Tissue-specific expression of TIPE2 provides insights into its function

Tumor necrosis factor-alpha-induced protein-8 like-2 (TNFAIP8L2, TIPE2) is a newly discovered negative regulator of innate immunity and cellular immunity. TIPE2 deficiency in mice causes fetal inflammatory diseases and TIPE2 downregulation in humans is associated with systemic autoimmunity. However, TIPE2 deficiency leads to a selective defect in humoral immunity. Due to the lack of a suitable antibody, the nature of cells and tissues that express TIPE2 protein has not been determined. In this study, we generated a highly specific antibody to TIPE2 and examined TIPE2 expression in various murine tissues by immunohistochemistry and RT-PCR. We found that TIPE2 was a cytoplasmic protein expressed preferentially in lymphoid tissues and a small group of non-lymphoid tissues. Within the lymphoid compartment, T cells appear to express high level of TIPE2 protein, while B cells and B cell zones of lymphoid organs were devoid of TIPE2. Within most of the non-lymphoid tissues, TIPE2 was not detected. However, several endocrine tissues and skeletal muscle expressed detectable TIPE2 protein and mRNA. Furthermore, high levels of TIPE2 were detected in monocyte/macrophage derived cell lines and ovarian adenocarcinoma cells, but not detectable or weakly expressed in most human carcinoma cell lines. These results indicate that TIPE2 may perform tissue-specific functions in both lymphoid and non-lymphoid compartments. They may also explain why TIPE2 deficiency enhanced cellular but not humoral immunity.     

The unique expression profile of human TIPE2 suggests new functions beyond its role in immune regulation

TIPE2 (tumor necrosis factor-α-induced protein 8-like 2, or TNFAIP8L2) is a newly identified negative regulator of innate and adaptive immunity. TIPE2 deficiency in mice leads to systemic inflammation and TIPE2 down-regulation in humans is associated with autoimmunity. However, the nature of human TIPE2-expressing cells in various tissues has not been characterized due to the lack of suitable antibodies. In the present study, we generated specific rabbit antibodies against human TIPE2 and examined human TIPE2 expression in various tissues by Western blot, flow cytometry, and immunohistochemistry. We found that unlike murine TIPE2 that is preferentially expressed by hematopoietic cells, human TIPE2 was also expressed in a wide variety of non-hematopoietic cell types, including hepatocytes, neurons in brain and brainstem, squamous epithelial cells in esophagus and cervix, transitional epithelial cells in bladder and ureter, and glandular epithelial cells in stomach, colon and appendix. For squamous epithelium, the level of TIPE2 expression increased dramatically in terminally differentiated cells with the proliferating precursor cells completely devoid of TIPE2. The unique expressional profile of human TIPE2 suggests new functions beyond controlling innate and adaptive immunity.     

TIPE 家族生物活性及作用研究进展

TIPE (Tumor necrosis factor-alpha-induced protein 8 like )家族是新近报道的免疫和肿瘤调节因子。该家族拥有四个高度同源的成员:TNFAIP8 (Tumor necrosis factor-induced protein 8), TIPE1 (TNFAIP8L1), TIPE2 (TNFAIP8L2)和TIPE3 (TNFAIP8L3)。它们虽然结构相似,但在组织、器官上的表达不同,在生物学功能及疾病中的作用存在较大的差别。TNFAIP8 具有抑制细菌感染与促肿瘤迁移的作用;TIPE2 是一种免疫和炎症负调控因子,可抑制某些肿瘤的生长;TIPE1 可诱导细胞凋亡具有抑瘤效应;TIPE3 可特异性结合磷脂第二信使,促进肿瘤生成。随着研究的深入,TIPE 家族在多种疾病的发生发展过程中表现出重要的调控作用,然而其具体生物活性与分子机制有待进一步的研究。     

肿瘤坏死因子α诱导蛋白8家族的研究进展

肿瘤坏死因子α诱导蛋白8（TNFAIP8）是调控凋亡过程的重要分子之一.随着研究的不断深入,TNFAIP8蛋白在细胞凋亡、信号转导、肿瘤细胞增生、侵袭及转移等生命过程中具有重要的调节作用.TNFAIP8家族成员TIPE2是一个新发现的蛋白分子,通过对T细胞受体和Toll样受体信号途径实行负向调控,进而调节机体固有免疫应答和适应性免疫应答过程.TNFAIP8蛋白的多效应性及TIPE2在维持机体免疫动态平衡中的调控作用预示着TNFAIP8家族在免疫学及医学研究中可能极具应用价值.本文综述了TNFAIP8及TIPE2蛋白结构和生物学功能研究的最新认识,并介绍TNFAIP8及TIPE2表达异常与疾病发生的关系和临床意义.     

