NF-kappaB activation in CD27 signaling: involvement of TNF receptor-associated factors in its signaling and identification of functional region of CD27.

CD27 belongs to TNF receptor family, and it is unique in this family for its disulfide-linked homodimerization of 55-kDa monomers. In the present study we demonstrate that overexpression of CD27 in 293 cells induces a low level of NF-kappaB activation, and the ligation of the receptor by its corresponding ligand (CD70) augments this signal dramatically. Either TNF receptor-associated factor-2 (TRAF2) or TRAF3 binds to the CD27 molecule from the coimmunoprecipitation experiment. This NF-kappaB activation signal is inhibited by dominant negative TRAF2 or intact TRAF3, indicating that TRAF2 and TRAF3 works as a mediator and an inhibitor, respectively. The activated NF-kappaB complex contains at least two components, p50 and p65, but not p52. All these phenomena have also been observed in the TNF receptor type II, CD30 and CD40 signaling system, indicating that this receptor family uses the common or similar molecules for this signal. Finally, we identified the 13-amino acid alignment in the cytoplasmic region of the CD27 molecule (residues 238-250 amino acids), which is critical for the NF-kappaB activation signal and also for its association with TRAFs. This amino acid alignment contains the EEEG sequence, which is essential for interaction of CD30 or CD40 with TRAFs (TRAF1 and TRAF2, but not TRAF3), and also contains the PIQED sequence, which is similar to PXQXT that is known to be necessary for interaction of TNF receptor II and CD30 with TRAFs (TRAF1, 2, and 3).

Cloning and functional analysis of new members of STAT induced STAT inhibitor (SSI) family: SSI-2 and SSI-3.

Upon the corresponding ligand's stimulation, the cytokine receptors activate several signal pathways: JAK-STAT pathway, Ras-MAP kinase pathway and so on. Recently, we demonstrated that one of the STAT3 (signal transducer and activator of transcription-3) target genes could suppress the function of STAT3 and designated as SSI-1(STAT induced STAT inhibitor-1). SSI-1 is thought to play a critical role in negative feedback control of JAK-STAT signaling pathway. In the present study, we identified two novel human genes which products have homologous region in their SH2 domain and its COOH-terminal region to mouse SSI-1. Northern blotting analysis and functional studies demonstrated that SSI-2 and SSI-3 mRNA were also induced by cytokine stimulation and their forced expression in mouse myeloid leukemia cell, M1, suppressed the apoptotic effect of LIF, like SSI-1. We also demonstrated the structure of human SSI-1.

Structure and function of a new STAT-induced STAT inhibitor.

The signalling pathway that comprises JAK kinases and STAT proteins (for signal transducer and activator of transcription) is important for relaying signals from various cytokines outside the cell to the inside. The feedback mechanism responsible for switching off the cytokine signal has not been elucidated. We now report the cloning and characterization of an inhibitor of STAT activation which we name SSI-1 (for STAT-induced STAT inhibitor-1). We found that SSI-1 messenger RNA was induced by the cytokines interleukins 4 and 6 (IL-4, IL-6), leukaemia-inhibitory factor (LIF), and granulocyte colony-stimulating factor (G-CSF). Stimulation by IL-6 or LIF of murine myeloid leukaemia cells (M1 cells) induced SSI-1 mRNA expression which was blocked by transfection of a dominant-negative mutant of Stat3, indicating that the SSI-1 gene is a target of Stat3. Forced overexpression of SSI-1 complementary DNA interfered with IL-6- and LIF-mediated apoptosis and macrophage differentiation of M1 cells, as well as IL-6 induced tyrosine-phosphorylation of a receptor glycoprotein component, gp130, and of Stat3. When SSI-1 is overexpressed in COS7 cells, it can associate with the kinases Jak2 and Tyk2. These findings indicate that SSI-1 is responsible for negative-feedback regulation of the JAK-STAT pathway induced by cytokine stimulation.

