RESUMEN
Cells of the female reproductive tract (FRT) can present antigen to naive and memory T cells. However, the effects of oestrogen, known to modulate immune responses, on antigen presentation in the FRT remain undefined. In the present study, DO11.10 T-cell antigen receptor transgenic mice specific for the class II MHC-restricted ovalbumin (OVA) 323-339 peptide were used to study the effects of oestradiol and pathogen-associated molecular patterns on antigen presentation in the FRT. We report here that oestradiol inhibited antigen presentation of OVA by uterine epithelial cells, uterine stromal cells and vaginal cells to OVA-specific memory T cells. When ovariectomized animals were treated with oestradiol for 1 or 3 days, antigen presentation was decreased by 20-80%. In contrast, incubation with PAMP increased antigen presentation by epithelial cells (Pam(3)Cys), stromal cells (peptidoglycan, Pam(3)Cys) and vaginal cells (Pam(3)Cys). In contrast, CpG inhibited both stromal and vaginal cell antigen presentation. Analysis of mRNA expression by reverse transcription PCR indicated that oestradiol inhibited CD40, CD80 and class II in the uterus and CD40, CD86 and class II in the vagina. Expression in isolated uterine and vaginal cells paralleled that seen in whole tissues. In contrast, oestradiol increased polymeric immunoglobulin receptor mRNA expression in the uterus and decreased it in the vagina. These results indicate that antigen-presenting cells in the uterus and vagina are responsive to oestradiol, which inhibits antigen presentation and co-stimulatory molecule expression. Further, these findings suggest that antigen-presenting cells in the uterus and vagina respond to selected Toll-like receptor agonists with altered antigen presentation.
Asunto(s)
Presentación de Antígeno/inmunología , Estradiol/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Útero/inmunología , Vagina/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Antígeno B7-1/biosíntesis , Antígeno B7-1/inmunología , Antígenos CD40/biosíntesis , Antígenos CD40/inmunología , Células Cultivadas , Estradiol/farmacología , Femenino , Inmunomodulación , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/farmacología , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Útero/efectos de los fármacos , Vagina/efectos de los fármacosRESUMEN
Pulmonary infections and dysfunction are frequent outcomes during the development of immunodeficiency associated with human immunodeficiency virus type 1 (HIV-1) infection, and obtaining a better understanding of the immunologic changes that occur in lungs following HIV-1 infection will provide a foundation for the development of further intervention strategies. We sought here to identify changes in the pulmonary immune environment that arise during simian immunodeficiency virus (SIV) infection of rhesus macaques, which serves as an excellent model system for HIV-1 infection and disease. To examine the gene expression profiles of macaque lung tissues following infection with the pathogenic SIV/DeltaB670 isolate, we performed cDNA microarray hybridizations with lung total RNAs using two commercially available cDNA arrays and a custom-fabricated, immunologically focused macaque cDNA microarray. In situ hybridization and real-time RT-PCR were performed to provide additional analyses of gene expression. Among the genes exhibiting the highest level of induction in lung tissues were the IFN-gamma-inducible chemokines, CXCL10/IP-10 and CXCL9/Mig. In situ hybridization and real-time RT-PCR strongly supported these findings. Correlation analyses revealed that the levels of expression of IFN-gamma, CXCL9/Mig, and CXCL10/IP-10 mRNAs were all strongly positively correlated, and that CXCL10/IP-10 mRNA and Pneumocystis carinii rRNA were positively correlated. Taken together, these findings demonstrate that inflammatory chemokines are among the most differentially expressed mRNAs in macaque lung tissues during systemic SIV infection of rhesus macaques, and provide insight into the complicated events occurring in the lung tissues during HIV-1 infection in humans.
