抑制性受体LAIR-1对巨核谱系分化的调节作用

 目的 研究抑制性受体LAIR-1在巨核谱系细胞的表达及其对巨核细胞分化的调节作用。方法 采用流式细胞术和激光共聚焦显微镜检测LAIR-1在巨核谱系细胞的表达;免疫磁珠法纯化脐血CD34+细胞,体外无血清培养系统诱导CD34+细胞向巨核细胞分化,观察LAIR-1被抗体或配体交联激活后对巨核细胞分化的影响。结果 LAIR-1表达于人骨髓CD34+CD41a+和CD41a+CD42b+细胞以及脐血CD34+CD41a-和CD34+CD41a+细胞;脐血CD34+细胞向巨核细胞分化过程中,LAIR-1表达在倍性为2N和4N的不成熟CD41a+细胞上;交联激活LAIR-1分子能够抑制脐血CD34+细胞向巨核细胞的分化。结论 LAIR-1可能是参与巨核细胞早期分化发育的一种新的负调控分子。     

The inhibitory collagen receptor LAIR-1 (CD305)

The immune system protects the body from invaders such as viruses and bacteria. Immune cells must be activated in the correct context to function properly. It is critical that the receptors, costimulatory molecules, and cytokines that orchestrate this activation are carefully regulated to prevent uncontrolled inflammation and autoimmunity. Inhibitory receptors play an important role in regulation of immune cell function, usually upon interaction with ligands present on other cells. In contrast, the function of the inhibitory leukocyte-associated Ig-like receptor (LAIR)-1 can be regulated by extracellular matrix collagens. LAIR-1 is expressed on most cells of the immune system, and its function has been studied on multiple cell types. This review summarizes current literature about LAIR-1, a receptor that potentially is able to regulate multiple steps of an immune response.     

Regulated expression of the inhibitory receptor LAIR-1 on human peripheral T cells during T cell activation and differentiation

The leukocyte-associated Ig-like receptor-1 (LAIR-1) is capable of inhibiting immune cell function through interaction with collagens. LAIR is expressed on the majority of peripheral blood mononuclear cells. The abundant expression of both receptor and ligand calls for regulatory mechanisms to relieve the continuous interaction between collagens and LAIR-1. This regulation may occur at the expression level of the receptor. Here, we report that LAIR-1 is indeed differentially expressed during human T cell differentiation. Naive CD4(+) and CD8(+) T cells as well as CD8(+) T cells of the effector phenotype express higher levels of LAIR-1 compared to memory T cells. In vitro stimulation revealed a decrease in LAIR-1 expression upon activation, and the lower LAIR-1 expression on CD127(-) T cells suggests that activation-induced down-modulation of LAIR-1 may also occur in vivo. Furthermore, crosslinking of LAIR-1 on primary T cells results in an inhibition of T cell function. Our data suggest that regulated expression of LAIR-1 and the subsequent change in the threshold for activation may be a mechanism to modulate inhibition of the immune system.     

Tumor-expressed collagens can modulate immune cell function through the inhibitory collagen receptor LAIR-1

Many tumor types over-express collagens, what correlates with enhanced metastatic capacity and unfavorable clinical outcome. This is generally explained by the importance of collagens in creating a microenvironment that supports tumor cell survival and enhances cell migration. Importantly, collagens act as ligands for the inhibitory receptor LAIR-1, which inhibits the function of multiple types of immune cells. Here we propose a new role for tumor expressed collagens and show that these structural proteins can be exploited by tumor cells to inhibit immune responses through an interaction with LAIR-1. We show that both LAIR-1-Fc fusion proteins and LAIR-1 expressing cells bind to transmembrane collagens expressed by tumor cells. Interference with collagen expression by specific knock-down of prolyl 4-hydroxylase diminishes LAIR-1 binding to tumor cells, demonstrating the specificity of the interaction. Consistently, both transmembrane collagens and extracellular collagens produced by multiple tumor cell types can activate LAIR-1. Furthermore, overexpression of collagen XVII on target cells results in diminished NK cell cytotoxic activity. Thus tumor-expressed collagens can bind and trigger immune inhibitory signaling via LAIR-1, suggesting that collagens indeed may affect tumor immune evasion.     

