RESUMEN
Efforts to understand the mechanisms that govern how immunodominant T-cell epitopes are selected from protein antigens have focused mostly on differences in the efficiency of processing and presentation of peptide/major histocompatibility complex (MHC) complexes by antigen-presenting cells, while little attention has been directed at the role of the T-cell repertoire. In this report, the influence of the T-cell repertoire on immunodominance was investigated using transgenic mice that express the beta chain from a T-cell receptor specific for a cryptic Ek restricted epitope of hen-egg lysozyme, HEL85-96. In these mice, the frequency of HEL85-96-specific T-cell precursors is increased 10-20-fold over non-transgenic mice. Transgenic mice respond as well as non-transgenic controls to intact HEL, even though they respond poorly or not at all to a variety of other antigens, including the dominant H-2k restricted epitopes of HEL. Following immunization with native HEL, the only HEL peptide that could recall a response in vitro in the transgenic mice was HEL85-96. Therefore, this normally cryptic epitope is the sole immunodominant epitope in the transgenic mice, and this alteration in immune response is due solely to an increase in the frequency of specific T-cell precursors. An analysis of four additional H-2k restricted cryptic epitopes of HEL suggests that three are similarly limited by T-cell frequency, and that only one is consistent with a defect in efficient antigen presentation. This indicates that there are at least two different types of cryptic epitopes, one in which crypticity is caused by inefficient processing or presentation, and another in which the frequency of specific T-cell progenitors is limiting.
Asunto(s)
Epítopos de Linfocito T/inmunología , Muramidasa/inmunología , Fragmentos de Péptidos/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Epítopos Inmunodominantes/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Receptores de Antígenos de Linfocitos T alfa-beta/análisisRESUMEN
The B7-1 (CD80) molecule provides costimulatory function for the activation of T helper lymphocytes upon encounter with antigen. To investigate the role of this molecule in thymocyte maturation, we have generated transgenic (Tg) mice in which CD80 expression is driven by the keratin 14 promoter (K14). This overexpression of CD80 resulted in the loss of detectable cell surface CD28 expression on thymocytes and a significant reduction in both the surface T cell receptor expression and the ratio of CD4(+) to CD8(+) single-positive thymocytes in Tg animals compared to nontransgenic (non-Tg) controls. While many of these defects were transient, the significant decrease in CD4(+) versus CD8(+) T cell ratio persisted peripherally. Peripheral T cells from these Tg mice were found to be significantly hyporesponsive to T cell mitogens and in mixed leukocyte reaction, effects that our data indicate are due to reduced IL-2 production by Tg T cells upon activation. Despite these functional defects, immunization with both complex and simple protein antigens produced no differences in the proliferative or humoral responses to these antigens between Tg and non-Tg groups. These data indicate that thymic CD80 signaling results in the deletion of significant numbers of CD4(+) T cells but does not culminate in antigen-specific immunodeficiency.
Asunto(s)
Antígeno B7-1/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Timo/inmunología , Animales , Antígeno B7-1/genética , Antígenos CD28/biosíntesis , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , División Celular , Regulación hacia Abajo , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Expresión Génica , Hemocianinas/inmunología , Tolerancia Inmunológica , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T Colaboradores-Inductores/citología , Timo/citología , Timo/metabolismoAsunto(s)
Antígenos CD , Sialoglicoproteínas/fisiología , Humanos , Leucocitos/fisiología , LeucosialinaRESUMEN
The human prostate-specific antigen (PSA) and glandular kallikrein-1 (hGK-1, also known as hK2) genes are tandemly located on chromosome 19, separated by a 12-kb intergenic region. The coordinate regulation of these two genes suggests the presence of common regulatory elements responsible for tissue specificity and/or levels of expression within this region. To identify such regulatory elements, we generated two sets of transgenic mice, which had incorporated either the PSA gene alone or together with the intergenic region. Both sets of transgenics exhibit remarkably prostate-specific expression of the transgene. However, the presence of the intergenic region abrogates the dependence on high PSA gene copy-number for high levels of PSA expression. This suggests the existence of a positive regulatory element in the intergenic region. By using a previously identified distal enhancer element of PSA (termed DEE 1) as a probe, we identified a cross-hybridizing fragment, which we termed DEE 2, in the intergenic region. Sequence analysis shows that DEE 2 is 76% identical to DEE 1, and it includes a putative androgen-responsive element. Here, we propose a model to illustrate how the two enhancers may work to regulate the transcription of PSA and hK2.
