Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 regulates the induction of Langerhans cell maturation

Recently, we reported that Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) plays an important role in the migration of Langerhans cells (LC). Here, we show that SHPS-1 is involved in the maturation of LC. Immunofluorescence analysis on epidermal sheets for I-A or CD86 revealed that LC maturation induced by 2,4-dinitro-1-fluorobenzene (DNFB) or by TNF-alpha was inhibited by pretreatment with an anti-SHPS-1 monoclonal antibody (mAb) or with CD47-Fc fusion protein, a ligand for SHPS-1. Further, FACS analysis demonstrated that I-A(+) LC that had emigrated from skin explants expressed CD80 or CD86, whereas CD47-Fc protein reduced CD80(high+) or CD86(high+) cells. CD47-Fc protein also reduced the up-regulation of surface CD80 or CD86 by LC remaining in the skin explants. In SHPS-1 mutant mice, we observed that the up-regulation of surface CD86 and CCR7 by LC induced by DNFB as well as that of surface CD80 and CD86 by LC in skin explants was attenuated. Finally, contact hypersensitivity (CHS) response was suppressed in SHPS-1 mutant mice and in wild-type mice treated with an anti-SHPS-1 mAb. These observations indicate that SHPS-1 plays an important role in the maturation of LC ex vivo and in vivo, and that SHPS-1-CD47 interaction may negatively regulate CHS.     

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.     

Distinct binding patterns of HS1 to the Src SH2 and SH3 domains reflect possible mechanisms of recruitment and activation of downstream molecules

We previously identified a gene, LckBP1, which encodes a proteinthat binds to the Lck SH3 domain and is identical to murineHS1. Using unstimulated T lymphocytes, we further demonstratedthat Lck binds to HS1 in vivo and that HS1 is tyrosine phosphorylatedupon TCR stimulation. In the present report, we analyzed thebinding pattern of several src kinases and HS1 in greater detail.The Lck SH3 domain binds to HS1 constitutively, while the LckSH2 domain associates with HS1 only upon TCR stimulation. Asimilar binding pattern was observed with Lyn and HS1, but notwith Fyn and HS1, in which the Fyn SH3 region associates withHS1 upon TCR stimulation but the Fyn SH3 region does not associatewith HS1 regardless of TCR stimulation. Such distinct bindingpatterns of the src kinase SH2 and SH3 domains to HS1 may representa mechanism by which src family kinases select substrates andactivate particular downstream signaling pathways.     

Molecular and functional characterization of a CS1 (CRACC) splice variant expressed in human NK cells that does not contain immunoreceptor tyrosine-based switch motifs

CS1 (CRACC, novel Ly9) is a novel member of the CD2 family expressed on natural killer (NK), T and stimulated B cells. Although the cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motifs (ITSM), which enables to recruite signaling lymphocyte activation molecule (SLAM)-associated protein (SAP/SH2D1A), it activates NK cells in the absence of a functional SAP. CS1 is a self ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity. Here we have identified a novel splice variant of CS1 (CS1-S), which lacks ITSM. Human NK cells express mRNA for both wild-type CS1 (CS1-L) and CS1-S and their expression level remained steady upon various stimulations. To determine the function of each isoform, cDNA for CS1-L and CS1-S were transfected into the rat NK cell line RNK-16 and functionally tested using redirected cytotoxicity assays and calcium flux experiments. CS1-L was able to mediate redirected cytotoxicity of P815 target cells in the presence of monoclonal antibody against CS1 and a rise in intracellular calcium within RNK-16 cells, suggesting that CS1-L is an activating receptor, whereas CS1-S showed no effects. Interestingly, SAP associated with unstimulated CS1-L and dissociated upon pervanadate stimulation. These results indicate that CS1-L and CS1-S may differentially regulate human NK cell functions.     

IA-2 combined epitope assay: a new, highly sensitive approach to evaluate IA-2 humoral autoimmunity in type 1 diabetes

Islet tyrosine phosphatase 2 (IA-2) is one of the major autoantigens in type 1 diabetes. The aim of this work was to evaluate which IA-2 construct(s) among those usually employed has the highest sensitivity and specificity for detecting IA-2 autoantibodies in autoimmune diabetes and whether the combination of different IA-2 constructs into a single assay allows the detection of immunoreactivities otherwise not detectable by a single construct. For this purpose, we tested the single immunoreactivities of IA-2 FL(aa 1-979), IA-2(BDC)(aa 256-556:630-979), IA-2 IC(aa 605-979), IA-2(aa 256-760), IA-2(aa 761-928), and of 7 combinations of these fragments in the sera of 203 newly diagnosed type 1 diabetic patient (DM: 109 males,94 females, mean age 12.9 +/- 7.5 years) and 43 prediabetic subject (PDM: 20 males, 23 females, mean age 10.3 +/- 6.0 years) sera. IA-2 IC was the single construct that showed the highest sensitivity and specificity both in DM and PDM subjects; however, all of the other IA-2 constructs investigated detected additional immunoreactivities with respect to it. The combined use into the same assay of IA-2 IC, IA-2 FL, and IA-2 (256-760) constructs allowed detection of IA-2 Abs in additional 13.3% DM and 30.4% PDM subjects compared to the single IA-2 IC construct, suggesting this methodology as a new, highly sensitive approach to the study of IA-2 autoimmunity in type 1 diabetes.     

