Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro

Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guérin (BCG) or sonicated BCG (s-BCG). We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation ofl[³H]methionine. The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon (IFN). BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells. We termed these cytotoxic effectors BCG-activated killer (BAK) cells. In contrast to their cytotoxicity against bladder tumour cells. BAK cells did not differ from unstimulated PBMC in the killing of K562 cells. Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B. We could demonstrate the presence of the cytokines IFN, IL-2, tumour necrosis factor (TNF) and TNFß in the supernatants harvested during the generation of BAK cells. Monoclonal antibodies neutralizing IFN were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN in the induction of BAK cells. Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations. The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset. CD4+ cells and macrophages did not exhibit cytolytic activity. Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.     

Suppressing IL-32 in monocytes impairs the induction of the proinflammatory cytokines TNFα and IL-1β

Targeting major proinflammatory cytokines such as IL-1β and TNFα is of great interest in patients with chronic inflammatory diseases, including rheumatoid arthritis, colitis, and psoriasis. The cytokine Interleukin (IL)-32 induces proinflammatory cytokines such as TNFα, IL-1β, IL-6, and chemokines. We previously used an IL-32 ligand-affinity column to purify proteinase 3, which is abundantly expressed in neutrophil and monocytic leukocytes but not in other cell types, and found that IL-32 is mainly produced by monocytic leukocytes. This evidence suggested that silencing endogenous IL-32 by short hairpin RNA (shRNA) in monocytic cells might reveal the precise function of endogenous IL-32. Indeed, lipopolysaccharide (LPS)- or phorbol myristate acetate (PMA)-induced proinflammatory cytokine production was significantly inhibited in shRNA/IL-32 stable clones as compared to control clones. Furthermore, macrophages in PMA-differentiated shRNA/IL-32 stable clones displayed remarkably impaired LPS- and IL-1β-induced proinflammatory cytokine production. These data suggest that IL-32 is not only involved in host defense against pathogens, but also might play a role in chronic inflammatory diseases. IL-32 production leads to major proinflammatory cytokine production during the initial immune response.     

Induction of cardiac angiogenesis requires killer cell lectin-like receptor 1 and α4β7 integrin expression by NK cells

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice, myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6, accompanied by substantial expression of IL-2, TNF-α, and CCL2. In contrast, NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo, IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation, enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study, we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases.     

Natural killer cells control a T-helper 1 response in patients with Behçet's disease

Introduction

Behçet's disease (BD) is a multisystem inflammatory disorder, in which a T-helper 1 (Th1)-polarized immune response plays a major role in the pathogenic process. We evaluated the regulatory role of natural killer (NK) cells in Th1-biased immune responses in patients with BD.

Methods

We studied 47 patients with BD, including 10 with active disease (aBD) and 37 with inactive disease (iBD), and 29 healthy controls. The activation status and cytotoxic activity of NK cells were examined by flow cytometry. The levels of mRNAs for immune modulatory and cytotoxic molecules in NK cells were determined by quantitative PCR. The IL-12 signal strength in NK cells was determined by assessing the phosphorylation state of its downstream component, signal transducer and activator of transduction 4, by immunoblotting. Finally, NK cells' ability to modulate the Th1 response was evaluated by co-culturing NK cells and T cells without cell contact.

Results

CD69⁺-activated NK cells were significantly increased in aBD compared with iBD or control samples, although their cytotoxic activities were similar. The iBD NK cells showed downregulated IL-12 receptor β₂ mRNA levels compared with aBD or control NK cells. The increased IL-13 expression was detected in a subset of BD patients: most of them had iBD. The IL-13 expression level in iBD patients was significantly higher than the level in controls, but was not statistically different compared with the level in aBD patients. The gene expression profile in iBD patients was consistent with the NK type 2 phenotype, and the shift to NK type 2 was associated with disease remission. NK cells from iBD patients showed impaired IL-12-induced signal transducer and activator of transduction 4 phosphorylation. Finally, iBD, but not control, NK cells suppressed IFNγ expression by aBD-derived CD4⁺ T cells in vitro.

Conclusions

NK cells may control disease flare/remission in BD patients via NK type 2-mediated modulation of the Th1 response.

     

Neurite outgrowth of mature retinal ganglion cells and PC12 cells requires activity of CK1δ and CK1ε

Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI) enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth.     

p53-mediated induction of Noxa and p53AIP1 requires NFκB

The intricate regulation of cell survival and cell death is critical for the existence of both normal and transformed cells. Two factors central to these processes are p53 and NFκB, with both factors having ascribed roles in both promoting and repressing cell death. Not surprisingly, a number of studies have previously reported interplay between p53 and NFκB. The mechanistic basis behind these observations, however, is currently incomplete. We report here further insights into this interplay using a system where blockade of NFκB inhibits cell death from p53, but at the same time sensitizes cells to death by TNFα. We found in agreement with a recent report showing that NFκB is required for the efficient activation of the BH3-only protein Noxa by the p53 family member p73, that p53’s ability to induce Noxa is also impeded by inhibition of NFκB. In contrast to the regulation by p73, however, blockade of NFκB downstream of p53 decreases Noxa protein levels without effects on Noxa mRNA. Our further analysis of the effects of NFκB inhibition on p53 target gene expression revealed that while most target genes analysed where unaffected by blockade of NFκB, the p53-mediated induction of the pro-apoptotic gene p53AIP1 was significantly dependent on NFκB. These studies therefore add further insight into the complex relationship of p53 and NFκB and since both Noxa and p53AIP1 have been shown to be important components of p53-mediated cell death responses, these findings may also indicate critical points where NFκB plays a pro-apoptotic role downstream of p53.     

