Th-1 lymphocytes induce dendritic cell tumor killing activity by an IFN-γ-dependent mechanism

Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.     

Antitumor effects of human recombinant interleukin-1α and etoposide against human tumor cells: mechanism for synergismin vitro and activityin vivo

Recombinant human interleukin 1α (rh IL-1α) and etoposide (VP-16) synergize for direct growth inhibition of several human tumor cell linesin vitro. Our previous studies demonstrated that VP-16 increased the number of membrane-associated IL-1 receptors (IL-1Rs) and also enhanced the internalization of receptor-bound rh IL-1α. The purposes of this study were to test our hypothess that these events were critical to the synergy between rhIL-1α and VP-16, to determine whether rhIL-1α and VP-16 synergize to increase superoxide (SO) anion radical productionin vitro since SO anion has been implicated in the toxic effects of IL-1, and to investigate the antitumor efficacy of the combinaton against tumors in vivo. A375/C6 melanoma cells and OVCAR-3 ovarian carcinoma cells were tested with IL-1 receptor antagonist (IL-1ra) before exposure to rhIL-1α, VP-16 and rhIL-1α plus VP-16. The synergistic or antagonistic effects were assessed by MTT assay. SO production was measured by reduction of cytochrome C. Athymic female mice bearing the A375/C6 melanoma were treated by rhIL-1α, VP-16, and rhIL-1α+VP-16. The antitumor effects were evaluated by quantitating tumor growth and survival time. Pretreatment with the IL-1ra abrogated the synergistic effects of rhIL-1α and VP-16. The production of SO radical by A375/C6 cells was increased 2.5 fold by the combination of rhIL-1α and VP-16, and the addition of exogenous SOD blocked the synergy between rhIL-1α and VP-16. However, when A375/S0D15 cells which over-expressed manganese superoxide dismutase (MnSOD) after MnSOD cDNA transfecton were exposed to rhIL-1α and VP-16, in vitro antagonism was observed. In vivo studies demonstrated that the combination of rhIL-1α and VP-16 delayed tumor growth better than either agent alone, although long-term survival was not improved because of substantial toxicity. Our results suggest that the synergistic antitumor effects of IL-1α and VP-16 may be due to IL-1R modulation and increased internalization of IL-1-IL-1R complex by VP-16 treatment, as well as to a subsequent increase in SO anion radical production from the tumor cells exposed to both drugs. Thus, the combnation of IL-1α and VP-16 might prove useful for the treatment of malignant diseasein vivo, if the increased toxicity can be reduced or managed. The US Government’s right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged.     

Microbe-induced T cell apoptosis: subversion of the host defense system?

Cells of multicellular organisms are equipped with a self destruction program called apoptosis to ensure homeostasis of the organism. Contraction of the lymphocyte compartment following recovery from an infection is controlled by this mechanism. But apoptosis of lymphocytes might be an Achilles tendon accessible to microbes to subvert the immune system. Evidence is cumulating that microbes use this mechanism to destroy microbe-specific T cells. We present an overview of microbe-induced T cell apoptosis discussing the consequences for the pathogenesis of microbial infection. The conventional role of lymphocytes during infection is to impose apoptotic threat to infected cells, the subject of this review highlights the opposite, lymphocytes as targets of microbe-induced death.     

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).     

Destination 'lysosome': a target organelle for tumour cell killing?

Lysosomes and lysosome-related organelles constitute a system of acid compartments that interconnect the inside of the cell with the extracellular environment via endocytosis, phagocytosis and exocytosis. In recent decades it has been recognized that lysosomes are not just wastebaskets for disposal of unused cellular constituents, but that they are involved in several cellular processes such as post-translational maturation of proteins, degradation of receptors and extracellular release of active enzymes. By complementing the autophagic process, lysosomes actively contribute to the maintenance of cellular homeostasis. Proteolysis by lysosomal cathepsins has been shown to mediate the death signal of cytotoxic drugs and cytokines, as well as the activation of pro-survival factors. Secreted lysosomal cathepsins have been shown to degrade protein components of the extracellular matrix, thus contributing actively to its re-modelling in physiological and pathological processes. The malfunction of lysosomes can, therefore, impact on cell behaviour and fate. Here we review the role of lysosomal hydrolases in several aspects of the malignant phenotype including loss of cell growth control, altered regulation of cell death, acquisition of chemoresistance and of metastatic potential. Based on these observations, the lysosome is proposed as a potential target organelle for the chemotherapy of tumours. We will also present some recent data concerning the technologies for delivering chemotherapeutic drugs to the endosomal-lysosomal compartment and the strategies to improve their efficacy.     