Asunto(s)
Quimiocinas CXC/genética , Interferón gamma/inmunología , Pulmón/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Quimiocina CXCL10 , Quimiocinas CXC/biosíntesis , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Hibridación in Situ , Interferón gamma/biosíntesis , Pulmón/metabolismo , Macaca mulatta , Análisis de Secuencia por Matrices de Oligonucleótidos , Pneumocystis carinii/genética , ARN/análisis , ARN/aislamiento & purificación , ARN de Hongos/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome de Inmunodeficiencia Adquirida del Simio/genéticaRESUMEN
HIV-1 positive patients generate Nef-specific CTL response, indicating that Nef is a potent immunogen. However, Nef is also known to down regulate the expression of CD4 and MHC-I molecules, thereby protecting virally infected target cells. We compared the immunogenicity of non-functional nef vaccine constructs to wild type functional nef as potential immunogen. Mice were immunized with different nef constructs and assessed for their ability to induce cellular immune responses. Evaluation of T cell immune responses in mice showed that non-functional nef vaccine constructs are capable of inducing a significant T cell immune response measured by IFN-gamma ELISPOT. Further epitope mapping studies indicate that one of our attenuated constructs, Nef R-38, has multiple CTL epitopes spanning throughout the gene. Our results indicate that functionally attenuated Nef antigen might be a better candidate for future multiprotein HIV-1 vaccine.
Asunto(s)
Vacunas contra el SIDA/inmunología , Productos del Gen nef/genética , Infecciones por VIH/prevención & control , Sistema Inmunológico/inmunología , Vacunas de ADN/inmunología , Vacunas contra el SIDA/farmacología , Animales , Mapeo Epitopo , Epítopos/inmunología , Productos del Gen nef/inmunología , Humanos , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/metabolismo , Memoria Inmunológica/inmunología , Interferón gamma/efectos de los fármacos , Ratones , Vacunas de ADN/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.
Asunto(s)
Presentación de Antígeno , Genes MHC Clase I , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1 , Antígenos HLA-B/genética , Negro o Afroamericano/genética , Alelos , Aminoácidos/fisiología , Linfocitos T CD8-positivos/inmunología , Estudios de Cohortes , Progresión de la Enfermedad , Estudio de Asociación del Genoma Completo , Antígenos VIH/inmunología , Infecciones por VIH/etnología , Infecciones por VIH/virología , Sobrevivientes de VIH a Largo Plazo , VIH-1/inmunología , Antígenos HLA-A/química , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Antígenos HLA-B/química , Antígenos HLA-B/inmunología , Antígenos HLA-B/metabolismo , Antígenos HLA-C/química , Antígenos HLA-C/genética , Antígenos HLA-C/inmunología , Antígenos HLA-C/metabolismo , Haplotipos , Hispánicos o Latinos/genética , Humanos , Inmunidad Innata , Modelos Logísticos , Modelos Moleculares , Polimorfismo de Nucleótido Simple , Conformación Proteica , Carga Viral , Población Blanca/genéticaRESUMEN
PROBLEM: CCL20/MIP3alpha is a chemokine for immature dendritic cells as well as an antibacterial against gram-positive and gram-negative bacteria. The role of CCL20/MIP3alpha as an antiviral is unknown. In this study, we have examined the production of CCL20/MIP3alpha by epithelial cells from the upper female reproductive tract as well as its activity as an antiviral molecule. METHOD OF STUDY: Primary uterine and Fallopian tube epithelial cells were treated with Poly(I:C) and CCL20/MIP3alpha mRNA and protein was measured by Realtime RT-PCR and ELISA assays. Anti-HIV activity was determined using an indicator cell line TZM-bl and quantified by using a luminometer. RESULTS: Primary uterine and Fallopian tube epithelial cells produce CCL20/MIP3alpha constitutively and the production is enhanced following stimulation with viral double-stranded RNA mimic Poly(I:C). Recombinant CCL20/MIP3alpha was able to inhibit both T-cell-tropic X4/IIIB and macrophage-tropic R5/BaL HIV-1 when virus was directly incubated with CCL20/MIP3alpha but not when CCL20/MIP3alpha was added to cells either prior to infection or post-infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV-1 and CCL20/MIP3alpha. CONCLUSION: This study demonstrates that CCL20/MIP3alpha is an important endogenous anti-HIV-1 microbicide of the female reproductive tract.