The TIPE (TNFAIP8) family in inflammation, immunity, and cancer

Tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8 or TIPE) family are recently identified proteins which are important for maintaining immune homeostasis. The mammalian TNFAIP8 family consists of four members: TNFAIP8, the first identified member of this family, TNFAIP8L1 (TNF-alpha-induced protein 8-like 1, TIPE1), TIPE2, and TIPE3, which share high degrees of sequence homology and involve in proliferation, inflammation, and cell death. Among the members, TNFAIP8 is considered to be associated with carcinogenesis, TIPE2 is an essential negative regulator of both innate and adaptive immunity and the depletion of TIPE2 would cause serve inflammatory disease. Whereas, little is known about TIPE1 and TIPE3.     

Identification of A T lineage antigen in the catfish

A murine monoclonal antibody produced against catfish thymocytes and immunoglobulin-negative lymphocytes in the blood identified a catfish T cell antigen designated Cf T1. The Cf T1 antigen was found to be expressed on thymocytes, a subpopulation of the lymphoid cells in blood and other lympho-hemopoietic tissues, and a T cell line, but was not expressed by erythrocytes, thrombocytes, myeloid cells, B cells or macrophage cell lines. Stimulation of blood mononuclear cells with the T cell mitogen, concanavalin A, resulted in an increased frequency of Cf T1⁺ cells. Conversely, lipopolysaccharide stimulation increased the number of IgM⁺ B cells and decreased the frequency of Cf T1⁺ cells. The Cf T1 antigen was defined as a single chain protein of M_r 35 000 lacking N- and O-linked sugars. The Cf T1 molecule thus provides a T lineage-specific marker in this bony fish representative.     

Roles of TIPE2 in hepatitis B virus-induced hepatic inflammation in humans and mice

Hepatitis B virus (HBV)-induced hepatic inflammation afflicts hundreds of millions of people worldwide and is a leading cause of hepatic cancer. While the deleterious effect of the chronic hepatitis is well recognized, the molecular mechanisms underlying the pathogenesis of HBV-induced hepatic inflammation are not well understood. We report here that the tumor necrosis factor-alpha-induced protein-8 like-2 (TIPE2 or TNFAIP8L2), a newly identified regulator of immune receptor signaling, plays an important role in controlling HBV-induced hepatitis. Patients with chronic hepatitis B had significantly reduced levels of TIPE2 expression in their peripheral blood mononuclear cells (PBMCs) as compared to healthy individuals. The TIPE2 expression negatively correlated with the blood levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (Tbil) as well as the HBV load of the patients. Importantly, using a murine model of HBV-induced hepatitis, we found that TIPE2-deficient mice developed significantly more severe hepatic inflammation than wild type mice. These results indicate that TIPE2 plays an important role in taming HBV-induced hepatic inflammation.     

A cytotoxic monoclonal antibody that binds to human B cells, T cells, monocytes, and a subset of lymphomas

Monoclonal antibodies have been derived against a variety of antigens on human leukocytes. We describe a complement-fixing, IgG2a, murine monoclonal antibody that was derived against ARH-77, a human plasma cell line. In a Western blot of a lysate of ARH-77, anti-ARH-77 recognized a 155 kD protein. On normal B cells, the antibody co-capped with surface immunoglobulin. Analysis of blood and lymph nodes indicated that the determinant recognized by the antibody is expressed by most B cells, T cells, monocytes, and lymphomas with surface immunoglobulin. The determinant was not present on granulocytes, platelets, mantle zone lymphocytes, or serum immunoglobulin. We conclude that anti-ARH-77 is a cytotoxic monoclonal antibody that recognizes a determinant shared by human mononuclear leukocytes. The determinant may be an exposed part of a membrane anchor or bridging protein associated with receptors on mononuclear cells.     

MiRNA-15a inhibits proliferation,migration and invasion by targeting TNFAIP1 in human osteosarcoma cells

Recent data strongly suggest the important role of miRNAs in various cancer-related processes. Osteosarcoma is the most common type of primary malignant bone tumor and is characterized by complex genetic changes and resistance to conventional treatments. In this study, the role of miRNA-15a (miR-15a) in the progression and metastasis of osteosarcoma was investigated. The result demonstrated that the expression of miR-15a was down-regulated in osteosarcoma tissues and cell lines as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. In functional assays, miR-15a inhibited cell proliferation, migration and invasion in U2OS and MG-63 cells. Meanwhile, bioinformatic analysis combined with experimental confirmation demonstrated that tumor necrosis factor; α-induced protein 1 (TNFAIP1) gene is a potential target of miR-15a and can be directly regulated by miR-15a. Down-regulation of TNFAIP1 induced effects on osteosarcoma cell lines similar to those induced by miR-15a. Taken together, these data suggest that miR-15a may act as a tumor suppressor, which is commonly down-regulated in both osteosarcoma tissues and cells. TNFAIP1 plays an important role in mediating miR-15a dependent biological functions in osteosarcoma. Reintroduction of miR-15a may be a novel therapeutic strategy by down-regulating TNFAIP1 expression.     