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.     

Induction of apoptosis in host cells: a survival mechanism for Leishmania parasites?

Leishmania parasites invade host macrophages, causing infections that are either limited to skin or spread to internal organs. In this study, 3 species causing cutaneous leishmaniasis, L. major, L. aethiopica and L. tropica, were tested for their ability to interfere with apoptosis in host macrophages in 2 different lines of human monocyte-derived macrophages (cell lines THP-1 and U937) and the results confirmed in peripheral blood mononuclear cells (PBMC). All 3 species induced early apoptosis 48 h after infection (expression of phosphatidyl serine on the outer membrane). There were significant increases in the percentage of apoptotic cells both for U937 and PBMC following infection with each of the 3 species. Early apoptotic events were confirmed by mitochondrial membrane permeabilization detection and caspase activation 48 and 72 h after infection. Moreover, the percentage of infected THP-1 and U937 macrophages increased significantly (up to 100%) following treatment with an apoptosis inducer. Since phosphatidyl serine externalization on apoptosing cells acts as a signal for engulfment by macrophages, induction of apoptosis in the parasitized cells could actively participate in spreading the infection. In summary, parasite-containing apoptotic bodies with intact membranes could be released and phagocytosed by uninfected macrophages.     

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor α, β, or γ genes

To test the hypothesis that the T-cell receptor (Tcr) gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16⁺ lymphocytosis were analyzed for rearrangements and expression of the human Tcr , , and genes. Two of the clones displayed distinct rearrangements of their Tcr and genes and expressed mature Tcr , , and l RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr and gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.     

Pseudomonas aeruginosa-induced production of free radicals by IFNgamma plus TNFalpha-activated human endothelial cells: mechanism of host defense or of bacterial pathogenesis?

We have previously shown that human umbilical vein endothelial cells (HUVEC) can be activated by IFNgamma plus TNFalpha to kill intracellular (IC) Pseudomonas aeruginosa through production of reactive oxygen intermediate, but the cumulative effects of cytokine activation and bacterial infection on host cells has not been extensively addressed. In this study we investigated the fate of IFNgamma plus TNFalpha-activated HUVEC that have harboured IC bacteria for up to 24 h. At 10 h, the endothelial cell killing of P. aeruginosa isolates exceeded 90%. IC bacteria enhanced the expression of inducible nitric oxide synthase (iNOS) and induced overproduction of NO and superoxide by infected HUVEC. P. aeruginosa IC infection also induced a slight decrease in the cellular level of reduced glutathione (GSH). Overproduction of NO correlated with a marked peroxidation of plasma membrane lipids and decline in HUVEC viability. Treatment of cells with the antioxidant alpha-lipoic acid significantly increased the survival of infected cells. Our data suggest that with the failure of adequate scavenger mechanisms, oxidant radicals overproduced in response to bacterial infection were highly toxic to host cells. Therefore, instead of contributing to defence against infectious agents, the upregulation of free radicals production by endothelial cells in response to cytokine activation would be detrimental to the host.     

Effects of temperature on maximum clinging ability in a diurnal gecko: evidence for a passive clinging mechanism?

The thermal dependence of performance of ectotherms, and particularly locomotor performance in lizards, has received much attention. However, only a single study has examined the effects of temperature on adhesive clinging ability in geckos, despite the importance of adhesion for many pad-bearing lizards and invertebrates. We set out to characterize the thermal response of clinging ability in the diurnal gecko, Phelsuma dubia in the temperature range of 15-35 degrees C. Our findings indicate that there is no significant trend in clinging ability for P. dubia with temperature and that there is high variation about the mean at all temperatures. These findings differ from other whole-organism studies of clinging performance and are suggestive of a passive clinging mechanism that is dominated by intermolecular van der Waals forces. These findings also suggest that clinging ability in this species is not under selective pressures resulting from thermal variation, and that P. dubia does not need to regulate body temperature closely to maximize clinging ability.     

