p55 IL-2 receptor mRNA precursors in murine T lymphocyte nuclei

An unusual family of cDNA clones homologous to human p55 IL-2R sequences was isolated from the murine HT-2 Th cell line. These clones were mapped, partially sequenced, and compared with previously published human and mouse IL-2R sequences. They appear to consist of various combinations of exons and introns, suggesting that they are derived from p55 IL-2R mRNA precursors. The configuration of exons in the splicing intermediates indicates that the murine and human gene organizations are similar and that the 3' end of intron 3 is well conserved between the two species. RNA mapping experiments using nuclear, cytoplasmic, and total RNA and probes derived from various parts of the p55 IL-2R gene support and extend the sequence data. They indicate that detectable amounts of immature p55 IL-2R mRNA are found specifically in the cell nucleus of the HT-2 cell line. Similar data were obtained for the Th cell clone 52.3 and the cytotoxic T cell line CTLL. All these results indicate that the T cell nucleus contains significant amounts of immature p55 IL-2R mRNA.     

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.     

In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels

The localization of mRNA encoding preproatrial natriuretic peptide (ANP) was investigated in cultured human umbilical vein endothelial cells (HUVEC) and tissue preparations of umbilical vein and artery. The techniques used were in situ hybridization and in situ hybridization combined with immunocytochemistry, using ³²P-radiolabelled and non-radioactive digoxigenin labelled complementary RNA probes. Human ANP mRNAs are mainly localized in the endothelial cells of the umbilical vein and, to a lesser extent, in the endothelial cells of the umbilical artery. The autoradiographic labelling and the intensity of digoxigenin staining were significantly reduced by treatment with RNase before in situ hybridization. This study provides unequivocal evidence for the expression of the ANP gene in the endothelial cells of human umbilical vessels, confirming that these endothelial cells have the ability to synthesize this peptide. The functional significance of the presence of the ANP mRNA in the endothelial cells of human umbilical vessels is discussed.     

IL-2 production in human T lymphotropic virus I-infected leukemic T lymphocytes analyzed by in situ hybridization

In situ hybridization studies were performed with 35S-labeled anti-sense RNA probes to study IL-2 mRNA expression in three human T lymphotropic virus I-infected T cell lines at the single cell level. In HuT 102, MT-2, and MT-4 cells, IL-2 mRNA-expressing cells were identified, occurring at frequencies of 2 x 10(-2), 8 x 10(-3), and 5 x 10(-3), respectively. In these cell lines, IL-2 mRNA was not detectable in RNA extracted from whole adult T cell leukemia cell populations because of dilution by other RNA species from the vast majority of cells that do not contain IL-2 mRNA. The data indicate the possibility of paracrine growth stimulation via IL-2 and its receptor even in those human T lymphotropic virus I-infected T cell populations that apparently lack IL-2 activity when analyzed by conventional assay procedures.     

CD1d-restricted NKT cells contribute to the age-associated decline of T cell immunity

NKT cells are known to regulate effector T cell immunity during tolerance, autoimmunity, and antitumor immunity. Whether age-related changes in NKT cell number or function occur remains unclear. Here, we investigated whether young vs aged (3 vs 22 mo old) mice had different numbers of CD1d-restricted NKT cells and whether activation of NKT cells by CD1d in vivo contributed to age-related suppression of T cell immunity. Flow cytometric analyses of spleen and LN cells revealed a 2- to 3-fold increase in the number of CD1d tetramer-positive NKT cells in aged mice. To determine whether NKT cells from aged mice differentially regulated T cell immunity, we first examined whether depletion of NK/NKT cells affected the proliferative capacity of splenic T cells. Compared with those from young mice, intact T cell preparations from aged mice had impaired proliferative responses whereas NK/NKT-depleted preparations did not. To examine the specific contribution of NKT cells to age-related T cell dysfunction, Ag-specific delayed-type hypersensitivity and T cell proliferation were examined in young vs aged mice given anti-CD1d mAb systemically. Compared with young mice, aged mice given control IgG exhibited impaired Ag-specific delayed-type hypersensitivity and T cell proliferation, which could be significantly prevented by systemic anti-CD1d mAb treatment. The age-related impairments in T cell immunity correlated with an increase in the production of the immunosuppressive cytokine IL-10 by splenocytes that was likewise prevented by anti-CD1d mAb treatment. Together, our results suggest that CD1d activation of NKT cells contributes to suppression of effector T cell immunity in aged mice.     

IL-4 regulates VIP receptor subtype 2 mRNA (VPAC2) expression in T cells in murine schistosomiasis.

In murine schistosomiasis, granuloma T cells express VPAC2 mRNA, whereas there is none in splenocytes. This suggests that T cell VPAC2 mRNA is inducible. To address this issue, splenocytes from schistosome-infected mice were incubated with anti-CD3 to induce VPAC2 mRNA, which only appeared when cell cultures also contained anti-IL-4 mAb. Granuloma cells expressed VPAC2 mRNA. This natural expression decreased substantially when cells were cultured 3 days in vitro. However, granuloma cells cultured with anti-IL-4 mAb strongly expressed VPAC2 mRNA. IL-4 KO mice were examined to further address the importance of IL-4 in VPAC2 regulation. Splenocytes and dispersed granuloma cells from IL-4 KO animals had substantially more VPAC2 mRNA than those in wild-type controls. VPAC2 mRNA content decreased when cells were cultured with rIL-4. VPAC2 mRNA localized to CD4+ T cells. Th1 cell lines expressed VPAC2 mRNA much stronger than Th2 cells. Anti-IL-4 mAb increased VPAC2 mRNA expression in Th2 cells cultured in vitro. However, rIL-4 could not suppress VPAC2 mRNA expression in Th1 cells. Thus, VPAC2 is an inducible CD4+ T cell receptor, and IL-4 down-modulates VPAC2 mRNA expression in Th2 cells.     