ERMAP is a B7 family-related molecule that negatively regulates T cell and macrophage responses

T cell activation and tolerance are tightly regulated by costimulatory and coinhibitory molecules. B7 family members play a crucial role in regulating immune responses. In this study, we identified erythroid membrane-associated protein (ERMAP) as a novel T cell inhibitory molecule. ERMAP shares significant sequence and structural homology with existing B7 family members in its extracellular domain. The ERMAP protein is expressed on the cell surface of resting and activated antigen-presenting cells (APCs) and in some tumor tissues. The putative ERMAP receptor is expressed on activated CD4 and CD8 T cells and macrophages. Both mouse and human ERMAP-IgG2a Fc (ERMAP-Ig) fusion proteins inhibit T cell functions in vitro. Administration of ERMAP-Ig protein ameliorates autoimmune diseases, including experimental autoimmune encephalomyelitis and type 1 diabetes, in mice. Anti-ERMAP antibody enhances macrophage phagocytosis of cancer cells in vitro. Furthermore, administration of an anti-ERMAP antibody inhibits tumor growth in mice likely by blocking the inhibitory effects of ERMAP on T cells and macrophages. Our results suggest that therapeutic interaction with the ERMAP inhibitory pathway may represent a novel strategy for treating patients with autoimmune disease or cancer.     

A novel prognostic factor TIPE2 inhibits cell proliferation and promotes apoptosis in pancreatic ductal adenocarcinoma (PDAC)

Background: Tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2 or TNFAIP8L2) is a newly discovered negative immune regulator. Studies have shown that TIPE2 causes significant malignant biological effects and is differentially expressed in various malignant tumors. However, the expression and roles of TIPE2 in pancreatic ductal adenocarcinoma (PDAC) are largely unknown.Materials and Methods: The expression of TIPE2 in PDAC tissues was assessed by immunohistochemistry, qPCR and western blot analysis and related clinicopathological parameters including survival time were analyzed. After overexpression of TIPE2, cell proliferation and apoptosis analysis were conducted, and the associated underlying molecular mechanism was also explored.Results: In the present study, TIPE2 was upregulated in early PDAC tissues, and TIPE2 expression decreased as the tumor progressed (P<0.001). TIPE2 expression was negatively associated with tumor size, TNM stage and metastasis of lymph nodes. Furthermore, as an independent risk factor, TIPE2 could be used to predict the survival of patients with PDAC (P=0.035). TIPE2 overexpression significantly suppressed the viability, proliferation and induced apoptosis of PDAC cells by inhibiting survivin and increasing the activity of caspase3/7.Conclusions: For the first time, this study demonstrated that TIPE2 is an independent prognostic factor in PDAC. TIPE2 inhibited the proliferation and induced apoptosis via regulating survivin/caspase3/7 signaling pathway. These results indicated that TIPE2 is a potential biomarker for predicting the prognosis of PDAC patients and plays a pivotal role in the progression of PDAC.     

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.     

抗人淋巴细胞T8抗原样McAb的研制和初步鉴定

用杂交瘤技术,将经人外周血E花环阳性细胞免疫小鼠脾细胞与NS-1骨髓瘤细胞融合后,产生一分泌IgG_1亚型McAb杂交瘤株。其所分泌抗体经微量放射免疫测定及间接免疫荧光法分析,表明它只能与T细胞系、76％的胸腺细胞及22％的外周血T淋巴细胞反应,不与其它各种不同细胞反应。将此抗体所识别入外周血T淋巴细胞亚群与抗Leu-2a识别T8~+细胞、抗Leu-3a识别T4~+细胞比较,发现此抗体与抗Leu-2a识别同一群细胞。此抗体能从T细胞表面沉淀出30KD(还原条件)或78KD(非还原条件)分子,并完全阻断FITC标记抗Leu-2a与T细胞的结合,说明此抗体是识别T8抗原样的McAb。     

Inhibition of in Vitro Generation of Cytotoxicity Against Tumor Cells by Monoclonal Antibody to Thy-1

The Thy-1 molecule on murine T lymphocytes has been suggested to play a role in cellular activation events leading to a variety of immunologic functions. We present evidence that this molecule may be involved in signals leading to the in vitro generation of cytotoxic T cells against several tumor cell lines used as stimulators in mixed tumor-lymphocyte culture. The presence of monoclonal antibody against a polymorphic determinant on the Thy-1 molecule markedly reduced the generation of cytotoxicity after three days of culture of murine splenocytes with stimulator tumor cells bearing low levels of Ia antigen. In contrast, no effect was seen when the stimulators were either allogeneic splenocytes, or a tumor cell line expressing large amounts of Ia. These results suggest that the Thy-1 molecule is critically involved in events leading to the generation of cytotoxic effectors under some, but not all conditions.     