Induction of megakaryocytic differentiation in primary human erythroblasts: a physiological basis for leukemic lineage plasticity

In myelodysplasias and acute myeloid leukemias, abnormalities in erythroid development often parallel abnormalities in megakaryocytic development. Erythroleukemic cells in particular have been shown to possess the potential to undergo megakaryocytic differentiation in response to a variety of stimuli. Whether or not such lineage plasticity occurs as a consequence of the leukemic phenotype has not previously been addressed. In this study, highly purified primary human erythroid progenitors were subjected to stimuli known to induce megakaryocytic differentiation in erythroleukemic cells. Remarkably, the primary erythroid progenitors rapidly responded with morphological and immunophenotypic evidence of megakaryocytic differentiation, equivalent to that seen in erythroleukemic cells. Even erythroblasts expressing high levels of hemoglobin manifested partial megakaryocytic differentiation. These results indicate that the lineage plasticity observed in erythroleukemic cells reflects an intrinsic property of cells in the erythroid lineage rather than an epiphenomenon of leukemic transformation.     

A role for G-CSF receptor signaling in the regulation of hematopoietic cell function but not lineage commitment or differentiation.

To investigate the specificity of cytokine signals in hematopoietic differentiation, we generated mice with a targeted mutation of their G-CSF receptor (G-CSFR) such that the cytoplasmic (signaling) domain of the G-CSFR is replaced with the cytoplasmic domain of the erythropoietin receptor. In homozygous mutant mice, expression of this chimeric receptor had no apparent affect on lineage commitment and was able to support the production of morphologically mature neutrophils. However, mutant neutrophils displayed reduced chemotaxis, and G-CSF-stimulated mobilization of neutrophils and hematopoietic progenitors from the bone marrow to blood was markedly impaired. Thus, the G-CSFR is generating unique signals that are required for certain specialized hematopoietic cell functions but are not required for granulocytic differentiation or lineage commitment.     

肿瘤患者外周血中抑制性受体LAIR-1表达升高

目的:研究LAIR-1在肿瘤患者中的表达,探讨其在抗肿瘤免疫应答中的作用.方法:应用夹心ELISA检测肿瘤患者血清中可溶型sLAIR-1的水平;应用免疫荧光染色和流式细胞术分析,观察LAIR-1在患者PBMC中NK细胞、CD4 T细胞、CD8 T细胞和B细胞上的表达.结果:肿瘤患者血清sLAIR-1水平为(4.6±3.2) μg/ L,明显高于正常人血清sLAIR-1 水平(3.9±3.0) μg/L,P<0.05.NK细胞,CD4 T细胞及CD8 T细胞表达LAIR-1水平上调.肺癌患者CD4/CD8比值明显低于正常人,B细胞百分率明显高于正常人.NK细胞、CD4 T细胞CD8 T细胞中LAIR-1的表达阳性率高于对照组.结论:在肿瘤患者LAIR-1分子表达水平高于正常人.肿瘤患者抑制性受体LAIR-1 表达水平的上调可能与肿瘤的免疫逃逸有关.     

微小RNA-16对K562细胞向巨核细胞系分化的影响

目的:观察微小RNA-16(microRNA-16,miR-16)对人白血病细胞K562向巨核细胞系分化的影响,并初步探索其中可能的机制。方法:在K562细胞中通过转染miR-16模拟物(mimics)或miR-16抑制物(inhibitor),实时荧光定量PCR检测miR-16的水平变化,通过流式细胞术检测巨核细胞系分化指标CD41、CD42b及CD61的表达。用Western blotting检测miR-16对下游白血病癌基因(myeloblastosis oncogene,MYB)蛋白水平的影响,进而利用流式细胞术检测miR-16是否通过影响MYB调控CD41、CD42b及CD61的表达。结果:转染miR-16-mimics可显著升高K562细胞中的miR-16水平并促进CD41、CD42b及CD61的表达(P0.05),而转染miR-16-inhibitor可明显抑制K562细胞中的miR-16水平同时降低CD41、CD42b及CD61的表达(P0.05);Western blotting证实miR-16可调控MYB蛋白水平,而沉默MYB可逆转miR-16对CD41、CD42b及CD61表达的调控作用。结论:miR-16可通过调控MYB的表达,调节人白血病细胞K562向巨核细胞系分化的能力。     

The inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1) is expressed at high levels by human naive T cells and inhibits TCR mediated activation

