RESUMO
WNT proteins constitute a very conserved family of secreted glycoproteins that act as short-range ligands for signaling with critical roles in hematopoiesis, embryonic development, and tissue homeostasis. These proteins transduce signals via the canonical pathway, which is ß-catenin-mediated and better-characterized, or via more diverse noncanonical pathways that are ß-catenin independent and comprise the planar cell polarity (PCP) pathway and the WNT/Ca++ pathways. Several proteins regulate Wnt signaling through a variety of sophisticated mechanisms. Disorders within the pathway can contribute to various human diseases, and the dysregulation of Wnt pathways by different molecular mechanisms is implicated in the pathogenesis of many types of cancer, including the hematological malignancies. The types of leukemia differ considerably and can be subdivided into chronic, myeloid or lymphocytic, and acute, myeloid or lymphocytic, leukemia, according to the differentiation stage of the predominant cells, the progenitor lineage, the diagnostic age strata, and the specific molecular drivers behind their development. Here, we review the role of Wnt signaling in normal hematopoiesis and discuss in detail the multiple ways canonical Wnt signaling can be dysregulated in acute leukemia, including alterations in gene expression and protein levels, epigenetic regulation, and mutations. Furthermore, we highlight the different impacts of these alterations, considering the distinct forms of the disease, and the therapeutic potential of targeting Wnt signaling.
Assuntos
Sinalização do Cálcio/imunologia , Epigênese Genética/imunologia , Regulação Leucêmica da Expressão Gênica/imunologia , Hematopoese/imunologia , Leucemia/imunologia , Via de Sinalização Wnt/imunologia , Doença Aguda , Animais , HumanosRESUMO
The S-layer is a (glyco)-proteinaceous envelope constituted by self-assembled subunits that form a two-dimensional lattice covering the surface of different species of Bacteria and Archaea. It could be considered as one of the most abundant biopolymers in our planet. Because of their unique self-assembly features, exhibiting repetitive identical physicochemical properties down to the subnanometer scale, as well as their involvement in specific interactions with host cells, the S-layer proteins (SLPs) show a high potential application in different areas of biotechnology, including the development of antigen carriers or new adjuvants. The presence of a glycosylated SLP on potentially probiotic Lactobacillus kefiri strains was previously described by our research group. In this study, we aim to investigate the role of carbohydrates present in the SLP from L. kefiri CIDCA 8348 (SLP-8348) in their internalization by murine macrophages, as well as to analyze their immunomodulatory capacity and their effect on LPS-stimulated macrophages. RAW 264.7 cells internalized the SLP-8348 in a process that was mediated by carbohydrate-receptor interactions since it was inhibited by glucose, mannose or EGTA, a Ca+2 chelating agent. These results correlated with the recognition of SLP-8348 by ConA lectin. We further show that while SLP-8348 was not able to induce the activation of macrophages by itself, it favored the LPS-induced response, since there was a significant increase in the expression of surface cell markers MHC-II, CD86 and CD40, as well as in IL-6 and IL-10 expression at both transcript and protein levels, in comparison with LPS-stimulated cells. The presence of EGTA completely abrogated this synergistic effect. Taken together, these results strongly suggest the involvement of both glycosidic residues and Ca+2 ions in the recognition of SLP-8348 by cellular receptors on murine macrophages. Moreover, these results suggest the potentiality of the SLP-8348 for the development of new adjuvants capable of stimulating antigen presenting cells by interaction with glycan receptors.
Assuntos
Cálcio/imunologia , Lactobacillus/metabolismo , Lipopolissacarídeos/administração & dosagem , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/administração & dosagem , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Células Cultivadas , Sinergismo Farmacológico , Lactobacillus/classificação , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Células RAW 264.7RESUMO
CD83 is a marker of mDCs directly related to their lymphostimulatory ability. Some data suggest that it has a central role in the immune system regulation, but how this function is performed remains to be determined. This work aimed to analyze the influence of CD83, present in mDCs, in the modulation of calcium signaling in T lymphocytes. Mo were differentiated into iDCs and activated with TNF-α. iDCs were treated, 4 h before activation, with siRNACD83, to reduce CD83 expression. Purified allogeneic T lymphocytes were labeled with the calcium indicator Fluo-4-AM, and calcium mobilization in the presence of mDCs was analyzed. CD83 knockdown mDCs induced lower calcium signal amplitude in T lymphocytes (29.0±10.0) compared with siRNAscr-treated mDCs (45.5±5.3). In another set of experiments, surface mDC CD83 was blocked with a specific mAb, and again, decreased calcium signaling in T lymphocytes was detected by flow cytometry and microscopy (fluorescence and confocal). In the presence of antibody, the percentage of responding T cells was reduced from 58.14% to 34.29%. As expected, anti-CD83 antibodies also reduced the proliferation of T lymphocytes (as assessed by CFSE dilution). Finally, in the absence of extracellular calcium, CD83 antibodies abrogated T cell signaling induced by allogeneic mDCs, suggesting that the presence of CD83 in mDC membranes enhances T lymphocyte proliferation by boosting calcium release from intracellular stores in these cells.
Assuntos
Antígenos CD/imunologia , Sinalização do Cálcio/imunologia , Membrana Celular/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Imunoglobulinas/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Anticorpos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Antígeno CD83RESUMO
In neutrophils, two receptors for IgG antibodies, namely FcgammaRIIA and FcgammaRIIIB are constitutively expressed, and a third one, FcgammaRI, can be upregulated by interferon-gamma. Whether FcgammaRIIIB is capable of triggering phagocytosis by itself is still controversial. The main role of FcgammaRI has not been clearly established in these cells. To address this problem, neutrophils were treated with interferon-gamma, and then phagocytosis mediated by each type of Fcgamma receptor was evaluated by flow cytometry. FcgammaRIIA was the most efficient receptor for phagocytosis. FcgammaRIIIB could mediate phagocytosis but much less efficiently than FcgammaRIIA. Both FcgammaRIIA- and FcgammaRIIIB-mediated phagocytosis were blocked by inhibitors of Src family kinases, Syk, PI 3-K, and ERK. In contrast, interferon-gamma-induced FcgammaRI was not able to mediate phagocytosis. Also, FcgammaRI did not activate ERK in the nucleus, but was however able to stimulate an efficient calcium rise. These data show that different neutrophil Fcgamma receptors possess different phagocytosis capabilities: FcgammaRIIA and FcgammaRIIIB, but not FcgammaRI, promote phagocytosis.
Assuntos
Neutrófilos/metabolismo , Fagocitose/imunologia , Receptores de IgG/metabolismo , Anticorpos , Sinalização do Cálcio/imunologia , Separação Celular , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Neutrófilos/imunologia , Neutrófilos/patologia , Receptores de IgG/imunologia , Quinases da Família src/metabolismoRESUMO
In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5-7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.