Asunto(s)
Antígeno Prostático Específico/biosíntesis , Calicreínas de Tejido/genética , Animales , Secuencia de Bases , Elementos de Facilitación Genéticos , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Modelos Genéticos , Datos de Secuencia Molecular , Próstata/metabolismo , Antígeno Prostático Específico/genética , TransgenesRESUMEN
Prostate-specific antigen (PSA) has been used clinically as a marker for the diagnosis and staging of prostate cancer due to its specific expression in prostate epithelial cells. In addition to its medical importance, its complex hormonal and tissue-specific regulation makes it an attractive model to study gene regulation. Two approaches have been applied to the identification of regulatory regions which confer this specific expression pattern. In vitro analysis of the regulatory regions of the human PSA gene using promoter reporter constructs and tumor cell lines has revealed a number of the DNA sequences involved in the hormone-dependent expression of PSA. We have pursued an alternative in vivo approach using transgenic animal technology, which is the focus of this review. Using this second approach, a transgenic mouse was generated using a 14 kilobase (kb) region of the human PSA gene encompassing the coding region and intervening sequences as well as 6 kb of upstream sequence and 2 kb of downstream sequence. This genomic DNA clone confers a PSA expression pattern in mice which appears to be very similar if not identical to that of humans, allowing us to investigate tissue-specificity and developmental regulation of PSA expression. In addition, these mice, for which PSA is a self-antigen, provide a model to test the feasibility and efficacy of PSA-directed immunotherapy for prostate cancer. The further identification of the PSA regulatory regions important for tissue-specificity may ultimately allow the design of new therapeutics for the treatment of prostate cancer.
Asunto(s)
Antígeno Prostático Específico/genética , Neoplasias de la Próstata/terapia , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Expresión Génica , Humanos , Inmunoterapia , Masculino , Ratones , Ratones TransgénicosRESUMEN
Transgenic (Tg) mice whose epidermal keratinocytes constitutively overexpress either B7-1 (CD80) or B7-2 (CD86) exhibited exaggerated cutaneous delayed type hypersensitivity (DTH) to haptens compared to non-Tg mice. To determine whether enhanced DTH in these Tg mice is seen in response to cutaneous fungal infections, a primary infection with Candida albicans was established by inoculating this organism on the occluded skin of Tg and non-Tg mice. These infections resolved 7 days after removal of occlusive dressing in all three groups of mice, without evidence of exaggerated inflammation in either the Tg or non-Tg mice. Only B7-2 Tg mice developed enhanced Th1-lymphocyte-mediated immune responses to C. albicans antigens after resolving this infection: enhanced footpad swelling in response to intradermal C. albicans antigens, enhanced production of mRNA encoding Th1 lymphokines in draining lymph nodes, and increased gamma interferon secreted into culture supernatants by lymph node T lymphocytes stimulated with Candida antigens in vitro. Lastly, Western blotting of sera from mice that had resolved this fungal infection indicated that only B7-2 Tg mice recognized a wide range of Candida-associated antigens. These data suggest that these two costimulatory molecules, when expressed by keratinocytes, do not deliver identical signals to C. albicans antigen-reactive Th1 lymphocytes. The enhanced immune response in B7-2 Tg mice to a cutaneous C. albicans infection demonstrates the importance of antigen presentation and costimulation in immune reactivity to fungi. Furthermore, B7-2 Tg mice may be useful in identification of protective Candida antigens.