In situ T cells in melanoma

During the past decade new insights have been gained into the role of T lymphocytes in the host's immune response to cancer in general and to melanoma in particular. Several melanoma-associated antigens (MAA) recognized by T cells have been characterized, and a number of HLA class I- and class II-restricted peptides have been identified. These antigens can be divided into three different groups: tumor-associated testis-specific antigens, melanocyte differentiation antigens, and mutated or aberrantly expressed antigens. These proteins give rise to several antigenic peptides. The number of known melanoma-associated peptides that can induce killing by cytotoxic T-lymphocytes (CTL) exceeds 30 and is still increasing. In line with these findings, clinical data indicate that the immune system is essential in the control of tumor growth. A brisk infiltration of lymphocytes is associated with a favorable prognosis, and complete or partial regression of primary melanoma occurs quite frequently. Furthermore, immunomodulatory therapies have accomplished complete or partial tumor regression in a number of patients. However, the immune response is in most cases inadequate to control tumor growth as tumor progression often occurs. Hence, the coexistence of a cellular immune response in melanoma lesions, demonstrated by the presence of clonally expanded T cells, remains a major paradox of tumor immunology. In the present paper we review current knowledge regarding tumor infiltrating lymphocytes (TIL) in melanoma and discuss possible mechanisms of escape from immune surveillance. Received: 20 March 1999 / Accepted: 3 March 1999     

In situ hybridization technique to localize rRNA and mRNA in mammalian neurons

In situ hybridization provides a method for identifying cells that contain specific nucleic acid sequences. This report outlines an in situ hybridization procedure for mammalian neural tissue. The method maintains morphological quality and produces excellent specificity. Seven tritiated nucleic acid probes were examined: two ribosomal RNA probes, a control pBR322 plasmid probe, two probes encoding portions of the gene for oxytocin, one probe each encoding a portion of vasopressin glycoprotein, and neurophysin. Using cryostat-cut rat brain sections, rRNA probes labeled the cytoplasm of all cells and the nucleoli of larger neurons. The plasmid probe failed to produce a strong signal. Oxytocin and vasopressin probes appropriately labeled the cytoplasm of hypothalamic magnocellular neurons. Vasopressin parvocellular neurons were not identified by the current method, and the shorter length neurophysin probe failed to produce a signal. Methodological variables were examined by counting autoradiographic grains in cells. The longer oxytocin probe produced a stronger signal than the shorter oxytocin and vasopressin probes, and higher probe concentrations resulted in stronger signal. Hybridization could be abolished by tissue pretreatment with RNAse A, and longer exposure time increased signal strength. The outlined fixation steps with fresh-frozen tissue produced a superior signal compared to paraformaldehyde-perfused tissue.     

薄芝糖肽对小鼠T细胞IL-2、IL-3 mRNA表达的影响

目的:探讨薄芝糖肽对小鼠T细胞IL-2、IL-3 mRNA表达的影响.方法:制备静息T淋巴细胞和活化T淋巴细胞,收集细胞并提取总mRNA,进行RT-PCR扩增,测定IL-2活性和IL-3活性,并对数据进行t测验.结果:小鼠IL-2、IL-3 mRNA表达量随薄芝糖肽浓度的增加而升高,且有一定的浓度依赖性.结论:薄芝糖肽免疫增强和抗肿瘤作用的基础是与其在转录水平增强IL-2 mRNA的表达分不开的.T细胞是薄芝糖肽作用的靶细胞,而且说明了增强T细胞中两种细胞因子mRNA的表达是薄芝糖肽免疫调节的重要作用机制.     

In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors

Summary The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with ³⁵S-labeled oligonucleotide probes in formalin-fixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the mRNA for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of mRNA expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.     

Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization

Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.     

Regulation of tyrosine hydroxylase mRNA in catecholaminergic cells of embryonic rat: analysis by in situ hybridization

In situ hybridization was used to examine the appearance of mRNA specific for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (CA) biosynthesis, in neural crest derivatives of the rat embryo. These derivatives include sympathetic ganglia and transient catecholaminergic cells of embryonic intestine. Messenger RNA is first detected in sympathetic ganglia at E11.5, the age corresponding to the initial immunocytochemical expression of TH protein. In older embryos increased accumulation of TH-specific mRNA in sympathetic ganglia parallels the increase in TH immunoreactivity. By contrast, mRNA for TH is difficult to detect in embryonic intestines at E11.5 but is found instead in cells clustered at the dorsal boundaries of the pharynx and foregut. Cells expressing TH mRNA are infrequently found in embryonic intestines at any age, even though TH protein is immunohistochemically apparent. Treatment of pregnant rats with doses of reserpine, known to increase circulating levels of glucocorticoid hormones and prolong the expression of TH protein in embryonic gut cells, dramatically but transiently increases the number of gut cells at E12.5 with detectable TH mRNA. After E13.5 TH mRNA is undetectable even in reserpine-treated guts. Reserpine treatment also increases the labeling density in sympathetic ganglia. Taken together, these data are consistent with the hypothesis that the microenvironment of the embryonic intestine affects gene expression directly to alter phenotype. Moreover, although reserpine administration briefly increases TH mRNA levels, the effect is short-lived and does not alter neurotransmitter phenotypic conversion.