Transient expression of human interleukin-2 and interferon-γ genes is regulated by interaction between distinct cell subsets

The level of transient expression of human IL-2 and IFN-γ genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-γ gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-γ was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-γ gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-γ mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition.     

Priming of leukocytes selectively increases the level of some interferon-α subtypes and not others

The effect of pretreatment with interferon (IFN) (‘priming’) on the production of individual IFN subtypes was studied in subpopulations of human peripheral blood mononuclear cells and in the myeloid cell line KG-1. It was found that priming had a selective enhancing effect on the production of certain IFN-α subtypes (IFN-α20K and IFN-α21K) and not on others. KG-1 cells produce both IFN-α and -β; however, only the production of IFN-α was enhanced by priming with either IFN-α,β or γ.     

Somatic cell mapping of the bovine interferon-α receptor

The bovine interferon- receptor (BoIFN-R) mediates the activity of bovine IFN-s and IFN-. In addition, human IFN-s have uniformly high biological activity on bovine cells. A ³²P-labeled derivative of human recombinant IFN-A (HuIFN-A-P1) binds well and can form a characteristic 130-kDa complex on bovine cells, but not on hamster cells. We have, therefore, analyzed the binding and covalent crosslinking of [³²P]HuIFN-A-P1 to a panel of bovine-hamster somatic cell hybrids. Binding to several bovine-hamster hybrid cell lines was strong (about 30–50% of that seen with bovine MDBK cells) and specific. The binding correlated uniquely with bovine syntenic group U10. In several of the hybrid lines, the ability of human IFN-B to enhance the expression of endogenous MHC class I molecules correlated with the binding results. We thus conclude that the bovine IFN-R structural gene (locus designation IFNAR) localizes to syntenic group U10. This group includes a number of other genes whose homologs map to human Chromosome (Chr) 21.A summary of this work was presented at the annual meeting of the International Society for Interferon Research (November 1991, Nice, France) and appeared as an abstract for that meeting (Langer et al., J Interferon Res 11 (Suppl): S203, 1991).     

The effect of interferon-β on mouse neural progenitor cell survival and differentiation

Interferon-β (IFN-β) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-β on the central nervous system (CNS) are not well understood. To determine whether IFN-β has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-β and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFNα/β receptor (IFNAR). In response to IFN-β treatment, no effect was observed on differentiation or proliferation. However, IFN-β treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-β treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-β can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.     

The effect of unphosphorylated and phosphorylated sugar moieties on human and mouse natural killer cell activity: is there selective inhibition at the level of target recognition and lytic acceptor site?

A number of different sugars were investigated for their effect on human and mouse natural killer cell (NK)-mediated cytolysis. From the pool of nonphosphorylated sugars, D-mannose, N-acetyl-D-glucosamine (NAcGlc), D-glucose, and, to a lesser extent, beta-gentiobiose were found to inhibit human NK cytolysis. Mouse NK activity against YAC-1 target cells was reduced consistently in the presence of D-mannose and NAcGlc only. The sugars, NAcGlc, D-glucose, and beta-gentiobiose, were specifically inhibitory against NK-mediated cytolysis with no inhibitory effects being observed against ADCC, monocyte-mediated cytolysis, or CTL activity. Pretreatment and washing at either the target or effector cell level as well as direct target binding assays using Percoll-purified NK cells indicated that at least NAcGlc and beta-gentiobiose function at the recognition stage of NK cytolysis. D-Mannose, which was the most effective nonphosphorylated sugar inhibitor, was capable of inhibiting all cell-mediated cytotoxic mechanisms tested (NK, ADCC, monocyte, and CTL) and its action did not appear to be solely due to an impairment in the recognition event. All the phosphorylated sugars caused significant inhibition of human and mouse NK-mediated cytolysis, although repeated analyses of sugar titration curves consistently showed mannose-6-phosphate (Man-6-P) to be the most effective inhibitor. Inhibition with the phosphorylated sugars was apparent against all cytotoxic mechanisms investigated. It is possible that these sugars may function as general metabolic inhibitors or may activate a common signal which negatively regulates cell-mediated cytotoxic mechanisms. Nevertheless, the relative degree of inhibition with the majority of these sugars (particularly Man-6-P) was greater against NK and ADCC activity than against monocyte and CTL activity. Furthermore, studies with selected well-characterized human and mouse NK-resistant target cells strongly indicated that these sugars, particularly Man-6-P, compete at an acceptor site responsible for the uptake of the NK lytic factor, which is independent of the recognition structure(s).     

Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-α therapy

Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-α (IFN-α). Animal studies showed that IFN-α administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases (MAOs), and finally in a change in NO production due to the H₂O₂ generated by MAOs. We examined the potential relationship between NO production in plasma and MAO-B activity in platelets of 43 cancer patients during 8 weeks of treatment with IFN-α. NO synthesis was quantitated by measuring both the ratio of citrulline and arginine (CIT/ARG-ratio) and total nitrite/nitrate (NOx) levels. Compared to baseline, MAO activity and NOx increased, while the CIT/ARG-ratio decreased. No associations were found between NOx, MAO and CIT/ARG-ratio. Only few associations were observed between changes in the biochemical parameters and changes in psychopathology induced by IFN-α, of which the association between changes in CIT and lassitude was the most consistent. The results suggest that peripheral NO production and MAO activity are unrelated to each other, and that peripheral changes in these biochemical parameters induced by IFN-α are unlikely to contribute to definite psychiatric disturbance.     

Plasmacytoid dendritic cell dichotomy: identification of IFN-α producing cells as a phenotypically and functionally distinct subset

Plasmacytoid dendritic cells (pDC) produce large amounts of type I IFN in response to invading pathogens, but can also suppress immune responses and promote tolerance. In this study, we show that in mice, these functions are attributable to two distinct pDC subsets, one of which gives rise to the other. CD9(pos)Siglec-H(low) pDC secrete IFN-α when stimulated with TLR agonists, induce CTLs, and promote protective antitumor immunity. By contrast, CD9(neg)Siglec-H(high) pDC secrete negligible amounts of IFN-α, induce Foxp3(+) CD4(+) T cells, and fail to promote antitumor immunity. Although newly formed pDC in the bone marrow are CD9(pos) and are capable of producing IFN-α, after these cells traffic to peripheral tissues, they lose CD9 expression and the ability to produce IFN-α. We propose that newly generated pDC mobilized from the bone marrow, rather than tissue-resident pDC, are the major source of IFN-α in infected hosts.     

The role of ‘Norin 10’ dwarfing genes in photosynthetic and respiratory activity of wheat leaves

Summary A comparative analysis of eight cultivars of spring wheat (Triticum aestivum) classified by height as tall (T), semi-dwarf (D₁), dwarf (D₂) and very dwarf (D₃) was conducted to study their efficiency of oxygen exchange during photosynthesis and dark respiration. Two cultivars were included in each height group.Cultivars carrying Norin 10 dwarfing genes (D₁, D₂ and D₃) were found to have a significantly higher photosynthetic rate per unit leaf area than talls (T) that lack these genes. Among the Norin gene carriers, dwarf group (D₂) was most efficient, followed by very dwarf (D₃) and semi-dwarf (D₁).Photosynthetic rate and respiratory rate were found to have a positive relationship.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

Physiological activity and receptor binding of 9α fluorohydrocortisone

Hepatic gluconeogenic stimulation by 9αfluorocortisol was associated with saturation of GR₁ and GR₃ entities of the glucocorticoid specific receptor (GR), even in presence of spironolactone; renal glycogen levels were not altered. Binding to MR₁ and MR₂ components of the mineralocorticoid specific receptor (MR) in the kidney persisted even in presence of 100 fold excess of nonradioactive corticosterone although this was totally abolished by cold equimolar spironolactone. These data suggest that this fluorinated derivative may be particularly appropriate in studying organ specific responses.     

Effect of short-time treatment with TNF-α on stem cell activity and barrier function in enteroids

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1β, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function.