Induction of apoptosis in bacillus Calmette-Guérin-activated T cells by transforming growth factor-beta

In view of the critical role played by bacillus Calmette-Guérin (BCG) in the development and functional activation of protective T cells against tuberculosis, it has become important to understand the mechanisms by which cytokines regulate BCG-mediated immune responses. There is evidence that cytokine-mediated suppression of T cell function by mechanisms, including apoptosis, may reduce host resistance in tuberculosis. However, it is unclear whether cytokine-mediated suppression of antigen-responsive T cells through apoptotic mechanisms may be operating during human cellular activation induced by BCG. Here we present evidence, for the first time, that treatment of BCG-activated T cells with transforming growth factor-beta (TGF-beta) induces cellular apoptosis. These results were further supported by the fact that treatment of cells with a blocking mAb directed to TGF-beta significantly inhibited the percentage of apoptosis induced by TGF-beta. Interestingly, TGF-beta-mediated death of BCG-activated T cells in cultures containing interleukin (IL)-12 was observed. Moreover, our results demonstrated the induction of apoptosis by TGF-beta in BCG-activated T cells cultured in the presence of exogenous IL-12. In addition, our data indicated that TGF-beta significantly inhibited both BCG-induced cell growth determined by thymidine uptake and BCG-induced IFN-gamma secretion. Finally, TGF-beta-induced apoptosis in BCG-activated T cells correlated inversely with BCG-induced IFN-gamma secretion. Taken together, these findings indicate that TGF-beta induces apoptosis in human T cells activated with BCG and at the same time suggest that loss of BCG-reactive T cells through apoptotic mechanisms could contribute to an increased susceptibility to Mycobacterium tuberculosis infection.     

Opposite effects of serotonin and interferon-α on the membrane potential and function of human natural killer cells

Summary In an earlier article, we reported that serotonin (5-hydroxytryptamine, 5-HT) inhibits the natural killer cell (NK) cytotoxicity of human whole blood in a dose-dependent manner and that natural human interferon-α (IFN-α) partially eliminates this effect. Because natural IFN-α might contain factors other than IFN, we repeated these experiments with recombinant human interferon-α (rhIFN-α) and separated blood lymphocytes enriched with NK cells and then demonstrated that IFN really is responsible for this effect. Furthermore, this investigation was carried out to clarify the mechanisms of the action of 5-HT and of rhIFN-α on NK cells. The inhibition of the cytotoxicity was pronounced when 5-HT was added at the onset of the cytotoxic assay, whereas the pretreatment of lymphocytes for 18 h only led to a slight inhibition. Moreover, rhIFN-α applied 1 h before or 1 h after the addition of 5-HT decreased the inhibitory effect of 5-HT. Flow cytometric analysis involving the use of a voltage-sensitive dye, oxonol, revealed that 5-HT depolarized, whereas rhIFN-α hyperpolarized the plasma membrane of the lymphocytes. Thus, it seems likely that the inhibitory effect of 5-HT on the cytotoxicity of peripheral human lymphocytes is due to the depolarization on the plasma membrane of the effector cells and that rhIFN-α antagonizes this ability via its hyperpolarizing activity.     

The presence of Estrogen Receptor β modulates the response of breast cancer cells to therapeutic agents

Breast cancer is a leading cause of death for women. The estrogen receptors (ERs) ratio is important in the maintenance of mitochondrial redox status, and higher levels of ERβ increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERβ ratio and the response to cytotoxic treatments such as cisplatin (CDDP), paclitaxel (PTX) and tamoxifen (TAM). Cell viability, apoptosis, autophagy, ROS production, mitochondrial membrane potential, mitochondrial mass and mitochondrial functionality were analyzed in MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) breast cancer cell lines. Cell viability decreased more in MCF-7 when treated with CDDP and PTX. Apoptosis was less activated after cytotoxic treatments in T47D than in MCF-7 cells. Nevertheless, autophagy was increased more in CDDP-treated MCF-7, but less in TAM-treated cells than in T47D. CDDP treatment produced a raise in mitochondrial mass in MCF-7, as well as the citochrome c oxidase (COX) and ATP synthase protein levels, however significantly reduced COX activity. In CDDP-treated cells, the overexpression of ERβ in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERβ in T47D cells promoted the opposite effects. In TAM-treated cells, ERβ-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERβ in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERβ expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.     

Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-β(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.     

Implications of reiterative DNA—Metal ion complexes in the induction and development of neoplastic cells

Experimental data on the content in metal ions of DNA preparations from various neoplastic and healthy tissues are summarized: metal ions are preferentially bound to reiterative DNA sequences, where they may induce conformational variations and thus modify the binding of effector molecules such as repressors and polymerases. A model is described where essential and toxic metals are successively loaded on ligand acceptor groups of increasing affinity and thus may reach the final active sites: enzymes and reiterative DNA sequences (equated at least partially to regulative DNA sequences). The effects of some molecules, including peptides, antibiotics, growth factors, hormones, and antineoplastic substances, on DNA conformation could be explained in part by their chelating ability. The neoplastic state may be induced by a modification of metal ion transfer chains: quantitatively by a continuous derepression of genes coding for metal ligands, genes that are only temporarily derepressed during development in normal cells, and qualitatively by modifications of the nucleotidic sequence of structural genes leading to an increase of the chelating ability of the coded metal ligand.     

Steroid hydroxylation with free and immobilized cells of Penicillium raistrickii in the presence of β-cyclodextrin

Free and Ca-alginate-immobilized cells of Penicillium raistrickii i 477 were used for 15-hydroxylation of 13-ethyl-gon-4-en-3,17-dione. The product formation in the presence of -cyclodetrin, in comparison with reactions carried out in the presence of methanol. Application of -cyclodextrin led to increasing solubility of the steroid substrate. The fungus was able to utilize -cyclodextrin as a carbon source. Correspondence to: H.-P. Schmauder     

Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ

Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant Vα14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NKT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGFβ. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from Vα14 TCR Tg mice to naïve CD4⁺ T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFNγ neutralization, we showed that this reduction was mediated by IFNγ. Furthermore, the main source of IFNγ during iTreg differentiation was NK1.1⁻CD4⁺Foxp3⁻ T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFNγ.     

Adsorption of phage λ to Salmonella typhimurium lamB + requires the presence of lipopolysaccharide in the outer membrane

Summary Two S. typhimurium strains TA1534 (rfa ⁺) and TA1538 (rfaE) were transformed with the lamB expression plasmid pAMH70. Transposition events with placMu55 hybrid phage were successful only with TA1534/pAMH70 strain. Using SDS-PAGE, the LamB protein was present in the total cell proteins but not in the outer membrane proteins of the TA1538/pAMH70 strain. The LamB protein must linked to the LPS of the outer membrane to allow adsorption of phage in S. typhimurium.     

Curcumin ameliorates TNF-α-induced ICAM-1 expression and subsequent THP-1 adhesiveness via the induction of heme oxygenase-1 in the HaCaT cells

Adhesion molecules such as ICAM-1 are important in the infiltration of leukocytes into the site of inflammation. In this study, we investigated the inhibitory effects of curcumin on ICAM-1 expression and monocyte adhesiveness as well as its underlying action mechanism in the TNF-α-stimulated keratinocytes. Curcumin induced expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. In addition, curcumin induced Nrf2 activation in dose- and time-dependent manners in the HaCaT cells. Curcumin suppressed TNF-α- induced ICAM-1 expression and subsequent monocyte adhesion, which were reversed by the addition of tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, or HO-1 knockdown using siRNA. Furthermore, Nrf2 knockdown using siRNA reversed the inhibitory effect of curcumin on the TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that curcumin may exert its anti-inflammatory activity by suppressing the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes. [BMB Reports 2013;46(8): 410-415]     

Role of interleukin-2 and interferon-γ in induction of activated natural killer cells from mice primed in vivo and subsequently challenged in vitro with the streptococcal preparation OK432

Summary The natural-killer(NK)-cell-mediated cytotoxicity to syngeneic tumor cells can be augmented by in vivo priming and subsequent in vitro challenge with the streptococcal preparation OK432. Supernatants of cocultures of spleen cells with OK432 contained interleukin-2 (IL-2) and interferon (IFN), mainly IFN-. As the anti-(mouse IFN-) monoclonal antibody but not anti-(mouse IFN-) antibody inhibited the induction of activated NK cells with OK432, the IFN- participated in this response. The enhancement of NK cell activity and production of IL-2 were partially inhibited by the pretreatment of spleen cells with mitomycin C or irradiation, and were completely abolished by pretreatment with actinomycin D. The IL-2 activity after treatment with various metabolic inhibitors ran parallel to the NK activity in a system augmented with OK432. The activity of incubated spleen cells with IL-2 receptors was increased by OK432 treatment, and the NK cell and IFN activities of supernatants were also abrogated by the treatment with anti-(mouse IL-2 receptor) monoclonal antibody, to block the interaction between IL-2 and these receptors of effector cells. The panning method clarified that the incubated spleen cells with IL-2 receptors are responsible for the production of IFN-. These results suggest that IL-2 plays a major role in inducing the activated NK cells from murine spleen cells primed in vivo and subsequently challenged in vitro with OK432, by the production of IFN-.