Acquired erythropoietin responsiveness of interleukin-2-dependent T lymphocytes retrovirally transduced with genes encoding chimeric erythropoietin/interleukin-2 receptors.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TILs) causes regression of some human tumors. However, the sustained proliferation and antitumor activity of TILs requires the coadministration of potentially toxic amounts of interleukin-2 (IL-2). In an effort to overcome the requirement by T cells for IL-2, we have introduced alternative growth factor receptors that use the relatively nontoxic cytokine erythropoietin (Epo) as a ligand. In our model system, the coexpression of chimeric receptors consisting of the extracellular portion of the Epo receptor (EpoR) and the intracellular portions of the IL-2 receptor subunits, beta and gamma, conferred Epo responsiveness on a T-cell line. By contrast, cells expressing the wild-type EpoR did not proliferate in response to Epo. This suggested that Epo binding caused the activation of an IL-2 signal pathway mediated by the chimeric receptors. This approach can be used to minimize toxicity and potentially improve cancer immunotherapy with TILs.

Electrochemical determination for enzymic production of ultimate carcinogen from tryptophan pyrolysate by rat hepatic microsomes.

This study established a rapid and sensitive method of determining the level of the ultimate carcinogen from 3-amino-1-methyl-5H-prido[4,3-b]indole (Trp-P-2) produced by rat hepatic microsomes. An electrochemical detector (ECD) used with high-performance liquid chromatography (HPLC) gave a linear calibration curve for synthetic N-hydroxy-Trp-P-2 (the ultimate carcinogenic form) at concentrations ranging between 0.3 and 340 pmol. The enzymic production of N-hydroxy-Trp-P-2 from Trp-P-2 was also determined by the ECD with HPLC. Hepatic microsomes (0.2 mg as protein) from rats treated with methylcholanthrene (MC) and phenobarbital (PB) were incubated with Trp-P-2 for 5 min. The mixture was centrifuged with acetonitrile and the supernatant was then analyzed using HPLC. The ECD determined the level of N-hydroxy-Trp-P-2 to levels nearing 1 pmol, and the preparation before submission to the HPLC took such a short time (5 min) that N-hydroxy-Tr-P-2 did not have sufficient time to decompose. Then, the microsomal N-hydroxylation activity on Trp-P-2 was compared with five different sources of microsomes. The microsomes from rats treated with MC plus PB, MC, PB, or polychlorinated biphenyl showed an activity level (mol/min/mol P450 enzymes) of 2.41 +/- 0.19, 1.92 +/- 0.21, 0.048 +/- 0.017, and 1.79 +/- 0.15, respectively, and those from untreated rats showed no activity. This method was useful for evaluating the N-hydroxylation activity of P450 enzymes.

Interleukin-2-transduced lymphocytes grow in an autocrine fashion and remain responsive to antigen.

The maintenance of T lymphocytes in vivo after adoptive transfer for immunotherapy requires the systemic administration of interleukin-2 (IL-2), but prolonged administration of IL-2 is associated with substantial toxicity. The constitutive production of IL-2 by T cells may be an alternative method to prolong T-cell survival and potentially augment antitumor responses. To study the effects of constitutive production of IL-2 on the growth and antigen reactivity of a murine T cell, the sperm-whale myoglobin (SWM) specific T-cell line 14.1 was retrovirally transduced with the cDNA for IL-2. Cells that were transduced with vectors without an internal promoter were able to proliferate in the absence of exogenously added IL-2, and to grow in an autocrine fashion. These vectors used an internal ribosomal entry site (IRES) to allow translation of the neomycin phosphotransferase (neor) gene. In contrast, the cells transduced with an IL-2 vector in which the neor gene was under the transcriptional control of an internal SV-40 promoter failed to proliferate or grow in the absence of exogenously added IL-2. The proliferation of the cells growing without IL-2 could be inhibited with antibodies to the IL-2 receptor or to human IL-2, indicating that they were still IL-2 dependent. Despite their autocrine growth, no tumor formation was observed in syngeneic mice injected subcutaneously with the transduced cells, and the cells retained their antigen reactivity and specificity. These results suggest that autocrine growth of T cells for therapy will not interfere with effector function.

Characterization of the heterodimeric complex of human IL-2 receptor alpha.beta chains reconstituted in a mouse fibroblast cell line, L929.