Regulation of the cytotoxic effect of human ‘normal killer cells’ on tumor cell lines by neuraminidase-treated T-lymphocytes

Summary Subpopulations of peripheral blood lymhocytes (PBL) from healthy individuals were separated according to their capacity to form various rosettes and tested for their cytotoxic activity on cell lines of urinary bladder and breast carcinomas. The subpopulation exerting the highest natural cytotoxic activity was characterized by the presence of cell surface Fc-receptors and by the lack of receptors for sheep red blood cells and for C'3 on their surface. Treatment with vibrio cholera neuraminidase (VCN) increased the cytotoxicity of unseparated PBL to a level twice as high as that of untreated PBL. The attachment of T-lymphocytes to tumor monolayers was increased several fold after VCN-treatment, while the attachment of other lymphocyte subpopulations was not. Evidence is presented that the augmentation of the cytotoxicity of PBL following VCN-treatment results from the interaction of VCN-treated T-lymphocytes, attached to target cells, with normal killer cells. It is suggested that augmentation of the activity of killer cells by T-lymphocytes may play a role in antitumor defense mechanisms.Abbreviations CMC Cell-mediated cytolysis - E-rosettes Rosettes formed with sheep red blood cells - EA-rosettes Rosettes formed with red blood cells coated with antibody - EAC'-rosettes Rosettes formed with red blood cells coated with antibody and complement - FCS Heat inactivated fetal calf serum - PBL Peripheral blood lymphocytes - RBC Red blood cells - RF-TAL E-rosette forming, target-attached lymphocytes - SRBC Sheep red blood cells - VCN Vibrio cholera neuraminidase     

Wilms' tumor 1 (WT1) gene in hematopoiesis: a surrogate marker of cell proliferation as a possible mechanism of action?

BACKGROUND: Wilms' tumor 1 (WT1) gene expression is seen in a significant number of cases of human neoplasia; however, the mechanism of action remains to be clarified. We hypothesized that WT1 gene is a surrogate marker of proliferation in normal hematopoietic cells and leukemias. While we and others have recognized its value as a tool for the detection of minimal residual disease (MRD), the objective of this study was to confirm our hypothesis regarding normal. METHODS: Samples from healthy donors (n=16) and UC blood (n=9) were cultured in Methocult for 21 days. Colonies were analyzed on days 7, 14 and 21 by RT-PCR for WT1 gene expression. Our positive controls were samples from patients with leukemia (n=91). Negative controls were from normal volunteers without stimulation (n=26). RESULTS: Results showed a statistically significant difference (P<0.0001) between cultured groups, with the highest level of WT1 gene expression in the positive controls and on day 14, when cells are at their maximal proliferation. DISCUSSION: In conclusion, WT1 gene expression in the proliferating colonies was highest on day 14, although less than in leukemia samples, confirming our hypothesis that WT1 gene is a surrogate marker of proliferation, not only in leukemogenesis but also, to a lesser degree, in normal cell proliferation.     

The histopathology of a human mesenchymal stem cell experimental tumor model: support for an hMSC origin for Ewing's sarcoma?

Sarcomas display varied degrees of karyotypic abnormality, vascularity and mesenchymal differentiation. We have reported that a strain of telomerized adult human bone marrow mesenchymal stem cells (hMSC-TERT20) spontaneously evolved a tumorigenic phenotype after long-term continuous culture. We asked to what extent our hMSC-TERT20 derived tumors reflected events found in human sarcomas using routine histopathological procedures. Early versus late passage hMSC-TERT20 cultures persistently expressed mesenchymal lineage proteins e.g. CD105, CD44, CD99 and vimentin. However, late passage cultures, showed increased immunohistochemical staining for CyclinD1 and p21WAF1/Cip1, whereas p27Kip1 staining was reduced. Notably, spectral karyotyping showed that tumorigenic hMSC-TERT20 cells retained a normal diploid karyotype, with no detectable chromosome abnormalities. Consistent with the bone-forming potential of early passage hMSC-TERT20 cells, tumors derived from late passage cells expressed early biomarkers of osteogenesis. However, hMSC-TERT20 cells were heterogeneous for alpha smooth muscle actin (ASMA) expression and one out of six hMSC-TERT20 derived single cell clones was strongly ASMA positive. Tumors from this ASMA+ clone had distinctive vascular qualities with hot spots of high CD34+ murine endothelial cell density, together with CD34- regions with a branching periodic acid Schiff reaction pattern. Such clone-specific differences in host vascular response provide novel models to explore interactions between mesenchymal stem and endothelial cells. Despite the lack of a characteristic chromosomal translocation, the histomorphology, biomarkers and oncogenic changes were similar to those prevalent for Ewing's sarcomas. The phenotype and ontogenesis of hMSC-TERT20 tumors was consistent with the hypothesis that sarcomas may arise from hMSC, providing a unique diploid model for exploring human sarcoma biology.     