Asunto(s)
Antígenos CD/inmunología , Antígeno B7-1/inmunología , Candidiasis Cutánea/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Antifúngicos/inmunología , Antígenos CD/genética , Antígenos Fúngicos/inmunología , Antígeno B7-1/genética , Antígeno B7-2 , Candida albicans , Epidermis/inmunología , Epidermis/metabolismo , Hipersensibilidad Tardía , Interferón gamma/biosíntesis , Queratinocitos , Ganglios Linfáticos/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Células TH1/inmunologíaRESUMEN
Epithelial repair following acute lung injury involves proliferation and differentiation of existing Clara cells and type II cells. Other mechanisms of epithelial repair may be involved in particularly severe cases. We used epithelial cell-specific markers to examine changes in the mouse lung epithelium 28 days after bleomycin treatment. The spatial distribution of surfactant proteins A, B, C (SPA, SPB, SPC), and Clara cell-specific protein (CC10) mRNA was compared by in situ hybridization in serial lung sections. CC10 mRNA-containing airway cells were replaced in many areas by SPB mRNA-expressing, ciliated cells that did not contain CC10 mRNA. In distal airway regions, we observed a subpopulation of epithelial cells that appeared to express SPA, SPB, SPC, and CC10 mRNA, and speculated that they may represent a multipotential stem cell population. These cells were found in focal clusters, which suggests that they expanded from a common cell. CC10 mRNA-containing cells were seen in alveolar-like structures thought to be the result of Clara cell migration or outpocketing. Our data suggest that there are repair mechanisms involved in epithelial repair after severe injury that have not previously been described.
Asunto(s)
Bleomicina/toxicidad , Pulmón/efectos de los fármacos , Uteroglobina , Animales , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas/genética , Surfactantes Pulmonares/genética , ARN Mensajero/análisisRESUMEN
B7-1 (CD80) is a second signal molecule usually associated with "professional" APCs that prevents the induction of T-cell clonal anergy and induces IL-2 production during antigen presentation. Tg mice whose epidermal KC overexpress B7-1 exhibit exaggerated and persistent CHS to a variety of haptens that lasts up to 8 weeks after hapten challenge. These Tg mice also exhibit significantly enhanced ear-swelling responses to irritants that are not persistent. Exaggerated CHS was not reflected in the draining lymph node. T-lymphocyte proliferative responses after sensitization and local challenge with haptens, as there were no significant differences between the B7-1 Tg and the NTg mice. However, RT-PCR analysis of mouse ear skin at the hapten challenge site indicated that B7-1 Tg mice had an alteration in the kinetics of in situ lymphokine transcripts compared to NTg mice: IFN-gamma transcripts were first detectable in Tg mouse skin at 2 weeks versus 24 h for NTg mice. RNase protection assays to detect inflammatory cytokine transcripts at hapten application sites indicated that B7-1 Tg mice responded to hapten application with increased TNF-alpha, IL-6, and TNF-beta transcripts compared to NTg mice. Thus, hapten-induced ear swelling in these Tg mice may be mediated by enhanced inflammatory cytokines during the early phase (1-14 days). IFN-gamma-producing lymphocytes may be responsible for the late phase of the ear-swelling response (14-42 days). These data indicate that B7-1 overexpression by KC in mouse skin directly or indirectly affects the nature of cutaneous inflammation induced by haptens and irritants.
Asunto(s)
Antígeno B7-1/genética , Queratinocitos/inmunología , Piel/inmunología , Animales , Secuencia de Bases , Citocinas/metabolismo , Cartilla de ADN/genética , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Expresión Génica , Haptenos/administración & dosificación , Técnicas In Vitro , Inflamación/etiología , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Cinética , Activación de Linfocitos , Ratones , Ratones Endogámicos DBA , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Linfocitos T/inmunologíaRESUMEN
The influence of beta-chain diversity on the expressed T-cell receptor (TCR) alpha-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR beta-chain gene. Analysis of these mice using alpha-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded beta chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards alpha chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in beta-transgenic mice is exhibited almost exclusively by CD4+ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8+ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed beta chains can profoundly influence the selection of companion alpha chains expressed in the periphery, and that alpha-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3alpha domain participates in direct contact with the MHC.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Sondas de ADN/genética , ADN Complementario/genética , Femenino , Expresión Génica , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia MolecularRESUMEN
CD43 (leukosialin), a sialylated glycoprotein expressed on the surface of most hematopoietic cells, has been implicated in cell adhesion and signaling. However, its precise physiological function remains unclear. We used mouse CD43 (mCD43)-immunoglobulin enhancer-transgenic (TG) mice to study the role of mCD43 in vivo. Previous work revealed that mCD43 expression on mature B cells in these mice resulted in immunodeficiency to T-dependent (TD) antigens (Ag), possibly by impairing B-T cell interactions. In the present study we have immunized the TG mice with the T-independent (TI) Ag fluorescein-(Fl) lipopolysaccharide (LPS) (TI type 1 Ag) and Fl-Ficoll (TI type 2 Ag). Surprisingly, the mCD43-Ig enhancer expressing mice were impaired in their ability to mount humoral responses to both Fl-LPS and Fl-Ficoll, and had decreased numbers of cells responding to Ag in vivo. Flow cytometric analysis was performed on peritoneal B-1 cells, a population which often plays a major role in humoral responses to TI Ag such as bacterial Ag. This analysis revealed similar B220, IgM and CD5 expression patterns for the TG and nontransgenic (NTG) B-1 cells. In addition, purified peritoneal B-1 cells from TG and NTG mice were able to respond to LPS. Stimulation of splenic B cells in vitro with Fl-LPS and Fl-Ficoll revealed that, in contrast to NTG B cell responses, TG B cell responses could not be enhanced by co-culture with T cells. However, soluble T cell factor enhancement of the TG B cell responses was normal. These data suggest that the mCD43 expression on B cells may inhibit cell interactions that are important for enhanced TI Ag responses. The anti-adhesive forces of mucins in general may thus be critical in regulating both TD and TI humoral responses.