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antivirales/farmacología , Quimiocina CCL20/inmunología , Quimiocina CCL20/farmacología , Células Dendríticas/inmunología , Genitales Femeninos/inmunología , VIH-1/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Células Epiteliales/inmunología , Células Epiteliales/virología , Trompas Uterinas/inmunología , Femenino , Genitales Femeninos/virología , Humanos , Poli I-C/farmacología , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Útero/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
OBJECTIVE: To examine the expression of toll-like receptors (TLR) by primary human Fallopian tube epithelial cells (FTEC) and to determine whether exposure to the TLR3 agonist poly(I:C) induces an antiviral response. DESIGN: Tissue culture study. SETTING: University medical center. PATIENT(S): Premenopausal women undergoing hysterectomy. INTERVENTION(S): Primary human FTEC were grown to confluence and high transepithelial resistance and treated with TLR agonists. Conditioned media was collected and RNA was extracted and analyzed for the expression of cytokines, chemokines, and antimicrobial genes. MAIN OUTCOME MEASURE(S): The RNA was analyzed by real-time polymerase chain reaction and protein levels were assessed by enzyme-linked immunosorbent assay. RESULT(S): The FTEC were demonstrated to express TLR1-9 but not 10. Treatment of FTEC with TLR3 agonist poly(I:C) resulted in increased expression of interleukin-8, tumor-necrosis factor alpha, human beta-defensin 2, interferon beta, and interferon stimulated genes myxovirus resistance gene 1, 2',5'-oligoadenylate synthetase, and protein kinase R. Additionally, FTEC exposed to poly(I:C) also resulted in the induction of TLR2, TLR3, and TLR7. CONCLUSION(S): Our results suggest that FTEC are sensitive to viral infection and/or exposure to viral double-stranded RNA and can respond by secreting proinflammatory cytokines that mediate the initiation of an inflammatory response as well as expressing genes that can directly inhibit viral replication.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Trompas Uterinas/efectos de los fármacos , Poli I-C/farmacología , Receptor Toll-Like 3/agonistas , Virus/inmunología , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Células Cultivadas , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Trompas Uterinas/inmunología , Trompas Uterinas/metabolismo , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Proteínas de Resistencia a Mixovirus , ARN Mensajero/metabolismo , ARN Viral/farmacología , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Zimosan/farmacología , beta-Defensinas/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Lymphoid tissues are sites of soluble and cell-associated antigen sampling of peripheral tissues, and they are key compartments for the generation of cellular and humoral immune responses. Hilar lymph nodes (HiLNs), which drain the lungs, were examined to understand the effects of simian immunodeficiency virus (SIV) infection on this compartment of the immune system. Histologic and messenger RNA (mRNA) expression profiling approaches were used to determine the numbers, types, and distributions of SIV viral RNA cells and to identify differentially expressed genes in HiLNs during SIV infection. SIV RNA cells were found to be primarily CD68 and localized to paracortical and medullary regions early in infection, whereas they resided mainly in paracortex during AIDS. As SIV infection progressed, CXCL9, CXCL10, interferon-gamma, and Toll-like receptor 3 levels all increased. In contrast, CCL19 increased early in infection but decreased during AIDS, whereas CCL21 decreased progressively throughout infection. Finally, local levels of cellular activation were increased throughout infection. Taken together, these findings indicate that SIV infection leads to an inflammatory environment in lung-draining lymph nodes that is characterized by type 1 cytokines and chemokines and likely has an impact on the nature and strength of immune responses to pulmonary pathogens.