Host queen killing by a slave-maker ant queen: when is a host queen worth attacking?

A new colony of the slave-making ant Polyergus breviceps is initiated when a newly mated gyne invades a host nest and kills the resident queen. This process seems to result in chemical camouflage of the invading gyne and allows her to usurp the position of colony reproductive. Young, recently mated Formica gynes, however, are not attacked. To determine whether worker and/or immature presence is the basis for aggression, we placed eggs, larvae, pupae and workers from mature F. gnava queens with newly mated F. gnava queens and observed the responses of introducedP. breviceps queens. Because no newly mated gyne was attacked, we tested newly mated F. gnava queens (1) once they had produced eggs, (2) when the offspring reached the larval, pupal and callow stages of development, and (3) every 2 weeks until aggression ensued. Eventually all F. gnava queens were attacked but only 29 weeks after having mated. Thus, although offspring are the ultimate benefit from attacking an established F. gnava queen,P. breviceps queens detect mature queens using another time-dependent feature that is reliably indicative of reproductive status. The similarity of host queen hydrocarbon profiles, often correlated with reproductive status in other ant species, suggests that other compounds reflect queen fecundity and produce a kairomonal effect, or that another cue signals host queen and colony suitability. Our findings indicate P. breviceps gynes have evolved to respond aggressively to a host gyne cue that appears long after mating, preventing attacks on gynes without the workers necessary for colony founding.Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Effects of the virus satellite gene βC1 on host plant defense signaling and volatile emission

Tomato Yellow Leaf Curl China virus spreads together with its invasive vector, the silverleaf whitefly B biotype, which exhibits higher growth rates on infected plants. Previous studies indicate that the virus satellite gene βC1 accounts for the visible symptoms of infection and inhibits the constitutive expression of jasmonic acid (JA)—a phytohormone involved in plant defense against whiteflies—and of some JA-regulated genes. Here we present new details of the effects of on plant signaling and defense, obtained with (non-host) transgenic Arabidopsis thaliana and Nicotiana benthamiana plants. We found that JA induction in response to wounding was reduced in plants expressing βC1. This result implies that βC1 acts on conserved plant regulation mechanisms and might impair the entire JA defense pathway. Furthermore, transformed N. benthamiana plants exhibited elevated emissions of the volatile compound linalool, suggesting that βC1 also influences plant-derived olfactory cues available to vector and non-vector insects.     

Effect of S-nitrosoglutathione on renal mitochondrial function: a new mechanism for reversible regulation of manganese superoxide dismutase activity?

Mitochondria are at the heart of all cellular processes as they provide the majority of the energy needed for various metabolic processes. Nitric oxide has been shown to have numerous roles in the regulation of mitochondrial function. Mitochondria have enormous pools of glutathione (GSH≈5–10 mM). Nitric oxide can react with glutathione to generate a physiological molecule, S-nitrosoglutathione (GSNO). The impact GSNO has on mitochondrial function has been intensively studied in recent years, and several mitochondrial electron transport chain complex proteins have been shown to be targeted by GSNO. In this study we investigated the effect of GSNO on mitochondrial function using normal rat proximal tubular kidney cells (NRK cells). GSNO treatment of NRK cells led to mitochondrial membrane depolarization and significant reduction in activities of mitochondrial complex IV and manganese superoxide dismutase enzyme (MnSOD). MnSOD is a critical endogenous antioxidant enzyme that scavenges excess superoxide radicals in the mitochondria. The decrease in MnSOD activity was not associated with a reduction in its protein levels and treatment of NRK cell lysate with dithiothreitol (a strong sulfhydryl-group-reducing agent) restored MnSOD activity to control values. GSNO is known to cause both S-nitrosylation and S-glutathionylation, which involve the addition of NO and GS groups, respectively, to protein sulfhydryl (SH) groups of cysteine residues. Endogenous GSH is an essential mediator in S-glutathionylation of cellular proteins, and the current studies revealed that GSH is required for MnSOD inactivation after GSNO or diamide treatment in rat kidney cells as well as in isolated kidneys. Further studies showed that GSNO led to glutathionylation of MnSOD; however, glutathionylated recombinant MnSOD was not inactivated. This suggests that a more complex pathway, possibly involving the participation of multiple proteins, leads to MnSOD inactivation after GSNO treatment. The major highlight of these studies is the fact that dithiothreitol can restore MnSOD activity after GSNO treatment. To our knowledge, this is the first study showing that MnSOD activity can be reversibly regulated in vivo, through a mechanism involving thiol residues.