RESUMEN

Antibodies to the pan-leukocyte adhesion-GPCR CD97 efficiently block neutrophil recruitment in mice, thereby reducing antibacterial host defense, inflammatory disease, and hematopoietic stem cell mobilization. Here, we investigated the working mechanism of the CD97 antibody 1B2. Applying sterile models of inflammation, intravital microscopy, and mice deficient for the CD97L CD55, the complement component C3, or the FcR common γ-chain, we show that 1B2 acts in vivo independent of ligand-binding interference by depleting PMN granulocytes in bone marrow and blood. Granulocyte depletion with 1B2 involved FcR but not complement activation and was associated with increased serum levels of TNF and other proinflammatory cytokines. Notably, depletion of granulocytes by CD97 antibody required acute inflammation, suggesting a mechanism of conditional, antibody-mediated granulocytopenia.

Asunto(s)

Anticuerpos Bloqueadores/farmacología , Granulocitos/efectos de los fármacos , Granulocitos/inmunología , Inflamación/inmunología , Glicoproteínas de Membrana/inmunología , Receptores Fc/inmunología , Animales , Especificidad de Anticuerpos/efectos de los fármacos , Antígenos CD55/inmunología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Humanos , Inflamación/complicaciones , Inflamación/patología , Leucotrieno B4/farmacología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutropenia/complicaciones , Neutropenia/inmunología , Neutropenia/patología , Peritonitis/complicaciones , Peritonitis/inmunología , Peritonitis/patología , Receptores Acoplados a Proteínas G , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

CD97 is a member of the EGF-TM7 family of adhesion class receptors, with a proposed role in inflammatory cell recruitment. Neutralization of murine CD97 with the anti-mCD97 mAb 1B2 was efficacious in prevention of murine collagen-induced arthritis, a model with features resembling rheumatoid arthritis. Here, the therapeutic potential of neutralizing CD97 in arthritis was studied with emphasis on the 1B2 pharmacokinetics. Mice with established arthritis were treated with anti-mCD97 or anti-TNF-alpha serum. Ab pharmacokinetics and biodistribution were studied in diseased and nondiseased mice using labeled 1B2. The impact of CD97 expression on Ab pharmacokinetics was studied using CD97 knockout mice. Treatment with 1B2 showed an efficacy comparable to anti-TNF-alpha treatment. Pharmacokinetic analysis of 1B2 in wild-type and CD97 knockout mice indicated a dose-dependent Ab clearance, due to specific interaction with CD97. Biodistribution studies showed accumulation of 1B2 in spleen and lung. In vitro studies using murine splenocytes revealed that CD97 when bound to Ab was internalized. Moreover, soluble CD97 was detected in the supernatant, suggesting Ag shedding. Finally, in arthritic mice, higher levels of soluble CD97 were found and 1B2 treatment led to specific targeting of inflamed paws, resulting in a higher clearance rate of 1B2 in arthritic mice than in wild-type mice. In conclusion, our data support a therapeutic value of CD97 neutralization in experimental arthritis. The pharmacokinetic profile of the 1B2 Ab illustrates the complexity of Ab elimination from an organism and stresses the importance of understanding Ag-Ab interactions when developing therapeutic mAbs.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Monoclonales/farmacocinética , Antígenos , Relación Dosis-Respuesta a Droga , Pulmón/metabolismo , Ratones , Ratones Noqueados , Farmacocinética , Receptores Acoplados a Proteínas G , Bazo/metabolismo , Distribución Tisular

RESUMEN

The heptahelical receptor CD97 is a defining member of the EGF-TM7 family of adhesion class receptors. In both humans and mice, CD97 isoforms are expressed with variable numbers of tandemly arranged N-terminal epidermal growth factor-like domains that facilitate interactions with distinct cellular ligands. Results from treatment of mice with mAbs in various disease models have suggested a role for CD97 in leukocyte trafficking. Here, we aimed to thoroughly characterize the expression profile of CD97, and delineate its biological function. To this end, we applied a novel polyclonal Ab, which is the first antiserum suitable for immunohistochemistry, and combined this analysis with the study of Cd97-lacZ knock-in mice. We show that similar to the situation in humans, hematopoietic, epithelial, endothelial, muscle, and fat cells expressed CD97. Despite this broad expression pattern, the Cd97(-/-) mouse that we created had no overt phenotype, except for a mild granulocytosis. Furthermore, granulocyte accumulation at sites of inflammation was normal in the absence of CD97. Interestingly, application of CD97 mAbs blocked granulocyte trafficking after thioglycollate-induced peritonitis in wild-type but not in knock-out mice. Hence, we conclude that CD97 mAbs actively induce an inhibitory effect that disturbs normal granulocyte trafficking, which is not perturbed by the absence of the molecule.