GTP酶激活蛋白(Src同源结构域3)结合蛋白1在乳腺癌细胞MDA-MB-231体外迁移中的作用

目的:研究GTP酶激活蛋白(Src同源结构域3)结合蛋白1[Ras-GTPase activating protein(Src homology domain 3)binding protein 1,G3BP1]在乳腺癌细胞MDA-MB-231体外迁移过程中的作用.方法:人乳腺癌细胞MDA-MB-231培养于RPMI1640培养基中;Western blot检测无表皮生长因子(Epidermal growth factor,EGF)刺激,以及10 ng/ml EGF分别刺激30秒、1分钟、2分钟和5分钟条件下乳腺癌细胞内G3BP1、蛋白激酶Cζ(Protein kinase Cζ,PKCζ)、Akt、pPKCζThin410/403及pAktSer473的表达水平;免疫共沉淀法检测乳腺癌细胞内G3BP1与PKCζ的相互作用;转染小干扰RNA抑制内源性G3BP1的表达;趋化实验和侵袭实验分别检测G3BP1抑制前后MDA-MB-231细胞穿过10 μm聚碳酸酯膜及人工基底膜的细胞数.结果:Western blot检测显示G3BP1在MDA-MB-231细胞内过表达;无EGF刺激时MDA-MB-231细胞内G3BP1,PKCζ与Akt均过表达,pPKCζThe410/403及pAktSer473不表达或低于Western blot检测下限;10 ng/ml EGF刺激后G3BP1、PKCζ与Akt表达水平不变,pPKCζThr410/403及pAktSer473的表达刺激时间增加而升高,至5分钟时达到峰值;在乳腺癌细胞内G3BP1与PKCζ具有相互作用;G3BP1表达抑制后乳腺癌细胞的趋化能力和侵袭能力均显著下降(P ＜0.05,P＜0.05).结论:G3 BP1通过与PKCζ相互作用,在乳腺癌细胞MDA-MB-231的体外迁移过程中发挥重要作用.     

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/NKG2-A⁺ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E⁺ 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via FcϵRI. Remarkably, interaction of CD94/NKG2-A⁺ RBL cells with the HLA-E⁺ .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.     

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAb) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAb against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogeneic, as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence on B7-1 expression.     

Interleukin 2 induces a transient downregulation of protein phosphatase 1 and 2A activity in human T cells

Stimulation of human CD4* T cell lines with interleukin 2 (IL-2) induces tyrosine, serine and threonine phosphorylation of a series of proteins involved in the IL-2 receptor (IL-2R) signaling pathway. Here, we examined whether IL-2 induces changes in the activity of protein serine/threonine phosphatases in antigen specific, CD4* human T cell lines. Using inhibitors of protein phosphatases 1 (PP1), PP2A, and PP2B, we provide evidence, that IL-2 induces a downregulation of PP activity in the cytoplasmic/membrane fraction. Thus, IL-2R ligation for 30 min triggers a 16 percent decrease in total PP2A activity (p<0.00005, n =17) and a seven percent decrease in PP1 activity (p<0.00005, n =17). Cytokine-induced downregulation of PP2A activity reaches a maximum 60 min after IL-2R ligation, and returns to baseline levels within two hours. Downregulation of PP1 activity reaches a maximum after 30 min and is largely reversed one hour after IL-2 stimulation. As determined from immunoblotting experiments using a specific anti-PP1 or anti-PP2A antibody, the amount of PP1 and PP2A recovered from cytosolic/membrane fraction remains unchanged after IL-2 treatment suggesting that the drop in PP1/PP2A activity might be due to a regulatory change rather than to a change in the amount of PP1 and PP2A. In conclusion, we provide evidence, for the first time, that IL-2 induces a transient downregulation of PP2A activity in T cells. In addition, our findings indicate that cytoplasmic PPl activity is transiently downregulated following IL-2R ligation in antigen-specific, human CD4* T cells.     