Asunto(s)
Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Animales , Proliferación Celular , Quimiocina CCL19/genética , Quimiocina CXCL9/genética , Hibridación in Situ , Ganglios Linfáticos/patología , Linfadenitis/inmunología , Linfadenitis/virología , Macaca fascicularis , Macaca mulatta , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Sexually transmitted infections are a major worldwide public health problem affecting millions of people. A number of bacteria, fungi, viruses, and protozoa can infect reproductive tissues, resulting in varying degrees of pathology ranging from little discomfort to death. The female reproductive tract has evolved innate and adaptive immune mechanisms that protect from microbial infection, thereby reducing infection and disease. Central to this protection are the epithelial cells that line the female reproductive tract. In the uterus, columnar epithelial cells provide a physical barrier to microbial infection, possess toll-like receptors that detect pathogens and secrete a number of constitutive and induced factors that directly or indirectly hinder infection. For example, uterine epithelial cells secrete peptides that destroy pathogenic microbes. In addition, epithelial cells produce chemokines and cytokines that attract and activate innate immune cells and serve as a link to the adaptive immune system. Further, uterine epithelial cells serve as a conduit for secretory antibodies to enter the lumen and can present antigen to T cells. These protective mechanisms contribute to an environment in the uterus that is generally considered sterile, unlike the environment in the lower female reproductive tract. The uterine environment is in constant flux due to the concentration changes in sex hormones that occur in preparation for reproduction. The sex hormones estrogen and progesterone alter the local immune system to prepare for conception, influence how well the immune system will tolerate antigenic sperm and a semi-allogeneic fetus and yet provide a network of protective immune mechanisms against microbial pathogens. Understanding how sex hormones influence uterine epithelial cell function will provide a basis for immune protection in the uterus.
RESUMEN
PROBLEM: Epithelial cells lining the uterine lumen are the first line of defense against pathogenic microbes. The objective of this study was to examine the expression of Toll-like receptors (TLRs), defensins and secretory leukocyte protease inhibitor (SLPI) in the mouse uterus and vagina and in primary uterine epithelial cells and to determine whether TLR agonists induce TLR and defensin expression. METHOD OF STUDY: The mRNA expression of alpha- and beta-defensins (AD1, 2 and 5 and BD1, 2 and 4) and SLPI was examined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) along with the secretion of macrophage chemotactic protein-1 (MCP-1), measured by enzyme-linked immunosorbent assay. RESULTS: Expression of TLR1-9 as well as beta-defensins 1, 2 and 4 and SLPI by uterine and vaginal tissues was demonstrated by RT-PCR. beta-Defensins and SLPI expression was greater in the vagina than in the uterus. Comparison of fresh and polarized uterine epithelial cells indicated that TLR2-6 expression was unaffected by culture. Incubation of polarized epithelial cells with TLR agonists [lipopolysaccharide (LPS), Pam3Cys, Poly (I:C) or PGN] induced TLR5 and TLR9 expression but had no effect on TLR4, defensins or SLPI. Furthermore, exposure to LPS, Pam3Cys, Poly (I:C) or PGN, induced MCP-1 secretion by polarized epithelial cells in culture. CONCLUSION: These results indicate that the uterus and vagina as well as uterine epithelial cells are responsive to bacterial and viral pathogens. Not only do epithelial cells respond to TLR agonists by releasing MCP-1, which mediates inflammatory responses, but they also influence the expression of selected TLR genes to further enhance innate immune protection.