Detection of Thy-1 on cell surface of human T lymphoid cell lines by a monoclonal antibody

A mouse IgG2b(kappa) monoclonal antibody (MAb) HB-2S-1 against human brain Thy-1 was secreted by a hybridoma clone after fusion of mouse myeloma cells with spleen cells from a mouse that went through a prolonged immunization procedure before fusion. When tested against isolated human Thy-1 by the enzyme-linked immunosorbent assay (ELISA), MAb HB-2S-1 in culture supernatant showed a titer of over 100,000, and a titer of over 10 million in ascites of a mouse injected with the hybrid clone. By immunoblotting, this antibody was found to bind a doublet of protein bands of approximately 25,000 daltons among all proteins solubilized by deoxycholate (DOC) from membrane of human brain cells. When tested on human lymphoid cell lines by immunofluorescence, MAb HB-2S-1 strongly stained four T lymphoma cell lines, C91-Pl, HUT-102, HUT-78, and C5-MJ; and weakly two leukemia cell lines, MOLT-3 and Jurkat(clone E6-1). It did not stain a third T leukemia cell line, CCRF-CEM; a human B cell line, Raji; a plasmacytoma cell line, HMy2; or a myelomonocytic cell line, HL-60. Peripheral blood lymphocytes from ten normal human adults and the viable T cells isolated from another normal individual were also negative.     

Ber-ACT8: new monoclonal antibody to the mucosa lymphocyte antigen.

Using a newly established HTLV-1 positive T cell line as an immunogen, a new monoclonal antibody, Ber-ACT8, was produced. It reacts with in vitro activated T cells and a small subset of normal resting T cells, but not with resting B cells or any of the 29 established human permanent cell lines tested. Immunohistological analysis of a wide spectrum of human tissues showed that Ber-ACT8 reactivity is restricted to a few T cells in the peripheral blood, the extrafollicular areas of lymph nodes and tonsils, and splenic red pulp. In the gut Ber-ACT8 labelled most intraepithelial T cells and up to 50% of lamina propria T cells. The antibody also immunostained T cells present in the oral and bronchial mucosa. Double labelling on splenic cells, fresh blood lymphocytes, and in vitro activated T cells showed that most Ber-ACT8 positive cells coexpressed CD8. Ber-ACT8 did not react with any of the 14 Hodgkin's lymphomas nor any of the 172 non-Hodgkin's lymphomas tested, with the exception of 10 cases of T cell lymphomas, five of which were located in the jejunum and associated with coeliac disease, and one B cell lymphoma, and most cases of hairy cell leukaemia tested. Parallel immunostainings with Ber-ACT8, anti-TCR-beta (beta F1), and anti-TCR-delta showed that most Ber-ACT8 positive T cells carry the TCR of alpha beta type. Comparison of Ber-ACT8 with HML-1, B-ly7, and LF61 showed essentially the same reactivity and an identical molecular target. The molecular structure recognised seems to be a trimeric molecule with components of 150, 125 and 105 kilodaltons, with the Ber-ACT8 epitope localised on the 150 kilodalton chain. The 150 kilodalton molecule contains an 0-linked carbohydrate moiety of about 10 kilodaltons. Because of its very selective distribution, the trimeric antigen is a powerful reagent for the diagnosis of gut T cell-derived T cell lymphomas and other extranodal T cell lymphomas, as well as hairy cell leukaemia.     

Tissue distribution of the T cell activation antigen Ta1. Serological, immunohistochemical and biochemical investigations.

The recently described Ta1 antigen is expressed by activated T cells in vitro and in vivo, as observed in patients with certain immune-mediated diseases, such as multiple sclerosis. In this paper we report on the tissue distribution of the Ta1 antigen. Serological testing of human tumour cell lines and immunohistochemical analysis of human tissue sections revealed a reactivity of the anti-Ta1 antibody with normal and malignant tissues of the upper gastro-intestinal tract, the biliary tract, exocrine pancreas and kidney. SDS-PAGE analysis of immunoprecipitates from 125I-labelled cells, employing the anti-Ta1 antibody, yielded a 113-115 kD band from three serologically Ta1 positive tumour cell lines, from a serologically Ta1 negative human EBV-transformed B lymphoblastoid cell line, from peripheral blood mononuclear cells (PBMC) and, as previously described, a 105 kD band from PHA activated T cells (Fox et al., 1984). After endoglycosidase F treatment similar bands of 85 kD were precipitated from activated T cells and from tumour cell lines. It is therefore likely that very similar glycoproteins, which differ only modestly in the size of carbohydrate chains, bear the Ta1 epitope on Ta1 positive tissues.