Asunto(s)
Antígenos CD , Antígenos T-Independientes/inmunología , Linfocitos B/inmunología , Sialoglicoproteínas/fisiología , Animales , Formación de Anticuerpos , Elementos de Facilitación Genéticos , Ficoll/inmunología , Genes de Inmunoglobulinas , Leucosialina , Lipopolisacáridos/inmunología , Activación de Linfocitos , Cooperación Linfocítica , Ratones , Ratones Transgénicos , Cavidad Peritoneal/citologíaRESUMEN
The contribution of T-cell-receptor beta-chain diversity to the T-cell antigen-specific repertoire was investigated using single-chain T-cell-receptor transgenic mice. Animals that express the rearranged beta-chain gene from a T hybridoma with specificity for a hen egg lysozyme peptide, designated HEL (85-96) were analysed for their ability to respond to a panel of diverse antigens. Transgenic mice exhibited a significantly elevated response to HEL (85-96) which was shown to be due to an increased frequency of HEL (85-96)-specific T-cell progenitors. This increased frequency of specific progenitors resulted in the ability of transgenic mice to respond to the peptide in the absence of antigen priming. Conversely, transgenic mice failed to respond to any other antigen tested. Furthermore, this apparent deficiency was associated with a significant decrease in the frequency of antigen-specific T-cell progenitors in transgenic mice. Surprisingly, the ability to launch an alloresponse was unaffected by the exclusive expression of the transgene-derived beta-chain. These results indicate that beta-chain diversity is crucial for the ability of the T-cell population to elicit a rapid and robust response to the profusion of different antigen/major histocompatibility complex (MHC) ligands potentially encountered by an individual. Furthermore, these results suggest a lesser role for beta-chain diversity in contributing to allorecognition, and support a model in which the direct recognition of peptide-mediated conformational MHC forms is the major contributor to the alloreactive response exhibited by the majority of T cells.
Asunto(s)
Epítopos/inmunología , Isoantígenos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Animales , División Celular/inmunología , Grupo Citocromo c/inmunología , Antígenos H-2/inmunología , Tolerancia Inmunológica , Memoria Inmunológica , Ratones , Ratones Transgénicos , Muramidasa/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
Human prostate-specific antigen (PSA) has been widely used as a serum marker for cancer of the prostate. The cell type-specific expression of PSA also makes it a potential tumor antigen for prostate cancer immunotherapy. Study of the immunological aspects of PSA within either normal or malignant prostate tissue has been hampered by the lack of a mouse model, because no PSA counterpart has been identified in mice. Using a 14-kb genomic DNA region that encompasses the entire human PSA gene and adjacent flanking sequences, we generated a series of human PSA transgenic mice. In the six independent lines of transgenic mice generated, the expression of the human PSA transgene, driven by its own cis-acting regulatory elements, is specifically targeted to the prostate. Tissue distribution analysis demonstrated that PSA transgene expression closely follows the human expression pattern. Immunohistochemical analysis of the prostate tissue also showed that the expression of the PSA transgene is confined to the ductal epithelial cells. Despite expressing PSA as a self-antigen in the prostate, these transgenic mice were able to mount a cytotoxic immune response against PSA expressed by tumor cells, indicating that expression of the transgene has not resulted in complete nonresponsiveness. This transgenic mouse model will provide a well defined system to gain an insight into the mechanisms of nonresponsiveness to PSA, ultimately leading to strategies for immunotherapy of human prostate cancer using PSA as the target antigen.
