Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor.

Soluble forms of the interleukin (IL)-2, IL-4 and IL-7 receptors which lack the transmembrane domain have been described. IL-6 is a growth factor important in the final differentiation of B-cells into plasma cells and in the pathogenesis of multiple myeloma. To determine whether the receptor for IL-6 may exist as a soluble molecule, RNA was analysed from the transformed B-cell lines U266, CESS and Daudi, from bone marrow from two myeloma patients, and from normal leukocytes. Using polymerase chain reaction, oligonucleotide primers which flank the transmembrane domain were selected to generate a 339 bp fragment. All samples produced equivalent amounts of the expected 339 bp fragment plus a smaller 245 bp fragment except Daudi which exhibited virtual absence of both. Sequence analysis of the smaller fragments from each of the five samples demonstrated the deletion of the entire transmembrane region from codons 356 (G-TG) to 387 (AG-G). The boundaries of this deletion were identical in all cases. Partial sequence analysis of the ligand-binding domain for U266 demonstrated identical sequences for the membrane-bound and soluble forms of the IL-6 receptor cDNAs. In summary, an mRNA which encodes a soluble form of the IL-6 receptor is expressed in both normal and myeloma cells.     

The Lin28/let-7a/c-Myc pathway plays a role in non-muscle invasive bladder cancer

We investigate the role of the Lin28/let-7a/c-Myc pathway in non-muscle invasive bladder cancer (NMIBC). Using RT-PCR, western blot and immunohistochemistry techniques, the levels of pre-let-7a, let-7a, Lin28 and c-Myc RNA and/or proteins were determined in samples of normal bladder tissue and bladder cancer. Expression of pre-let-7a was found to be negatively correlated with the pathological grade of bladder cancer, while let-7a showed a positive correlation with bladder cancer pathological grade. Expression of Lin28 RNA and protein was not significantly different between normal bladder tissue and low-grade transitional cell carcinoma of bladder (TCC) but the expression levels in high-grade TCC were remarkably increased. Expression of c-Myc RNA and protein was significantly higher in bladder cancer samples in comparison to normal bladder tissue without correlation with cancer differentiation. Expression of all the above RNAs and proteins showed no significant difference in Ta and T1 stages. The Lin28/let-7a/c-Myc pathway plays an important role in NMIBC. In particular, expression levels of let-7a correlate with the degree of cancer differentiation but not cancer stage.     

Adult human liver contains CD8pos T cells with naive phenotype, but is not a site for conventional alpha beta T cell development

Normal adult human liver (AHL) contains populations of unconventional lymphocytes that have been shown in the mouse to mature locally. The presence of lymphoid progenitors together with IL-7, recombinase-activating gene, and pre-TCR-alpha expression in AHL suggests similar local T cell development activity in humans. Flow cytometry was used to characterize potentially naive hepatic alphabeta-T cells. We looked for evidence of TCR-alphabeta cell development in AHL by quantifying delta deletion TCR excision circles (TRECs) in CD3(pos) populations isolated from the liver and matched blood of eight individuals. Phenotypic analysis of hepatic T cells suggests the presence of Ag-inexperienced populations. TRECs were detected in all blood samples (mean, 164.10 TRECs/ micro g DNA), whereas only two hepatic samples were positive at low levels (59.40 and 1.92). The relatively high level of CD8(pos) T cells in these livers with a naive phenotype suggests that in addition to its role as a graveyard for Ag-specific activated CD8(pos) T cells, naive CD8(pos) T cells may enter the liver without prior activation. The almost complete absence of TRECs suggests that normal AHL is not a site for the development of conventional alphabeta T cells.     

Evidence for the Presence of Nontranslated T7 Late mRNA in Infected F′(PIF+) Episome-Containing Cells

The inability of T7 to develop in cells of Escherichia coli containing F(+) or substituted F' episomes is a result of the failure to synthesize late proteins; no in vivo translation of mRNA species synthesized by the T7 RNA polymerase occurs. Further experiments have been performed to measure the amount of late mRNA in T7-infected F'(PIF(+)) cells. (We have designated the property of phage inhibition of F factors as PIF; the wild-type episome is therefore F'[PIF(+)].) T7 late proteins were synthesized in vitro by using a system programed with RNA extracted from T7-infected F(-) and F'(PIF(+)) cells. The T7 lysozyme, product of gene 3.5, and the gene 10 head protein were assayed. The following results were obtained: (i) mRNA capable of supporting in vitro synthesis of lysozyme and the gene 10 head protein is present in T7-infected F'(PIF(+)) cells; (ii) lysozyme mRNA extracted from T7-infected F'(PIF(+)) cells is present at 70 to 75% of the level found in T7-infected F(-) cells; (iii) gene 10 mRNA is present at 35 to 78% of the level found in T7-infected F(-) cells. No in vivo synthesis of either lysozyme or gene 10 protein can be detected in T7-infected F'(PIF(+)) cells although normal synthesis of these proteins occurs in F(-) cells. These findings confirm that the block in T7 development in F'(PIF(+)) cells results from the failure to translate late classes of T7 RNA.     