Asunto(s)
Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Útero/efectos de los fármacos , Útero/inmunología , Vagina/efectos de los fármacos , Vagina/inmunología , Animales , Secuencia de Bases , Células Cultivadas , Quimiocina CCL2/metabolismo , Defensinas/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Inmunidad Innata , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias , Receptores Toll-Like/genética , Útero/metabolismo , Vagina/metabolismoRESUMEN
The uterine endometrium coordinates a wide spectrum of physiologic and immunologic functions, including endometrial receptivity and implantation as well as defense against sexually transmitted pathogens. Macrophages and epithelial cells cooperatively mediate innate host defense against bacterial invasion through the generation of immunologic effectors, including cytokines and antimicrobial peptides. In this study, we demonstrate that stimulation of peripheral blood monocytes and uterine macrophages with bacterial LPS induces the production of biologically active proinflammatory IL-1beta. High doses of estradiol enhance LPS-induced IL-1beta expression in an estrogen receptor-dependent manner. Furthermore, both peripheral blood monocyte- and uterine macrophage-derived IL-1beta induce secretion of antimicrobial human beta-defensin 2 by uterine epithelial cells. These data indicate dynamic immunologic interaction between uterine macrophages and epithelial cells and implicate a role for estradiol in the modulation of the immune response.
Asunto(s)
Células Epiteliales/metabolismo , Interleucina-1/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Regulación hacia Arriba/inmunología , Útero/citología , Útero/inmunología , beta-Defensinas/biosíntesis , Adyuvantes Inmunológicos/farmacología , Adulto , Caspasa 1/metabolismo , Línea Celular , Línea Celular Tumoral , Células Epiteliales/inmunología , Estradiol/farmacología , Femenino , Humanos , Interleucina-1/sangre , Interleucina-1/fisiología , Macrófagos/enzimología , Macrófagos/metabolismo , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Útero/enzimologíaRESUMEN
BACKGROUND: During pregnancy, the placenta may become exposed to micro-organisms, such as viruses, which may pose a substantial threat to the embryo/fetus well-being. Recent insight into the immunological capabilities of the trophoblast suggests that the placenta may function as an active barrier by recognizing and responding to pathogens through Toll-like receptors (TLRs). METHODS: The objective of this study was to determine whether the engagement of TLR-3 with viral dsRNA by first-trimester trophoblast could induce the production of factors necessary to generate an antiviral response. Therefore, trophoblast cells were exposed to the TLR-3 agonist, Poly(I : C). RESULTS: We report that following stimulation with Poly(I : C), first-trimester trophoblast cells produce interferon beta (IFNbeta) and secretory leukocyte protease inhibitor (SLPI), as well as the intracellular factors 2',5'-oligoadenylate synthetase (OAS), Myxovirus-resistance A (MxA) and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G). This response is TLR-3 specific because the TLR-4 ligand, lipopolysaccharide (LPS), had no effect on the production of these antimicrobial factors. Furthermore, we describe a positive feedback mechanism in which IFNbeta enhances the antiviral response by promoting the production of OAS, MxA and APOBEC3G. CONCLUSIONS: These findings suggest that trophoblast cells are able to recognize and specifically respond to viral products in a highly regulated fashion and that the placenta may be pivotal in the control of viral infections at the maternal-fetal interface.
Asunto(s)
Antivirales/farmacología , Poli I-C/farmacología , Receptor Toll-Like 3/agonistas , Trofoblastos/metabolismo , Apolipoproteínas B/química , Femenino , Humanos , Ligandos , Lipopolisacáridos/metabolismo , Embarazo , Primer Trimestre del Embarazo , ARN Bicatenario/química , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.
