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1.
J Exp Med ; 187(2): 245-51, 1998 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-9432982

RESUMO

Efficient loading of major histocompatibility complex class II molecules with peptides requires the invariant chain (Ii) and the class II-like molecule H-2M. Recent in vitro biochemical studies suggest that H2-M may function as a chaperone to rescue empty class II dimers. To test this hypothesis in vivo, we generated mice lacking both Ii and H-2M (Ii-/-M-/-). Antigen presenting cells (APCs) from Ii-/-M-/- mice, as compared with APCs from Ii-/- mice, exhibit a significant reduction in their ability to present self-peptides to a panel of class II I-Ab-restricted T cells. As a consequence of this defect in the loading of self peptides, CD4(+) thymocyte development is profoundly impaired in Ii-/-M-/- mice, resulting in a peripheral CD4(+) T cell population with low levels of T cell receptor expression. These findings are consistent with the idea that H-2M functions as a chaperone in the peptide loading of class II molecules in vivo.


Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos H-2/genética , Antígenos H-2/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/fisiologia , Peptídeos/imunologia , Peptídeos/metabolismo , Animais , Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/genética , Autoantígenos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/fisiologia , Ligação Proteica/genética
2.
J Immunol ; 144(4): 1295-303, 1990 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-2137488

RESUMO

The internalization and intracellular transport of IL-1 and its receptor were examined in the murine T cell line EL-4. For 4 h after internalization intracellular 125I-IL-1 alpha remains bound to its receptor without degradation. Electron microscope autoradiography demonstrates that internalized IL-1 accumulates in purified nuclei. The IL-1 extracted from these nuclei is still bound to receptor. As no receptors for IL-1 were detected in untreated nuclei, these results suggest IL-1 driven translocation of the cell surface IL-1R complex to the nucleus. IL-1R internalization was correlated with IL-1 signal transduction events required to induce growth factor production from several subclones of EL-4 cells. The subsequent transport of the internalized IL-1R complex to the nucleus suggests the possibility for a nuclear site for IL-1R signaling.


Assuntos
Interleucina-1/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Compartimento Celular , Linhagem Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Camundongos , Dados de Sequência Molecular , Peso Molecular , Receptores de Interleucina-1 , Transdução de Sinais , Fatores de Tempo
3.
Eur J Immunol ; 26(12): 3185-93, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8977321

RESUMO

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide: MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of E alpha52-68: I-A(b) and class II-associated invariant chain peptide (CLIP): I-A(b) complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of E alpha52-68: I-A(b) complexes in thymic epithelial cells is approximately 5-10% of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A(b) complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.


Assuntos
Complexo Antígeno-Anticorpo/biossíntese , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Biossíntese Peptídica , Timo/metabolismo , Animais , Antígenos CD/biossíntese , Epitélio/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
4.
Immunity ; 7(2): 197-208, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9285405

RESUMO

The role of self-peptides in positive selection of CD4+ T cells has been controversial. We show that some self-peptides are presented by the MHC class II molecule I-A(b) in mice lacking Ii or H-2M but not in mice expressing a transgene-encoded peptide fused to I-A(b). In experiments using specific antibodies to block selection, these low-abundance self-peptides were implicated in the positive selection of some CD4+ T cells in H-2M-/- mice. However, all three mutant backgrounds failed to positively select two class II-restricted transgenic T cell receptors. Our findings suggest that minor components of the self-peptide repertoire can contribute to positive selection of a significant number of CD4+ T cells. In addition, the data suggest that T cell receptor repertoires selected in wild-type mice and in mice displaying limited spectra of self-peptides are distinct.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Epitélio/imunologia , Epitélio/metabolismo , Feminino , Antígenos H-2/genética , Antígenos H-2/metabolismo , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Timo/imunologia , Timo/metabolismo , Transgenes/imunologia
5.
J Biol Chem ; 266(33): 22661-70, 1991 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-1658005

RESUMO

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.


Assuntos
Interleucina-1/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Cátions Bivalentes/farmacologia , Linhagem Celular , Ativação Enzimática , Humanos , Células KB , Cinética , Camundongos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato
6.
Immunity ; 1(7): 585-94, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7600287

RESUMO

To define the intracellular site of assembly of endogenous peptide-MHC class II complexes, an immunochemical approach was undertaken employing a monoclonal antibody specific for an endogenous peptide-class II complex in combination with subcellular fractionation. Here, we show that newly synthesized MHC class II molecules, upon exit from the Golgi, are delivered into a dense endocytic compartment (MIIC) distinct from late endosomes and lysosomes. Endogenous peptide-class II complexes are initially formed in this compartment and subsequently traffic through late endosomal vesicles prior to cell surface expression. Exogenous antigen delivered via immunoglobulin receptors is targeted to MIIC en route to lysosomes after passing through early and late endosomes. Processing of an endocytosed antigen was observed in this compartment. Our results suggest a specific role for MIIC in the processing of endogenous and exogenous proteins as well as the assembly of peptide-MHC class II complexes.