The receptor for IL-2 has been known to exist in three forms on the basis of their affinities to IL-2: high, intermediate, and low affinity forms. Two IL-2R components have been identified as IL-2R alpha (p55, Tac Ag) and IL-2R beta (p70-75) chains, both bind IL-2 with low and intermediate affinities, respectively. Recently, we cloned human IL-2R beta chain cDNA and demonstrated that the cDNA product binds IL-2 with intermediate affinity and forms high affinity IL-2R with coexpressed IL-2R alpha chain in a human T cell line, Jurkat. In this study, we report the establishment of the mouse fibroblast transformants expressing either the IL-2R beta chain alone or both the IL-2R alpha and IL-2R beta chains. In contrast to lymphoid cells, significant IL-2 binding was not detected in the transformants expressing the IL-2R beta chain alone at IL-2 concentrations (50 pM to 10 nM) generally utilized. Nonetheless, the transformants expressing both IL-2R alpha and IL-2R beta chains displayed two forms of the IL-2R with high and low affinities to IL-2. However, neither IL-2 internalization nor signal transduction via the high affinity IL-2R complex were observed in the L929 transformants. Those findings suggest that the interaction of the IL-2R beta chain with the IL-2R alpha chain occurs in the absence of additional lymphoid specific component(s) to form high affinity IL-2R, but that this interaction is insufficient for IL-2 internalization and signal transduction just as observed in lymphoid cells. The experimental approach described here may allow further dissection of the molecular architecture of the IL-2R complex in the ligand binding, internalization, and signal transduction.

Interleukin 2 receptor beta chain expressed in an oligodendroglioma line binds interleukin 2 and delivers growth signal.

Interleukin 2 (IL-2) is a potent growth factor for T lymphocytes, playing a crucial role in the immune response. In view of the considerable evidence that the immunoregulatory cytokines (or lymphokines) also play a role in the growth and differentiation of cells in the central nervous system (CNS), we examined the operation of the IL-2 system in a cell line of CNS origin by expressing a cDNA encoding the beta chain of the human IL-2 receptor (IL-2R beta, a 75-kDa protein). When the cDNA was expressed in a human oligodendroglioma cell line, ONS-21, the IL-2R beta bound IL-2 with an affinity similar to that in lymphoid cells (Kd, approximately 2 nM). Furthermore, cell proliferation ([3H]thymidine incorporation) was stimulated by IL-2. These results demonstrate that the same cytokine receptor is functional in cells of the immune system and CNS and point to a molecular mechanism that is similar for growth-signal transduction between lymphoid and neural cells but that may be different in other cells, such as fibroblasts.

The human interleukin-2 receptor beta-chain gene: genomic organization, promoter analysis and chromosomal assignment.

The chromosomal gene for the human interleukin-2 receptor beta-chain (IL-2R beta) was isolated and characterized. The entire IL-2R beta gene is composed of ten exons spanning about 24.3 kilobases, in which the protein is encoded by the exons 2-10. The cysteine rich extracellular region which displays a significant evolutionary resemblance to other cytokine receptors, as well as growth hormone and prolactin receptors, is encoded primarily by exons 3 and 4, whereas the membrane proximal, cysteine poor domain showing a homology with type III modules of fibronectin is encoded by exon 7. Sequence analysis of the 5'-flanking region revealed the presence of potential binding sites for transcription factors such as Octamer binding factors, AP-1, AP-2 as well as the 'GC-clusters'. At least five potential cap sites were identified by S1 mapping analysis. The 850 bp DNA sequence of the 5'-flanking region exhibited constitutive promoter activity when it was linked upstream of the HSV-tk reporter gene and then transfected into YT cells, a human leukemic cell line. By applying the RFLP linkage analysis, the IL-2R beta gene has been assigned to chromosome 22q12-13.

Murine interleukin 2 receptor beta chain: dysregulated gene expression in lymphoma line EL-4 caused by a promoter insertion.