Control of T cell development in vivo by subdomains within the IL-7 receptor alpha-chain cytoplasmic tail

IL-7/IL-7R signaling functions in both growth and differentiation during T cell development. In this study, we examined the extent these activities were controlled by signaling associated with distinct IL-7R alpha cytoplasmic domains by transgenic expression of wild-type or cytoplasmic deletion mutants of IL-7R alpha in the thymi of IL-7R alpha(-/-) mice. We show an essential requirement for the tyrosine-containing carboxyl-terminal T domain in restoring thymic cellularity, pro-/pre-T cell progression, and survival. In contrast, the functional differentiation of TCR alpha beta cells and the development of TCR gamma delta cells are partially independent of the T domain. Thus, separate cytoplasmic domains of the IL-7R alpha chain differentially control distinct functions during T cell development, whereas normal IL-7R-dependent thymic development requires the integrated activity of all these domains.     

CD7 augments T cell proliferation via the interleukin-2 autocrine pathway.

The role of CD7, a T cell differentiation antigen, in T cell function is not known at present; this study evaluates the effect of anti-CD7 mAb in PMBC cultures activated with suboptimal concentrations of lectins, antigens, and anti-CD3 mAb. We found that the inclusion of anti-CD7 resulted in increased IL-2 production and IL-2R-alpha expression in these cultures. H-7, a protein kinase C (PKC) inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, significantly suppressed the proliferation of T cells in comitogenic assays. This suggested that the comitogenic effect mediated by CD7 molecule involved both the PKC and the PTK pathways of T cell activation. These drugs appeared to affect the CD7-mediated effects by inhibiting the IL-2 autocrine pathway, especially the up-regulation of IL-2R-alpha since inhibition was not relieved with exogenous rIL-2. Taken together, our results suggest that CD7 augments T cell function by up-regulating IL-2R-alpha expression and IL-2 production via multiple pathways of protein phosphorylation.     

Presence of a 20 bp insertion/deletion in the ITS1 region of Verticillium lecanii

Polymerase chain reaction (PCR) amplification of the complete ribosomal RNA internally transcribed spacer (ITS) region of 36 isolates of Verticillium lecanii and related species gave a single 620 bp product in 31 isolates. Five isolates received as V. lecanii, however, gave a single product of 600 bp. Restriction fragment analysis of the PCR products from all isolates gave consistent patterns for the 31 isolates with a 620 bp product. The five isolates with the 600 bp product showed only minor discrepancies to these, generally related to the size of only one restriction fragment. The total ITS region was sequenced from 10 typical 620 bp isolates and one 600 bp isolate. Sequence variation between the isolates varied from 0 to 14.5%, and the 20 bp size discrepancy was found to relate to an insertion or deletion in the centre of the ITS1 region.     

Human IL-7: a novel T cell growth factor

IL-7 is a hemopoietic growth factor that induces the proliferation of early B lineage cells. In the course of studies to determine its effect on human bone marrow cells, we noted a marked outgrowth of mature T cells. When T cells from the circulation were cultured with IL-7, a dose-dependent proliferative response was observed. The target cells included both the CD4+ and CD8+ subpopulations of T cells, but the memory T cells (CD45R-) were better responders than unprimed T cells (CD45R+). IL-7 induced the expression of receptors for IL-2 and transferrin and higher levels of the 4F2 activation Ag. Although T cell responses to suboptimal concentrations of IL-7 were enhanced by the addition of IL-2, the proliferative response to IL-7 was not inhibited by neutralizing antibody to the IL-2R (Tac), nor was IL-2 secretion detected in this response. This response pattern of mature T cells suggests an important role for IL-7 in normal T cell physiology in humans.     

IL-7 enhances the survival and maintains the size of naive T cells.

T cells require continual presence of extrinsic signals from their in vivo microenvironment to maintain viability. T cells removed from these signals and placed in tissue culture atrophied and died in a caspase-independent manner. Atrophy was characterized by smaller cell sizes, delayed mitogenic responses, and decreased glycolytic rate. Bcl-2 expression remained constant in vitro despite ongoing cell death, indicating that endogenous Bcl-2 expression is insufficient to explain the life span and size control of lymphocytes in vivo and that cell-extrinsic signals provided may be required to maintain both cell viability and size in vivo. One such signal, IL-7, was found to maintain both the size and survival of neglected T cells in vitro. IL-7 was not unique, because the common gamma-chain cytokines IL-2, IL-4, and IL-15, as well as the gp130 cytokine IL-6, also promoted both T cell survival and size maintenance. IL-7 did not induce resting T cells to proliferate. Instead, IL-7 stimulated neglected T cells to maintain their metabolic rate at levels comparable to freshly isolated cells. The survival and trophic effects of IL-7 could be separated because IL-7 was able to promote up-regulation of Bcl-2 and maintain cell viability independent of phosphatidylinositol 3-kinase and mammalian target of rapamycin activity but was unable to prevent cellular atrophy when phosphatidylinositol 3-kinase and mammalian target of rapamycin were inhibited. These data demonstrate that T cells require the continuous presence of extrinsic signals not only to survive but also to maintain their size, metabolic activity, and the ability to respond rapidly to mitogenic signals.     

Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.     

T cell-specific negative regulation of transcription of the human cytokine IL-4.

IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells, B cells, and mast cells. We investigated the upstream regulatory elements of the human IL-4 promoter. A novel T cell-specific negative regulatory element (NRE) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene: -311CTCCCTTCT-303 (NRE-I) and -288CTTTTTGCTT-TGC-300 (NRE-II). A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II, respectively, were identified. Furthermore, a positive regulatory element was found 45 bp downstream of the NRE. The enhancer activity of the PRE was completely suppressed when the NRE was present. These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element. These data may have implications for the stringent control of IL-4 expression in T cells.