Human leukocyte-associated Ig-like receptor-1 (LAIR-1) is a transmembrane glycoprotein with a single extracellular Ig-like domain and a cytoplasmic tail containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs). It is constitutively expressed on the majority of human mononuclear leukocytes and functions as an inhibitory receptor. In this study, we show that freshly isolated peripheral blood T cells are heterogeneous in their expression levels of LAIR-1. We have found that naive T cells express the highest levels of LAIR-1, even more than memory cells. The cross-linking of LAIR-1 inhibits T cell receptor (TCR) mediated signals in freshly isolated human naive T cells and whole populations of CD4⁺ or CD8⁺ T cells. TCR cross-linking increased cell surface expression of LAIR-1 in a process that requires p38 MAP kinase and ERK signaling. Altogether, these results indicate that LAIR-1 is capable of negatively regulating T cell functions, and its high level of expression by naive T cells suggests that it may function at an early stage in the development of an immune response.     

p40/LAIR-1 regulates the differentiation of peripheral blood precursors to dendritic cells induced by granulocyte-monocyte colony-stimulating factor

p40/LAIR-1, a member of the immunoglobulin superfamily, is a surface molecule broadly distributed among leukocytes which has been shown to down-regulate T and NK cell activation. In this study, we show that p40/LAIR-1 is highly expressed in CD14⁺ peripheral blood mononuclear cells (PBMC). When cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10 – 14 days, CD14⁺ cells acquired morphologic and phenotypic features ( i.e. loss of CD14 and expression of CD80^bright and CD86^bright ) typical of dendritic cells (DC) and lost the expression of p40/LAIR-1. Engagement of p40/LAIR-1 (but not of CD58) by specific monoclonal antibodies prevented CD14⁺ PBMC differentiation into DC; when cultured in the presence of GM- CSF upon p40/LAIR-1 cross-linking, the resulting cells were CD14⁺ CD80^dull CD86^dull and displayed a macrophage-like morphology. We have recently demonstrated that peripheral blood CD14⁺ cells co-expressing the CD34 progenitor marker represent the circulating precursors of CD83⁺ DC. Herein we show that cross-linking of p40/LAIR-1 prevented the maturation of CD14⁺ CD34⁺ cells into CD83⁺ DC. This effect appears to be consequent to the impairment of GM-CSF receptor-mediated activation signaling. Indeed, triggering of GM-CSF receptors in both CD14⁺ and CD14⁺ CD34⁺ cells led to increases in the intracellular free calcium concentrations which were inhibited by p40/LAIR-1 engagement. Taken together, these data suggest a possible regulating role played by p40/LAIR-1 in the process of differentiation from peripheral blood precursors into DC induced by GM-CSF.     

The interleukin-1 receptor associated kinase 1 contributes to the regulation of NFAT

IRAK-1 is a critical modulator regulating innate immunity signaling processes. However, the physiological substrates for IRAK-1 remain poorly defined. In this report, we have demonstrated that IRAK-1 is a kinase responsible for the constitutive phosphorylation and inactivation of the Nuclear Factor of Activated T-cell (NFAT). Expression of IRAK-1 suppressed NFAT reporter activity. Correspondingly, the levels of both nuclear NFATc1 and NFATc4 were constitutively elevated in IRAK-1(-/-) cells. Furthermore, the phosphorylation of NFATc4 at the S(168)PS(170)P site was significantly diminished in IRAK-1(-/-) cells. Mechanistically, we observed that IRAK-1 interacted with NFATc4 via the C-terminus of IRAK-1 and the N-terminal NHR region of NFATc4. IRAK-1 mutants that ablated either its kinase activity or its interaction with NFATc4 failed to suppress NFAT reporter activity. The expression level of COX2, which is under the control of NFAT, was elevated in IRAK-1(-/-) cells. Functionally, ApoE(-/-)/IRAK-1(-/-) mice were protected from high-fat-diet-induced hypertension and atherosclerosis. Taken together, our findings reveal NFAT molecules as novel physiological targets for IRAK-1.     

The role of p53 in megakaryocyte differentiation and the megakaryocytic leukemias of Down syndrome

Sequence analysis of the p53 tumor suppressor gene was performed on diagnostic blood and bone marrow samples from nine children with Down syndrome and acute megakaryoblastic leukemia. p53 sequence alterations were not observed in any of seven cases of transient leukemia; however, samples from two of three patients with acute megakaryoblastic leukemia harbored sequence alterations. The acquisition of both mutations (Gly245Val and Arg72Pro) in the transformation from transient leukemia to overt acute megakaryoblastic leukemia suggests a functional role of mutant p53 in the evolution of this disease.