). At 15 min, Au-OVA was evident in the early compartments resembling the compartment of uncoupling of receptor and ligand. At 30 min, multivesicular bodies were labeled with Au-OVA, and by 60 min Au-OVA was present in the primary and secondary lysosomes. The FITC-labeled Ags colocalized with an early endosomal marker (anti-cathepsin D), a late endosomal marker (M6PR), a lysosomal marker (CD63), and with 3-(2, 4-dinitroanilino)-3'-aminomethyldipropylamine, a marker of acidic vesicles. The BEAS-2B and A549 cells, and NEC and AEC, expressed surface Fcgamma receptor and internalized IgG immune complexes. The NEC and AEC also expressed the costimulatory molecules CD80 and CD86 as determined with flow cytometry, the reverse transcription-polymerase chain reaction for RNA, and immunohistochemistry, and T-cell proliferation could be blocked by treating NEC and AEC with anti-CD80 and anti-CD86 antibodies. Our findings suggest that respiratory epithelial cells may have a role in local Ag presentation.
Asunto(s)
Antígenos/inmunología , Bronquios/inmunología , Bronquios/fisiología , Mucosa Nasal/inmunología , Mucosa Nasal/fisiología , Complejo Antígeno-Anticuerpo/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos CD/inmunología , Antígeno B7-1/inmunología , Antígeno B7-2 , Complejo CD3/inmunología , Línea Celular , Células Cultivadas , Endocitosis , Células Epiteliales/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Cinética , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Microscopía Confocal , Microscopía Electrónica , Receptores de IgG/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.