RESUMO
During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.
Assuntos
Diferenciação Celular/imunologia , Inflamação/imunologia , MAP Quinase Quinase 6/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Animais , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/imunologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HL-60 , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/farmacologia , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/imunologia , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.
Assuntos
Adesões Focais/metabolismo , Filamentos Intermediários/metabolismo , Mecanotransdução Celular/fisiologia , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Ácido Okadáico/farmacologia , Plectina/metabolismo , Vimentina/metabolismoRESUMO
Environmentally exposed epithelial Langerhans cells (LCs) encounter diverse innate stress signals, which lead to the activation of complex intracellular signaling cascades. Among these, p38 MAPK is consistently phosphorylated. For which aspects of LC activation triggering of p38 signaling is sufficient remains to be elucidated. We show that conditional induction of a dominant active form of MAPK kinase 6 (d.a.MKK6), a direct upstream kinase of p38, in LCs efficiently induces the up-regulation of costimulatory molecules and enhances their T-cell stimulatory capacity. These immediate effects showed no or only a minor requirement for classical NF-kappaB signaling. Concomitant with LC activation, d.a.MKK6 induced the alternative NF-kappaB member RelB, whose nuclear localization marks mature DCs. Specific inhibition of nuclear RelB during d.a.MKK6-induced LC activation further enhanced their maturation state. This observation was validated using the p38 activator anisomycin, thus suggesting a novel LC intrinsic control mechanism regulated by RelB.
Assuntos
Células de Langerhans/citologia , MAP Quinase Quinase 6/fisiologia , Fator de Transcrição RelB/fisiologia , Anisomicina/farmacologia , Apresentação de Antígeno , Antígeno B7-1/biossíntese , Antígeno B7-1/genética , Antígeno B7-2/biossíntese , Antígeno B7-2/genética , Caspase 1/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Meios de Cultura Livres de Soro , Citocinas/farmacologia , Doxiciclina/farmacologia , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Genes Dominantes , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Células de Langerhans/metabolismo , MAP Quinase Quinase 6/genética , Modelos Imunológicos , NF-kappa B/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/genética , Linfócitos T/imunologia , Células U937 , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Neutrophil granulocytes (Gs) represent highly abundant and short-lived leukocytes that are constantly regenerated from a small pool of myeloid committed progenitors. Nuclear receptor (NR) family members are ligand-activated transcription factors that play key roles in cellular proliferation and differentiation processes including myelopoiesis. Retinoid X receptor alpha (RXRalpha) represents the predominant NR types I and II homo- and heterodimerization partner in myeloid cells. Here we show that human myeloid progenitors express RXRalpha protein at sustained high levels during macrophage colony-stimulating factor (M-CSF)-induced monopoiesis. In sharp contrast, RXRalpha is down-regulated during G-CSF-dependent late-stage neutrophil differentiation from myeloid progenitors. Down-regulation of RXRalpha is critically required for neutrophil development since ectopic RXRalpha inhibited granulopoiesis by impairing proliferation and differentiation. Moreover, ectopic RXRalpha was sufficient to redirect G-CSF-dependent granulocyte differentiation to the monocyte lineage and to promote M-CSF-induced monopoiesis. Functional genetic interference with RXRalpha signaling in hematopoietic progenitor/stem cells using a dominant-negative RXRalpha promoted the generation of late-stage granulocytes in human cultures in vitro and in reconstituted mice in vivo. Therefore, our data suggest that RXRalpha down-regulation is a critical requirement for the generation of neutrophil granulocytes.
Assuntos
Células Progenitoras Mieloides/citologia , Neutrófilos/citologia , Receptor X Retinoide alfa/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Regulação para Baixo/genética , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Mielopoese/efeitos dos fármacos , Transdução GenéticaRESUMO
Langerhans cells (LCs) are highly abundant dendritic cells (DCs) in epidermal and mucosal tissues. The transcription factors PU.1 and Id2 have been implicated as positive regulators of LC development from hematopoietic progenitor cells. LC differentiation from progenitors is absolutely dependent on transforming growth factor beta 1 (TGF-beta1) in vitro as well as in vivo; however, downstream mechanisms are poorly defined. We found that both PU.1 and Id2 are induced by TGF-beta1 in human CD34+ monocyte/LC (M/LC) progenitor cells, and that neither ectopic PU.1 or Id2 alone, nor both together, could replace TGF-beta1 in its instructive function on LC commitment. However, both factors critically contributed to LC differentiation by acting at 2 distinct intersection points. Ectopic PU.1 strongly enhanced TGF-beta1-dependent LC development. Additionally, Notch-induced generation of interstitial-type DCs was associated with PU.1 up-regulation. Thus, PU.1 is generally increased during myeloid DC development. Ectopic Id2 inhibits the acquisition of early monocytic characteristics by cells generated in the absence of TGF-beta1 and also inhibits monocyte induction by alternative stimuli. Since TGF-beta1 represses a default monocyte pathway of common progenitor cells, PU.1 and Id2 seem to modulate lineage options of M/LC precursors, downstream of TGF-beta1.
Assuntos
Proteína 2 Inibidora de Diferenciação/fisiologia , Células de Langerhans/imunologia , Células-Tronco/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Antígenos CD/imunologia , Antígenos CD34/imunologia , Técnicas de Cultura de Células , Primers do DNA , Sangue Fetal/fisiologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Recém-Nascido , Reação em Cadeia da Polimerase , Células-Tronco/imunologia , Fator de Crescimento Transformador beta1RESUMO
In humans, epithelial Langerhans cells (LCs) and monocyte-derived/interstitial dendritic cells (DCs) constitute 2 myeloid DC sublineages. Molecular mechanisms involved in their development from common myeloid progenitors remain poorly defined. Here we demonstrate that the nuclear factor-kappaB (NF-kappaB) transcription factor RelB regulates the generation of monocytic CD14(+)CD11b(+) precursors of interstitial DCs from human hematopoietic progenitors. RelB overexpression promoted, whereas endogenous RelB inhibition (by p100DeltaN) blocked, precursor cell development along this DC subset pathway. RelB inhibition specifically arrested precursor progression from CD14(lo)CD11b(-) to CD14(+)CD11b(+) stages. Precursors were still capable of LC and granulocyte differentiation but were defective in macrophage-colony-stimulating factor (M-CSF)-dependent monocyte/macrophage differentiation. RelB inhibition markedly differed from classical NF-kappaB signaling inhibition because IkappaBalpha superrepressor (IkappaBalpha-SR), but not p100DeltaN, impaired LC/DC differentiation, DC adhesion, and progenitor cell proliferation. Although RelB up-regulation and nuclear translocation are regarded as hallmarks of human myeloid DC maturation, ectopic RelB overexpression failed to promote DC maturation. Our results suggest that RelB regulates human monopoiesis and monocyte-derived DC subset development.
Assuntos
Células Dendríticas/citologia , Monócitos/citologia , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas I-kappa B/farmacologia , Células Progenitoras Mieloides/citologia , Inibidor de NF-kappaB alfa , Fator de Transcrição RelBRESUMO
The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe skin blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause skin blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.