Asunto(s)
Antígeno Prostático Específico/biosíntesis , Antígeno Prostático Específico/genética , Transcripción Genética , Animales , Línea Celular , Citotoxicidad Inmunológica , Cartilla de ADN , Terapia Genética/métodos , Genitales Masculinos/metabolismo , Humanos , Terapia de Inmunosupresión/métodos , Inmunoterapia/métodos , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Antígeno Prostático Específico/inmunología , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , Transfección , Células Tumorales CultivadasRESUMEN
To test the potential for genetically transferring foreign sequences into autologous cells for specific modulation of immunity, we have generated transgenic mice that express an engineered peptide-IgG construct in the peripheral B cell compartment. B cells from these mice express and can be stimulated to secrete a murine IgG1 chain grafted with residues 12-26 from bacteriophage A cI repressor protein in-frame at the heavy chain N terminus. As expected, 12-26-IgG transgenic mice are profoundly tolerant to the peptide at both the T and B cell levels. Importantly, the injection of transgenic whole spleen, purified B cells, or even bone marrow cells into normal, immunocompetent adults results in profound peptide-specific T cell tolerance, as well as partial B cell tolerance. Injection of LPS-activated peptide-Ig-expressing B cells was uniquely effective at diminishing an ongoing humoral immune response typical of both Th1 and Th2 help. Since fixed transgenic B cells were tolerogenic, this suggests that secretion of the fusion protein is not required for tolerogenicity. These results show that an engineered self Ig, as well as B lymphocytes expressing epitopes from such a fusion protein, can regulate both cellular and humoral immune responses. Moreover, these studies provide the basis for expressing foreign epitopes on engineered IgG for the induction of gene-transferred tolerogenesis in autoimmune states.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Tolerancia Inmunológica , Inmunoglobulina G/genética , Activación de Linfocitos , Fragmentos de Péptidos/genética , Factores de Edad , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos B/metabolismo , Médula Ósea/efectos de la radiación , Epítopos/análisis , Femenino , Inmunidad Celular/genética , Inmunización , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/biosíntesis , Interfase/inmunología , Tejido Linfoide/trasplante , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/inmunología , Ingeniería de Proteínas , Quimera por Radiación , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Clinical use of the antineoplastic agent bleomycin is restricted due to pulmonary toxicity. Murine models of bleomycin-induced pulmonary fibrosis have been developed in an attempt to understand the mechanisms involved in the fibrotic process. Studies have shown that the alveolar epithelium is damaged early after bleomycin treatment. The purpose of this study was to evaluate the pattern of gene expression in airway and alveolar epithelial cells after bleomycin exposure in mice that vary in susceptibility to bleomycin-induced fibrosis. Surfactant protein C (SPC) and Clara cell-specific protein (CC10) mRNA were used as cell-specific markers of alveolar type II cells and airway Clara cells, respectively. Mice were treated with a single intratracheal dose of bleomycin and the pattern of SPC and CC10 transcripts was examined by in situ hybridization. The pattern of SPC mRNA 28 days after treatment was uniform in controls and resistant mice but exhibited a patchy appearance in sensitive mice. Bleomycin treatment also resulted in a strain-dependent loss of CC10 mRNA-expressing cells. In sensitive mice 28 days after treatment, SPC mRNA was ectopically expressed in the distal bronchiolar epithelium in a morphologically distinct cell type. Serial sections revealed that these cells either coexpressed CC10 mRNA or were located adjacent to CC10 mRNA-containing cells. This unique cell population may represent a progenitor cell type important in epithelial repair. The strain-dependent changes in CC10 and SPC gene expression after bleomycin treatment are suggestive of a role for the epithelium in pulmonary fibrosis versus repair.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Inhibidores Enzimáticos/metabolismo , Pulmón/efectos de los fármacos , Biosíntesis de Proteínas , Proteolípidos/biosíntesis , Surfactantes Pulmonares/biosíntesis , Uteroglobina , Animales , Antibióticos Antineoplásicos/administración & dosificación , Biomarcadores/análisis , Bleomicina/administración & dosificación , Células Epiteliales , Epitelio/efectos de los fármacos , Hibridación in Situ , Intubación Intratraqueal , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas/análisis , Proteolípidos/análisis , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Surfactantes Pulmonares/análisis , Especificidad de la EspecieRESUMEN