Asunto(s)
Células Epiteliales/inmunología , Células Epiteliales/virología , Proteínas Inflamatorias de Macrófagos , Glicoproteínas de Membrana/agonistas , Poli I-C/farmacología , Receptores de Superficie Celular/agonistas , Útero/inmunología , Útero/virología , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/metabolismo , Quimiocina CCL4 , Quimiocinas/metabolismo , Quimiocinas CC , Citocinas/metabolismo , Células Epiteliales/metabolismo , Femenino , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Interferón beta/biosíntesis , Interferón beta/genética , Interferón beta/fisiología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/inmunología , Proteínas/metabolismo , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/inmunología , Receptor Toll-Like 1 , Receptor Toll-Like 10 , Receptor Toll-Like 3 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Útero/citología , Útero/metabolismo , beta-Defensinas/biosíntesis , beta-Defensinas/genéticaRESUMEN
PROBLEM: Uterine epithelial cells produce cytokines that stimulate leukocytes in response to a microbial insult. The goals of this study were to determine if uterine epithelial cells produce the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), and to see if toll-like receptor (TLR) agonists stimulate MIF secretion. METHODS OF STUDY: Human uterine epithelial cells were isolated and grown in cell culture inserts. Levels of MIF secretion were examined by ELISA and MIF messenger RNA (mRNA) expression was examined using real time RT-PCR. RESULTS: Uterine epithelial cells constitutively secrete MIF and exposure to the TLR3 agonist poly (I:C) resulted in enhanced apical secretion of MIF. MIF secretion appeared to be from pre-formed intracellular stores, since exposure of epithelial cells to poly (I:C) had little effect on the expression of MIF-mRNA. CONCLUSIONS: These results demonstrate that uterine epithelial cells constitutively produce MIF and stimulation with poly (I:C) results in enhanced MIF production. This suggests that MIF secretion by uterine epithelial cells may play a critical role in innate immune responses against viral pathogens mediated through TLR3.
Asunto(s)
Endometrio/efectos de los fármacos , Endometrio/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Poli I-C/farmacología , Receptor Toll-Like 3/agonistas , Adulto , Células Cultivadas , Femenino , Humanos , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/fisiología , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptor Toll-Like 3/fisiologíaRESUMEN
The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.
Asunto(s)
Estradiol/farmacología , Inflamación/prevención & control , Interleucina-1/farmacología , Receptores de Interleucina-1/genética , Útero/efectos de los fármacos , Útero/inmunología , Células Cultivadas , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Estradiol/administración & dosificación , Femenino , Humanos , Inflamación/etiología , Inflamación/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Receptores Tipo I de Interleucina-1 , Útero/citología , Útero/metabolismo , beta-Defensinas/biosíntesis , beta-Defensinas/genéticaRESUMEN
The objective of this study was to examine the expression of toll-like receptors (TLRs) by the uterine epithelial cell line ECC-1 and to determine if stimulation of the expressed TLRs induces changes in cytokine and/or chemokine secretion. The expression of TLR1 to TLR9 by ECC-1 cells was demonstrated by reverse transcription polymerase chain reaction, with only TLR10 not being expressed. Stimulation of ECC-1 cells using agonists to TLR2, TLR4 and TLR5 induced the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1), as well as the pro-inflammatory cytokine IL-6, and occurred in a dose-dependent manner. In response to zymosan and flagellin, pathogen-associated molecular patterns (PAMP) that are recognized by TLR2 and TLR5 respectively, ECC-1 cells secreted significantly more IL-8, MCP-1 and IL-6 than in response to other TLR agonists. In contrast, agonists to TLR3, TLR7, and TLR9 had no effect on the secretion of the 13 cytokines or chemokines analysed. These results indicate that uterine epithelial cells are important sentinels of the innate immune system. Further it indicates that all but one of the known TLRs are expressed by ECC-1 cells and that stimulation through specific TLRs mediates changes in the expression of key chemokines and pro-inflammatory cytokines that aid in the defence of the uterus against potential pathogens.