What goes up must come down: A tripartite Dok‐3/Grb2/SHIP1 inhibitory module limits BCR signaling

Properly regulated immunity requires precise integration of activating and inhibitory signals. As for other lymphocytes, B cells express an antigen‐specific activating receptor, the B‐cell antigen receptor (BCR), and inhibitory receptors (e.g. FcγRIIb) that exercise checkpoint control on B‐cell activation. Moreover, following BCR engagement, CD19 recruits proteins that amplify BCR signaling, while CD22 initiates a negative feedback loop by recruiting proteins that inhibit BCR signaling. Initial BCR signaling is mediated by protein tyrosine kinases and lipid kinases; inhibitory receptors directly antagonize the actions of these enzymes by recruiting protein tyrosine phosphatases and lipid phosphatases and positioning them close to actively signaling BCRs. Previously it was thought that inhibitory receptors such as FcγRIIb and CD22 were essential for bringing these phosphatases near the BCR. In this issue of the European Journal of Immunology, Manno et al. show that a tripartite inhibitory module consisting of the adaptor proteins Dok‐3 and Grb2 and the lipid phosphatase SHIP1 binds directly to activated BCRs and limits the Ca²⁺ mobilization that is required for B lymphocyte activation. This reveals that the BCR can be both an activating and inhibitory receptor, one that activates signaling enzymes while initiating a negative feedback loop that prevents excessive signaling.     

LRCH1维持狼疮肾炎模型小鼠调节性T细胞的功能

目的 探讨富含亮氨酸的重复序列和钙调理蛋白同源域1(LRCH1)在狼疮肾炎模型小鼠调节性T细胞(Tregs)中的表达和功能.方法 6月龄C57BL/6小鼠和MRL/lpr小鼠,用流式细胞仪分选脾脏和肾脏内的CD45+CD4+CD25+CD127low Tregs,RT-qPCR检测Tregs LRCH1 mRNA水平....     

不同剂量培哚普利对缺血性心功能障碍家兔心功能及ACE2/Ang-(1-9)/Ang-(1-7)轴的影响

 目的: 探讨不同剂量培哚普利对缺血性心功能障碍家兔心功能的影响。方法: 采用结扎冠状动脉前降支的方法制作缺血性心功能障碍家兔模型。利用随机数字表法将30只家兔随机分为培哚普利大、小剂量组和心功能障碍组。大、小剂量组分别给予培哚普利生理盐水溶液(浓度分别为1 g/L、0.33 g/L)2 mL·kg^-1·d^-1灌胃;心功能障碍组给予等量生理盐水灌胃。4周后心脏超声测定心功能;real-time PCR检测血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)和血管紧张素2型受体(angiotensin type 2 receptor,AT2R) mRNA表达;ELISA检测家兔血清血管紧张素(angiotensin,Ang)-(1-9)和Ang-(1-7)水平。结果: 与心功能障碍组相比,不同剂量培哚普利均可改善心功能(P<0.01),大剂量培哚普利比小剂量培哚普利改善心功能的效果显著(P<0.05);心功能障碍家兔应用培哚普利后,血清Ang-(1-9)和Ang-(1-7)水平均增高(P<0.01),ACE2和AT2R mRNA表达均增加(P<0.01);与小剂量组相比,大剂量组心肌ACE2和AT2R mRNA表达均增高(P<0.01),血清Ang-(1-9)水平增高(P<0.05),血清Ang-(1-7)水平无明显增加。相关性分析发现,左室射血分数与血清Ang-(1-9)、ACE2及AT2R水平呈正相关关系(P<0.01),与血清Ang-(1-7)水平无相关关系。结论: 大剂量培哚普利较小剂量培哚普利可以更有效改善缺血性心功能障碍家兔心功能,其心功能的改善可能与Ang-(1-9)水平增多,引起AT2R活化相关。     

Differential expression, shedding, cytokine regulation and function of TNFR1 and TNFR2 in human fetal astrocytes

Tumor necrosis factor (TNF)-alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF-alpha receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF- receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.     

Differential involvement of reactive oxygen species in apoptosis caused by the inhibition of protein phosphatase 2A in Jurkat and CCRF-CEM human T-leukemia cells

A better understanding of dysregulated signaling pathways in cancer cells may suggest novel strategies to prevent tumor development and/or progression. Here we show that Jurkat and CCRF-CEM human T-leukemia cell lines were more sensitive than normal human T cells to the cytotoxic effect of inhibiting protein phosphatase 2A (PP2A). Inhibition of PP2A by okadaic acid (OA) caused T-leukemia cells to die by apoptosis, as indicated by DNA fragmentation, caspase-3 activation, loss of mitochondrial membrane potential (DeltaPsi(m)), and changes in nuclear morphology that were consistent with apoptosis. PP2A might therefore be a useful intracellular target for the treatment of T cell-derived leukemias. We also observed that reactive oxygen species (ROS) were generated in response to PP2A inhibition in T-leukemia cells. However, loss of DeltaPsi(m) that resulted from PP2A inhibition was not prevented by exogenous antioxidants (glutathione and N-acetyl-cysteine), indicating that OA-induced changes in mitochondrial membrane permeability were not a consequence of ROS production. Moreover, exogenous antioxidants protected CCRF-CEM T-leukemia cells from apoptosis caused by PP2A inhibition but failed to prevent OA-induced apoptosis in Jurkat T-leukemia cells, indicating a differential role for ROS in apoptosis caused by PP2A inhibition in two different human T-leukemia cell lines.