Assuntos
Apresentação de Antígeno , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Peptídeos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Células Apresentadoras de Antígenos/ultraestrutura , Compartimento Celular , Fracionamento Celular , Linhagem Celular , Membrana Celular/metabolismo , Endossomos/metabolismo , Citometria de Fluxo , Cinética , Lisossomos/metabolismo , Camundongos , Modelos Imunológicos , Testes de Precipitina , Fatores de Tempo
7.
Proc Natl Acad Sci U S A ; 86(9): 3045-9, 1989 May.
Artigo em Inglês | MEDLINE | ID: mdl-2470087

RESUMO

We have cloned a cDNA encoding a receptor identical to the native Mr 80,000 glycoprotein that binds interleukin (IL) 1 alpha and -beta in murine T cells. Chinese hamster ovary (CHO) cells transfected with this T-cell IL-1 receptor (IL-1R) [CHO(IL-1R)] cDNA express approximately 100,000 IL-1Rs per cell, compared to the less than 100 receptors present on control CHO cells. For two functional responses to IL-1, prostaglandin synthesis and cytokine secretion, CHO(IL-1R) cells were 1000 times more sensitive to IL-1 alpha than were control CHO cells. Northern blot analysis and antibody precipitation demonstrated that one of the cytokines induced was granulocyte colony-stimulating factor and that mRNA levels for this cytokine were increased in CHO(IL-1R) cells by IL-1 alpha concentrations that had no effect on control cells. To establish the role of the recombinant receptors in signal transduction, an IL-1R cDNA modified by deletion of the predicted cytoplasmic domain was expressed in the CHO cell line termed CHO(IL-1R delta CT). CHO(IL-1R delta CT) cells expressed approximately 100,000 high-affinity IL-1 binding sites per cell, but these cells were less sensitive than control lines to IL-1, as measured by prostaglandin and cytokine release. These results show that the IL-1R cDNA encodes the entire functional receptor and that the cytoplasmic domain is required for signal transduction but not ligand binding.


Assuntos
DNA/genética , Regulação da Expressão Gênica , Interleucina-1/fisiologia , Receptores Imunológicos/genética , Linfócitos T/metabolismo , Animais , Linhagem Celular , Fatores Estimuladores de Colônias/biossíntese , Fatores Estimuladores de Colônias/genética , Cricetinae , Dinoprostona/biossíntese , Fator Estimulador de Colônias de Granulócitos , Cinética , Camundongos , RNA Mensageiro/metabolismo , Receptores de Interleucina-1 , Proteínas Recombinantes , Transdução de Sinais , Transfecção
8.
Eur J Immunol ; 26(2): 385-93, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8617308

RESUMO

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP:MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP:MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Li peptide bound to the peptide-binding groove.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/química , Linfócitos B/imunologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/química , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/química
9.
Proc Natl Acad Sci U S A ; 88(13): 5809-13, 1991 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-1829529

RESUMO

Granulocyte/macrophage colony-stimulating factor-interleukin 3 (GM-CSF-IL-3) fusion proteins were generated by construction of a plasmid in which the coding regions of human GM-CSF and IL-3 cDNAs were connected by a synthetic linker sequence followed by subsequent expression in yeast. Both GM-CSF-IL-3 and IL-3-GM-CSF fusion proteins were purified to homogeneity and shown to bind to cell-surface receptors through either their GM-CSF or IL-3 domains. The fusion proteins exhibited enhanced receptor affinity, proliferative activity, and hematopoietic colony-stimulating activity compared with either IL-3 and/or GM-CSF alone. This suggests that GM-CSF-IL-3 fusion proteins may hold future promise as therapeutic agents.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Hematopoese/efeitos dos fármacos , Interleucina-3/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Bases , Ligação Competitiva , Clonagem Molecular , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta a Droga , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Humanos , Técnicas In Vitro , Interleucina-3/química , Dados de Sequência Molecular , Oligonucleotídeos/química , Conformação Proteica , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes de Fusão/química
10.
Eur J Immunol ; 31(5): 1485-94, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11465105

RESUMO

We report a strategy for high through-put sequence analyses of large MHC class II-bound peptide repertoires which combines automated electrospray ionization tandem mass-spectrometry with computer-assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I-Ab in activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H-2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II-bound self peptides derived from cytosolic proteins exists in vivo.


Assuntos
Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Autoantígenos/química , Autoantígenos/imunologia , Citosol/química , Antígenos de Histocompatibilidade Classe II/imunologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Algoritmos , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/metabolismo , Automação/métodos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Cromatografia Líquida de Alta Pressão , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Hibridomas/imunologia , Tolerância Imunológica/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/imunologia , Proteínas/metabolismo , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia
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