Asunto(s)
Citocinas/inmunología , Células Epiteliales/inmunología , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Útero/inmunología , Análisis de Varianza , Línea Celular , Quimiocina CCL2/inmunología , Citocinas/análisis , Relación Dosis-Respuesta a Droga , Femenino , Flagelina/farmacología , Humanos , Interleucina-6/inmunología , Interleucina-8/inmunología , Ligandos , Glicoproteínas de Membrana/genética , ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 1 , Receptor Toll-Like 10 , Receptor Toll-Like 2 , Receptor Toll-Like 3 , Receptor Toll-Like 4 , Receptor Toll-Like 5 , Receptor Toll-Like 7 , Receptor Toll-Like 9 , Receptores Toll-Like , Zimosan/farmacologíaRESUMEN
Chemokines are small chemoattractant cytokines involved in normal and pathological immune processes. Although extensive nucleotide sequence data are available for human and murine chemokine cDNA sequences, very few data are currently available regarding rhesus macaque sequences. To increase our understanding of immune function in nonhuman primates, we have used reverse-transcription polymerase chain reaction (RT-PCR) to clone and sequence rhesus macaque cDNAs from each of the C, CC, CXC, and CX3C groups of chemokines. Relative to the respective human chemokines, these 25 chemokine cDNA sequences were from 77% to 98% identical. Of the amino acid differences between the rhesus macaque and human chemokines, 51% were species-specific when compared together with the respective murine chemokine sequences. These studies of rhesus macaque chemokine sequences demonstrate that chemokine genes are highly conserved across species, and provide a large foundation for the study of chemokine biology and genetics in nonhuman primates.
Asunto(s)
Quimiocinas/genética , Macaca mulatta/genética , Macaca mulatta/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Quimiocinas/clasificación , Quimiocinas C/genética , Quimiocinas CC/genética , Quimiocinas CX3C/genética , Quimiocinas CXC/genética , Clonación Molecular , Secuencia Conservada , ADN Complementario/genética , Evolución Molecular , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Toll-like receptor (TLR) signal transduction is a central component of the innate immune response to pathogenic challenge. Although recent studies have begun to elucidate differences in acquired immunity in tissues of the human female reproductive tract, there is a relative paucity of work regarding innate defense mechanisms. We investigated TLR mRNA and protein expression in tissues of the human female reproductive tract. Constitutive mRNA expression of TLRs 1 to 6 was observed in fallopian tubes, uterine endometrium, cervix, and ectocervix. Furthermore, transcripts of the signaling adapter MyD88 and the accessory molecule CD14 were also detected in all tissues assayed. Quantitative analysis of TLR2 mRNA levels revealed highest expression of this molecule in fallopian tube and cervical tissues, followed by endometrium and ectocervix. In contrast to TLR2, TLR4 expression declined progressively along the tract, with highest expression in the upper tissues (fallopian tubes and endometrium), followed by cervix and ectocervix. In addition to mRNA, protein expression of TLR2 and TLR4 was also documented in these tissues. These data suggest that TLRs are differentially expressed in distinct compartments of the female reproductive tract and may provide insight regarding the regulation of inflammation and immunity within the tract.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genitales Femeninos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Antígenos de Diferenciación/genética , Antígenos de Superficie/genética , Femenino , Humanos , Receptores de Lipopolisacáridos/genética , Antígeno 96 de los Linfocitos , Persona de Mediana Edad , Factor 88 de Diferenciación Mieloide , Especificidad de Órganos , Posmenopausia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Inmunológicos/genética , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface proteins that has been implicated as a progression factor in a number of pathologic conditions from chronic inflammation to cancer to Alzheimer's disease. In such conditions, RAGE acts to facilitate pathogenic processes. Its secreted isoform, soluble RAGE or sRAGE, has the ability to prevent RAGE signaling by acting as a decoy. sRAGE has been used successfully in animal models of a range of diseases to antagonize RAGE-mediated pathologic processes. In humans, sRAGE results from alternative splicing of RAGE mRNA. This study was aimed to determine whether the same holds true for mouse sRAGE and, in addition, to biochemically characterize mouse sRAGE. The biochemical characteristics examined include glycosylation and disulfide patterns. In addition, sRAGE was found to bind heparin, which may mediate its distribution in the extracellular matrix and cell surfaces of tissues. Finally, our data indicated that sRAGE in the mouse is likely produced by carboxyl-terminal truncation, in contrast to the alternative splicing mechanism reported in humans.