Leukosialin (CD43 or sialophorin) is a cell surface sialoglycoprotein implicated in cell adhesion and proliferation whose tightly regulated expression in B lymphocytes is likely important for their normal development and/or function. To examine the physiologic role of mouse CD43 (mCD43) in vivo, we exploited transgenic (TG) mice whose developmental expression of mCD43 was extended during B cell differentiation so that mCD43 was now expressed on peripheral B cells. Despite having increased B cells, localization of lymphocytes in the TG spleens appeared normal by immunocytochemistry with anti-CD4, anti-CD8, and anti-B220 mAbs. However, the numbers of splenic germinal centers and the resting sera Ig levels were decreased in the TG mice compared with littermate controls. TG mice had decreased humoral responses to the T-dependent Ags keyhole limpet hemocyanin and OVA, as well as reduced Ag-specific B cell numbers. In contrast, in vitro LPS stimulation of purified TG or control B cells resulted in similar proliferation and IgM responses. Thus, the alteration of B cell mCD43 expression that resulted in profound immunodeficiency in vivo was not due to absolute defects in B cell development or Ab production. However, TG B cells had a decreased ability to homotypically aggregate and to present Ag to the T cell hybridoma B3Z. These data suggest that the immunodeficiency seen in vivo is due to the anti-adhesive forces of mCD43 preventing normal T-B cell interaction. This likely reflects a general property of mucins in regulating cell interactions.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/inmunología , Síndromes de Inmunodeficiencia/etiología , Sialoglicoproteínas/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD/genética , Linfocitos B/patología , Adhesión Celular/genética , Adhesión Celular/inmunología , Diferenciación Celular , Expresión Génica , Hemocianinas/inmunología , Inmunoglobulina M/biosíntesis , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Leucosialina , Lipopolisacáridos/farmacología , Activación de Linfocitos , Cooperación Linfocítica/genética , Cooperación Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Modelos Biológicos , Ovalbúmina/inmunología , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Bazo/inmunología , Bazo/patologíaRESUMEN
Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans.
Asunto(s)
Neoplasias Experimentales/inmunología , Antígeno Prostático Específico/fisiología , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas/inmunología , Animales , Epítopos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Masculino , Sarcoma de Mastocitos/genética , Sarcoma de Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Antígeno Prostático Específico/biosíntesis , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , TransfecciónRESUMEN
Although fibroblasts are traditionally described as static cells providing framework and support for tissues, there is an accumulating body of evidence showing that fibroblasts are a dynamic cell type which exist in functionally and morphologically heterogeneous subpopulations. Fibroblast subsets have been shown to play a critical role in the production and regulation of extracellular matrix components, in wound repair and regeneration, and have been implicated in the pathogenesis of fibrotic conditions. We have reviewed the evidence supporting heterogeneity of fibroblasts from pulmonary, periodontal, and dermal tissues. In addition, we will explore the role fibroblast subpopulations may play in the complex process of wound repair and regeneration.
RESUMEN
Leukosialin (also known as Ly48, CD43, and sialophorin) is a major cell surface sialoglycoprotein found on a variety of hematopoietically derived cells. The precise function of this molecule is poorly understood but it has been implicated in cell proliferation and intercellular adhesion. We developed a transgenic mouse model to assess leukosialin's function in vivo. Our approach was to alter mouse CD43 (mCD43) expression in the B-cell lineage where it is tightly regulated, by expressing it in peripheral B cells where it is normally absent. To drive expression of leukosialin in mature B cells, the immunoglobulin heavy chain enhancer was fused to the mCD43 gene. mCD43-immunoglobulin heavy chain enhancer transgenic mice display splenomegaly due to increased numbers of B cells. Transgenic B cells show a striking increase in their ability to survive in vitro compared to B cells from nontransgenic control mice. This prolonged survival is reflected in a decreased susceptibility to apoptosis. These observations suggest that mCD43 plays an important role in the regulation of B-cell survival. The alteration of the temporal expression, or "disregulation," of a gene in transgenic mice provides a general strategy for elucidating the in vivo role of other molecules involved in cell signaling and adhesion.