Asunto(s)

Anticuerpos/administración & dosificación , Inhibición de Migración Celular/inmunología , Regulación de la Expresión Génica/inmunología , Marcación de Gen , Granulocitos/citología , Granulocitos/inmunología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Animales , Anticuerpos/fisiología , Inhibición de Migración Celular/genética , Femenino , Marcación de Gen/métodos , Granulocitos/metabolismo , Inmunofenotipificación , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Leucocitosis/genética , Leucocitosis/inmunología , Leucocitosis/patología , Pulmón/citología , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Receptores Acoplados a Proteínas G , Bazo/citología , Bazo/inmunología , Bazo/metabolismo

RESUMEN

The TLX1/HOX11 homeobox gene is frequently activated in T-cell acute lymphoblastic leukaemia (T-ALL) by the t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations or by as yet unknown transcriptional mechanisms in the absence of 10q24 cytogenetic abnormalities. Almost all TLX1(+) T-ALLs exhibit a CD4(+)CD8(+) double-positive (DP) phenotype. To investigate the role of TLX1 as an initiating oncogene in T-ALL pathogenesis, we assessed the consequences of retroviral vector-directed TLX1 expression during the differentiation of murine and human thymocytes in fetal thymic organ cultures. Interestingly, enforced expression of TLX1 disrupted the differentiation of murine fetal liver precursors and human cord blood CD34(+) stem/progenitor cells prior to the DP thymocyte stage. Although differentiation arrest was associated with an increased percentage of apoptotic thymocytes, it could only be partially bypassed by coexpression of transgenic BCL2. Mutation of the invariant asparagine residue at position 51 of the homeodomain - which is required for efficient DNA binding - released the block, consistent with the notion that TLX1 inhibits thymocyte differentiation and promotes T-cell oncogenesis by functioning as a transcription factor. The relevance of these findings is discussed in the context of activating NOTCH1 mutations and the other genetic lesions implicated in the multistep transformation process of TLX1(+) T-ALL.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de Homeodominio/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas Proto-Oncogénicas/genética , Timo/inmunología , Animales , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Genes Homeobox , Genes bcl-2/inmunología , Vectores Genéticos , Proteínas de Homeodominio/metabolismo , Humanos , Inmunofenotipificación , Leucemia-Linfoma de Células T del Adulto/inmunología , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas/metabolismo , Retroviridae/genética , Transducción Genética

RESUMEN

Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context-specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

Asunto(s)

Linfocitos/fisiología , Proteínas Proto-Oncogénicas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/genética , Animales , Linfocitos B/patología , Linfocitos B/fisiología , Transformación Celular Neoplásica/genética , Metilación de ADN , Regulación de la Expresión Génica , Linfocitos/patología , Linfoma de Células T/genética , Linfoma de Células T/patología , Ratones , Ratones SCID , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Células Madre/fisiología , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Timo/crecimiento & desarrollo , Timo/fisiología , Transactivadores/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo

RESUMEN

Differentiation of hematopoietic stem and progenitor cells is under strict control of a regulatory network orchestrated by lineage-specific transcription factors. A block in normal differentiation is a major contributing factor in the development of solid tumors and leukemias. Cells from patients with acute myeloid leukemia (AML) frequently harbor mutated or dysregulated transcription factor genes, suggesting their involvement in leukemogenesis. As a consequence, these alterations diminish the pool of available molecules of a small number of critical transcription factors, such as CCAAT enhancer binding proteins, PU.1, GATA-1, and AML-1. In this review, we focus on the mechanisms of how this functional pool of transcription factors is maintained during normal and malignant hematopoiesis, including direct protein-protein interactions, competition for DNA binding, and the control of transcription factor genes by proximal and distal regulatory elements. Results of recent studies of mice carrying hypomorphic PU.1 alleles have indicated that reduction in the expression of a single transcription factor is capable of predisposing mice to AML. The implications of these findings for the study of hematopoiesis in the future as well as novel approaches to more disease-specific therapies are discussed.

Asunto(s)

Hematopoyesis , Leucemia/genética , Factores de Transcripción/fisiología , Factor de Transcripción Activador 2 , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Leucemia/patología , Ratones , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

T-cell development requires cytokines and intimate contact with stromal cells provided exclusively by the thymus. Consequently, an in vitro model of thymocyte differentiation, fetal thymic organ culture (FTOC), has been developed. FTOC recapitulates the normal development of T-cells derived from both mouse and human progenitor populations, providing a more rapid means to study T-cell development compared with alternative in vivo approaches. Furthermore, FTOC is easily amenable to genetic manipulation using retroviral gene transfer. In this chapter, we outline the basic FTOC technique and describe several applications, including retroviral transduction of mouse thymocyte subsets and human CD34+ stem/progenitor cells.