     

Structural and functional variability among DQ β alleles of DR2 subtypes

Homozygous lymphoblastoid cell lines representing various Dw subtypes of DR2 were examined for polymorphism at the DQ locus by molecular and cellular techniques. The subtypes studied included Dw2, Dw12, and a group heterogenous by cellular typing that we shall refer to as non-Dw2/non-Dw12. Restriction fragment length polymorphism analysis of cell lines representing these subtypes revealed DQ -specific patterns consistent with cellular typing. Two-dimensional gel electrophoresis of DQ molecules from representative cell lines revealed a structural polymorphism of DQ among the three subtypes. The DQ chain migrated to a position that was unique to each subtype and was consistent among various representative cell lines of each subtype. Nucleotide sequence analysis of cDNA clones of DQ from Dw2, Dw12, and non-Dw2/non-Dw12 lines confirmed that the variability resided at the genetic level. Variability was found in the form of numerous scattered nucleotide substitutions throughout the first domain of these alleles. The DQ gene of the non-Dw2/non-Dw12 cell line AZH was further found to be almost identical with the DQ gene of a DR1 line (Bell et al. 1985b), implicating a common evolutionary origin of these alleles. The only difference between these two sequences was due to an apparent gene conversion event at amino acid 57. T-cell cloning experiments resulted in the derivation of Epstein-Barr virus-specific, DQw1-restricted clones that proliferated against only those cell lines that exhibited the DQ gene common to AZH and the DR1 cell line. Thus, the polymorphism among DQ alleles within DR2 results in subtype-specific restriction.     

In vivo topoisomerase I inhibition attenuates the expression of hypoxia-inducible factor 1α target genes and decreases tumor angiogenesis

Topoisomerase I is a privileged target for widely used anticancer agents such as irinotecan. Although these drugs are classically considered to be DNA-damaging agents, increasing evidence suggests that they might also influence the tumor environment. This study evaluates in vivo cellular and molecular modifications induced by irinotecan, a topoisomerase I-directed agent, in patient-derived colon tumors subcutaneously implanted in athymic nude mice. Irinotecan was given intraperitoneally at 40 mg/kg five times every 5 d, and expression profiles were evaluated at d 25 in tumors from treated and untreated animals. Unexpectedly, the in vivo antitumor activity of irinotecan was closely linked to a downregulation of hypoxia-inducible factor-1α (HIF1A) target genes along with an inhibition of HIF1A protein accumulation. The consequence was a decrease in tumor angiogenesis leading to tumor size stabilization. These results highlight the molecular basis for the antitumor activity of a widely used anticancer agent, and the method used opens the way for mechanistic studies of the in vivo activity of other anticancer therapies.     

δ antagonist and κ agonist activity of naltriben: Evidence for differential κ interaction with the δ1 and δ2 opioid receptor subtypes

The selective δ₂ receptor antagonist Naltriben (NTB) has played an important role in the identification of subtypes of the δ opioid receptor, termed δ₁ and δ₂, and their role in antinociception. However, the majority of these studies have been conducted in the mouse. The present study determined the opioid receptor selectivity of subcutaneously (s.c.) administered NTB in the rat. Five minute pretreatment with 1 mg/kg s.c. NTB antagonized the increase in TFL produced by i.t. administration of equieffective doses of the δ₂ receptor agonist [D-Ala², Glu⁴]deltorphin (DELT) or the δ₁ receptor agonist [D-Pen², D-Pen⁵]enkephalin (DPDPE), but did not antagonize the μ receptor agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO). These data confirm previous reports that NTB is a selective δ opioid receptor antagonist. However, this dose of NTB antagonized DELT and DPDPE to an equivalent extent, suggesting that its selectivity for the δ₂ receptor is not maintained after s.c. administration in the rat. A lower dose of NTB (0.56 mg/kg s.c.) was ineffective. When the dose of NTB was increased to 3 mg/kg s.c. the antagonism of DELT and of DPDPE was unexpectedly lost. Pretreatment with the κ receptor antagonist nor-binaltorphimine (nor-BNI) partially restored the antagonism of DELT, but not DPDPE by this dose of NTB and did not modify the antagonism of DAMGO by NTB. These data suggest that high doses of NTB have κ receptor agonist-like activity and support the proposal that κ opioid agonists diminish the actions of δ receptor antagonists. They also suggest that nor-BNI-sensitive κ opioid receptors interact with δ₂, but not δ₁ opioid receptors in the spinal cord.