Asunto(s)
Receptores Inmunológicos/aislamiento & purificación , Empalme Alternativo , Animales , Cromatografía de Afinidad , Pulmón/metabolismo , Ratones , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The Nef protein of Simian immunodeficiency virus (SIV) associates with multiple T lymphocyte signaling proteins, including the T cell receptor (TCR) zeta chain. We demonstrate here that these interactions are conserved and highly specific. Nefs derived from genetically diverse strains of SIV (SIV(mac)239, SIV(smm)PBj, and SIV(smm)DeltaB670) all interacted with TCR zeta on two separate domains, referred to as SIV Nef interaction domains (SNIDs), as examined in both yeast two-hybrid and glutathione-S-transferase (GST) fusion protein pull-down assays. Multiple HIV-1 Nefs were examined and none interacted with TCR zeta. In contrast, HIV-2(UC1) Nef, similar to SIV Nef, interacted with TCR zeta on two domains, although only the SIV Nefs potently reduced cell-surface expression of the TCR/CD3 complex in T cells. In addition, we examined the abilities of SIV, HIV-2, and HIV-1 Nefs to interact with the cytoplasmic domains of other signaling molecules including CD3epsilon, CD3gamma, and FcepsilonRIgamma, which also contain YxxL motifs, and determined that SIV and HIV-2 Nefs interacted only with TCR zeta, whereas HIV-1 Nef did not interact with any signal-transducing cytoplasmic domain examined. Last, to gain further insight into the mechanism by which Nef down-modulates the TCR/CD3 complex, we mutated or deleted regions on Nef involved in endocytosis, localization of Nef to the plasma membrane, interaction with cellular kinases, or that were conserved among multiple strains of SIV. Mutation of the myristoylation site and a conserved region surrounding a putative PKC phosphorylation site were the only mutations that abrogated Nef-mediated down-modulation of the TCR/CD3 complex. These findings demonstrate there is a spectrum of associations between SIV, HIV-2, and HIV-1 Nefs, and the TCR/CD3 complex, and suggest that down-modulation of the TCR/CD3 complex occurs via association with subsets of cellular proteins that are different from those involved in CD4 and CD28 down-modulation.
Asunto(s)
Secuencia Conservada , Regulación hacia Abajo , Proteínas de la Membrana/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Citoplasma/metabolismo , Endocitosis , Productos del Gen nef/genética , VIH-1/metabolismo , VIH-2/metabolismo , Humanos , Células Jurkat , Mamíferos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tirosina , Proteínas Reguladoras y Accesorias Virales/genética , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Chemokines are important mediators of cell trafficking during immune inductive and effector activities, and dysregulation of their expression might contribute to the pathogenesis of human immunodeficiency virus type 1 and the related simian immunodeficiency virus (SIV). To understand better the effects of SIV infection on lymphoid tissues in rhesus macaques, we examined chemokine messenger RNA (mRNA) expression patterns by using DNA filter array hybridization. Of the 34 chemokines examined, the interferon gamma (IFN-gamma)-inducible chemokine CXC chemokine ligand 9/monokine induced by interferon-gamma (CXCL9/Mig) was one of the most highly up-regulated chemokines in rhesus macaque spleen tissue early after infection with pathogenic SIV. The relative levels of expression of CXCL9/Mig mRNA in spleen and lymph nodes were significantly increased after infection with SIV in both quantitative image capture and analysis and real-time reverse transcriptase-polymerase chain reaction assays. In addition, in situ hybridization for CXCL9/Mig mRNA revealed that the patterns of expression were altered after SIV infection. Associated with the increased expression of CXCL9/Mig were increased numbers of IFN-gamma mRNA-positive cells in tissues and reduced percentages of CXC chemokine receptor (CXCR) 3(+)/CD3(+) and CXCR3(+)/CD8(+) lymphocytes in peripheral blood. We propose that SIV replication in vivo initiates IFN-gamma-driven positive-feedback loops in lymphoid tissues that disrupt the trafficking of effector T lymphocytes and lead to chronic local inflammation, thereby contributing to immunopathogenesis.