Asunto(s)

Diferenciación Celular/fisiología , Linfopoyesis/fisiología , Retroviridae , Linfocitos T/fisiología , Timo/fisiología , Transducción Genética/métodos , Animales , Diferenciación Celular/genética , Células Madre Hematopoyéticas/fisiología , Humanos , Linfopoyesis/genética , Ratones , Técnicas de Cultivo de Órganos/métodos , Timo/citología

RESUMEN

The transcription factor C/EBP alpha is required for granulopoiesis and frequently disrupted in human acute myeloid leukemia (AML). Here, we show disruption of C/EBP alpha blocks the transition from the common myeloid to the granulocyte/monocyte progenitor but is not required beyond this stage for terminal granulocyte maturation. C/EBP alpha-deficient hematopoietic stem cells (HSCs) have increased expression of Bmi-1 and enhanced competitive repopulating activity. Bone marrow in adult C/EBP alpha-deficient mice was filled with myeloblasts, similar to human AML, supporting the notion that disruption of C/EBP alpha cooperates with other events in the development of leukemia. Therefore, C/EBP alpha is not only essential for granulocyte development but, in addition, is a regulator of hematopoietic stem cell activity.

Asunto(s)

Proteína alfa Potenciadora de Unión a CCAAT/deficiencia , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Envejecimiento/fisiología , Animales , Recuento de Células Sanguíneas , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Feto/metabolismo , Eliminación de Gen , Granulocitos/citología , Granulocitos/metabolismo , Hematopoyesis , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Noqueados

RESUMEN

HOX11 encodes a homeodomain protein that is aberrantly expressed in T-cell acute lymphoblastic leukemia as a consequence of the t(10;14) and t(7;10) chromosomal translocations. We previously reported that HOX11 immortalizes murine hematopoietic progenitors and induces pre-T-cell tumors in mice after long latency. It has been demonstrated in a number of studies that HOX11, similar to other homeodomain proteins, binds DNA and transactivates transcription. These findings suggest that translocation-activated HOX11 functions as an oncogenic transcription factor. Here we report that HOX11 represses transcription through both TATA-containing and TATA-less promoters. Interestingly, transcriptional repression by HOX11 is independent of its DNA binding capability. Moreover, a systematic mutational analysis indicated that repressor activity was separable from immortalizing function, which requires certain residues within the HOX11 homeodomain that make base-specific or phosphate-backbone contacts with DNA. We further showed that the pathologic action of HOX11 involves DNA binding-dependent transcriptional pathways that are distinct from those controlling expression of a chromosomal target gene (Aldh-1). We conclude that dysregulated expression of a particular set of downstream target genes by DNA binding via the homeodomain is of central importance for leukemia initiation mediated by HOX11.

Asunto(s)

Transformación Celular Neoplásica , ADN/metabolismo , Proteínas de Homeodominio/fisiología , Proteínas Oncogénicas/fisiología , Células 3T3 , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Femenino , Citometría de Flujo , Regulación de la Expresión Génica , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Inmunohistoquímica , Isoenzimas/genética , Leucemia-Linfoma de Células T del Adulto/genética , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Retinal-Deshidrogenasa , Relación Estructura-Actividad , Transcripción Genética , Transfección

RESUMEN

Dysregulation of homeobox (HB)-containing genes is becoming increasingly recognized as the underlying basis of many hematologic malignancies. Expression of clustered HB (HOX) genes within the hematopoietic system, and enforced overexpression and knockout studies have provided support for the concept that these homeodomain-containing transcription factors play a significant role in the developmental biology of hematopoietic cells. Diverged HB (non-HOX) genes have recently been identified as either cofactors and/or accelerators of leukemic disease mediated by HOX genes or as bona fide oncogenes. In this review, we examine the evidence that supports a central role for HB genes in normal and malignant hematopoiesis, paying particular attention to the non-HOX class and the possible mechanisms through which they contribute to leukemic transformation.

Asunto(s)

Transformación Celular Neoplásica/genética , Regulación Leucémica de la Expresión Génica/genética , Genes Homeobox/genética , Hematopoyesis/genética , Leucemia/genética , Animales , Diferenciación Celular/genética , División Celular/genética , Humanos , Leucemia/metabolismo , Leucemia/fisiopatología , Proteínas Represoras/genética , Activación Transcripcional/genética