The functional, high-affinity interleukin 2 receptor (IL-2R) consists of at least two receptor components, IL-2R alpha (p55) and IL-2R beta (p70-75). The cDNA encoding the murine IL-2R beta has been isolated by using the previously cloned cDNA for human IL-2R beta as a probe. Analysis of the cDNA revealed that the murine IL-2R beta shows a marked homology with the human IL-2R beta and that it is also structurally related to other cytokine receptors such as erythropoietin receptor. The cDNA-directed murine IL-2R beta formed high-affinity IL-2R in conjunction with the endogenous IL-2R alpha in a murine pro-B-cell line and could transduce IL-2-induced growth signal. In mouse lymphoma line EL-4, the IL-2R beta gene was found to be rearranged by the insertion of the long terminal repeat sequence of an intracisternal A particle, giving rise to constitutive expression of the IL-2R beta mRNA.

Human interleukin 2 (IL 2) receptor beta chain allows transduction of IL 2-induced proliferation signal(s) in a murine cell line.

Interleukin 2 (IL 2) delivers cell growth signal by virtue of its interaction with the high-affinity receptor complex, which consists of two distinct IL 2-binding molecules: the IL 2 receptor alpha (IL 2R alpha) and beta (IL 2R beta) chains. Unlike many known growth factor receptors, neither of the IL 2R chains seems to contain a tyrosine kinase domain. In this report, we have shown that the human IL 2R beta chain expressed in a murine IL 3-dependent, non-lymphoid cell line can transduce IL 2-induced cell proliferation signal(s) in combination with the autologous mouse IL 2R alpha chain. This observation should provide a tool to dissect IL 2-induced signal transduction pathway in lymphoid and non-lymphoid cells.

Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's.

Interleukin-2 (IL-2) binds to two distinct receptor molecules, the IL-2 receptor alpha (IL-2R alpha, p55) chain and the newly identified IL-2 receptor beta (IL-2R beta, p70-75) chain. The cDNA encoding the human IL-2R beta chain has now been isolated. The overall primary structure of the IL-2R beta chain shows no apparent homology to other known receptors. Unlike the IL-2R alpha chain, the IL-2R beta chain has a large cytoplasmic region in which a functional domain (or domains) mediating an intracellular signal transduction pathway (or pathways) may be embodied. The cDNA-encoded beta chain binds and internalizes IL-2 when expressed on T lymphoid cells but not fibroblast cells. Furthermore, the cDNA gives rise to the generation of high-affinity IL-2 receptor when co-expressed with the IL-2R alpha chain cDNA.

Ligand-dependent selection of the receptor gene: segregation of IL-2 binding activity and anti-Tac reactivity by a single amino acid alteration in the Tac antigen (p55).

The Tac antigen (p55, CD25) is a 55 kDa glycoprotein that binds interleukin 2 at low affinity (Kd congruent to 10-50 nM). Expression of the Tac antigen is induced in the activated human T cells to constitute the functional, high-affinity IL-2 receptors (IL-2Rs) (Kd congruent to 10 pM) in conjunction with p70-75. A monoclonal antibody, anti-Tac, recognizes this molecule and inhibits the binding of IL-2 to both high- and low-affinity IL-2Rs. This observation indicates that IL-2 and anti-Tac binding sites are located close to each other within the Tac molecule. In this report, by utilizing a novel approach, we selected cDNAs encoding the Tac antigen variants whose reactivity with anti-Tac is greatly reduced, while retaining their IL-2 binding activity. Each of the mutant cDNAs contained a point (G----A) mutation resulting in an amino acid substitution at the particular amino-terminal portion of the Tac molecule (Asp-4). These results demonstrate that N-terminal amino acid Asp-4 is involved in the epitope recognized by anti-Tac, and that IL-2 binding site and anti-Tac binding site are structurally separable from each other in the Tac molecule.

The effects of orally administered linoleic acid and its autoxidation products on intestinal mucosa in rat.