Asunto(s)
Antígenos CD , Linfocitos B/inmunología , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Sialoglicoproteínas/biosíntesis , Bazo/inmunología , Animales , Apoptosis , Recuento de Células , Diferenciación Celular , Supervivencia Celular , Elementos de Facilitación Genéticos/genética , Citometría de Flujo , Leucosialina , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/biosíntesis , Bazo/citologíaRESUMEN
Since mouse keratinocytes are tolerogenic antigen presenting cells for T cell activation, the expression of second signal molecules such as B7-1 was targeted to epidermal keratinocytes (KC) in vivo in transgenic mice. The expression vector used to create transgenic mice consisted of a keratin 14 promoter fused 5' to the full length open reading frame of the cDNA encoding mouse B7-1 (between 10 and 30 copies of the transgene per genome). Expression of B7-1 cell surface protein was assessed by in situ immunostaining of cryostat sections of tail skin with CTLA-4/Ig fusion protein, revealing high levels of cell surface expression of B7 by all epidermal KC of transgenic mice, and a lack of such expression in nontransgenic animals. The skin of such transgenic mice (derived from three different founder mice) was grossly and histologically normal, with normal numbers of Langerhans cells and dendritic epidermal T cells. Immunologic challenge of transgenic mice with epicutaneous haptens such as fluorescein isothiocyanate revealed enhanced and persistent delayed-type hypersensitivity responses, with an altered kinetics of resolution when compared with nontransgenic controls. These data indicate that in normal, nontransgenic mice, tolerogenic antigen presentation by KC plays an important physiologic role in damping T cell-mediated inflammation in the skin by competing with professional APC for TCR occupancy in antigen specific T-lymphocytes that migrate into the epidermis. This also implies that altered regulation of B7-1 gene expression by epidermal cells may account for skin "hyperresponsiveness" encountered in some chronic dermatologic disorders.
Asunto(s)
Antígeno B7-1/análisis , Hipersensibilidad Tardía/inmunología , Inmunoconjugados , Queratinocitos/inmunología , Abatacept , Animales , Antígenos CD , Antígenos de Diferenciación/metabolismo , Antígeno CTLA-4 , Tolerancia Inmunológica , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Linfocitos T/inmunologíaRESUMEN
Susceptibility of mice to the induction of pulmonary fibrosis by bleomycin sulfate is inbred strain dependent, with C57BL/6 mice exhibiting high sensitivity to the drug and BALB/c mice demonstrating a resistant phenotype. The lungs of bleomycin treated C57BL/6J and BALB/cBy mice were analyzed for their mRNA expression level of a panel of cytokines using a semi-quantitative polymerase chain reaction (SQ-PCR) assay. Transforming growth factor-beta 1 (TGF-beta 1) mRNA was found to increase sevenfold by 5 days after bleomycin treatment of C57BL/6J (sensitive) mice. BALB/cBy (resistant) animals demonstrated a lower level of TGF-beta 1 mRNA induction, approximately threefold, after bleomycin administration. Analysis of interleukin-1 beta (IL-1 beta) mRNA levels also revealed a difference between the two strains, with BALB/cBy mice expressing approximately fourfold higher IL-1 beta mRNA levels than C57BL/6J mice. This result suggested possible protection by IL-1 beta. Analysis of (C57BL/6JxBALB/cBy)F1 hybrids, which are shown in this report to be sensitive to bleomycin-induced fibrosis, revealed a high IL-1 beta mRNA level, similar to that in the resistant parent. Thus, the observed strain variation in the level of IL-1 beta mRNA is not associated with differences in susceptibility to the induction of pulmonary fibrosis. In contrast, strain variation in interleukin-6 (IL-6) mRNA levels was observed that was completely concordant with the segregation of susceptibility phenotypes between the parental and F1 strains. This result indicates a possible association between sensitivity to bleomycin-induced fibrosis and inducibility of IL-6 mRNA upon drug treatment. Analysis of TGF-beta 2, interferon-gamma, interleukin-2, interleukin-3, and interleukin-4 (IL-4) mRNA showed no detectable strain variation in steady state mRNA levels in the lung as a consequence of bleomycin treatment. In contrast, the level of IL-4 receptor mRNA was induced to a higher degree in both sensitive groups (C57BL/6J and F1) than in resistant mice (BALB/cBy). Therefore, modulation of the IL-4 response, not at the level of IL-4 but through regulation of the IL-4 receptor, may play a role in pulmonary fibrogenesis.