Linoleic acid and its autoxidation products, hydroperoxides and their secondary products, were orally administered to rats (350 mg each/rat). Hemorrhage was seen in the alimentary canal at 6 h after the dose of hydroperoxides. To examine their toxicities on intestinal mucosa, the activities of mucous enzymes (sucrase, maltase, and alkaline phosphatase) were measured. Hydroperoxides and their secondary products decreased the enzyme activities in jejunum at 6 h after the doses and increased them in both jejunum and ileum at 15 h, as compared to linoleic acid or saline solution. The decrease of enzyme activity was marked in the hydroperoxide group and the increase was marked in the secondary product group. Then, in in vitro experiments, the effects of autoxidation products on these enzymes were determined. Autoxidation products inactivated only alkaline phosphatase. Thus, the results in vivo disagreed with those in vitro. It was assumed that autoxidation products orally administered attacked a membrane of intestinal microvilli whereas in vitro they directly affected the enzymes.

Effect of orally administered 9-oxononanoic acid on lipogenesis in rat liver.

9-Oxononanoic acid, which is one of the major products of the autoxidation of linoleic acid, was administered orally to rats and its effect on hepatic lipid metabolism was investigated. The de novo synthesis of fatty acids was strongly reduced 30 h after the administration of 100 mg of 9-oxononanoic acid as compared to that in the saline-administered group. Activity of acetyl-CoA carboxylase decreased by 60% and the activity of carnitine palmitoyltransferase increased by 35% in the test group. The level of triacylglycerols in serum was low and the level of free fatty acids remained unchanged. Thus, the administration of 9-oxononanoic acid decreased hepatic lipogenesis. It is generally believed that the reduction in lipogenesis is facilitated by a decrease in the NADPH level. The ratio of NADPH/NADP in the test group, however, became high as compared to that in the control group, and the activities of glucose 6-phosphate and isocitrate dehydrogenases increased. On the other hand, the levels of CoA derivatives, especially long-chain acyl-CoA, were higher in the test group than in the control. Therefore, the reduction of hepatic lipogenesis in the 9-oxononanoic acid group could be attributed to the inhibition of acetyl-CoA carboxylase by the accumulated long-chain acyl-CoA.

Effect of orally administered secondary autoxidation products of linoleic acid on carbohydrate metabolism in rat liver.

The effects of orally administered secondary autoxidation products of linoleic acid in rat liver were investigated. Their administration led to two toxic effects on hepatic carbohydrate metabolism, as compared to the administration of saline or linoleic acid used as controls. One effect was depletion of glucose 6-phosphate and fructose 6-phosphate caused by the reduction of glycolysis and glycogenolysis, accompanied by decreases in glycogen synthesis and pentose phosphate cyclic activity. The reduction in these metabolic systems seems unlikely to occur because phosphofructokinase was regulated by ATP or citrate enzymatically, because their accumulation in the liver was not detected in the secondary products. Another toxic effect was the depletion of oxaloacetate and isocitrate caused by the reduction in enzyme activity of the mitochondrial citrate cycle. On the basis of these results, the hepatotoxic effects of secondary products are discussed as follows: the incorporated secondary products impaired the activities of hexokinase and phosphoglucomutase in the liver. The reduction in these enzyme activities resulted in the depletion of glucose 6-phosphate and fructose 6-phosphate, which led ultimately to decreases in the activities of phosphofructokinase, the pentose phosphate cycle, and glycogen synthesis. Moreover, the secondary products disturbed the mitochondrial membrane, resulting in a decrease in the activity of the citrate cycle, which was accompanied by depletion of its metabolites.

Transmembrane signaling of interleukin 2 receptor. Conformation and function of human interleukin 2 receptor (p55)/insulin receptor chimeric molecules.

Chimeric genes were constructed which gave rise to the expression of novel receptor molecules consisting of the extracellular domain of the human interleukin 2 receptor (IL-2-R; p55 or Tac antigen) joined to the transmembrane domain and either full-length or truncated cytoplasmic domain of the human insulin receptor (Ins-R). Expression studies using mouse T cell line EL-4 revealed that the chimeric receptors are able to manifest properties indistinguishable from the authentic IL-2-R. On the other hand, stimulation of the tyrosine kinase activity by IL-2 was not observed in the chimeric receptor with the entire cytoplasmic domain of the Ins-R. These findings thus shed light on the structural conformation and functioning of the IL-2-R complex.