Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell–based cancer vaccination

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated apoptotic tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I– and class II–restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-, and TNF- by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 K^b /SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN- and TNF- production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.Abbreviations DC Dendritic cell - FSC Forward scatter - SSC Side scatter - TC Tumor cells This work was supported by Grant 9853 from the Leukaemia Research Fund, UK; a JRC studentship from GKT; and the Lewis Family Research Trust     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

Chloroquine induces activation of nuclear factor-kappaB and subsequent expression of pro-inflammatory cytokines by human astroglial cells

Chloroquine, an antimalarial lysosomotropic base, is known for its anti-inflammatory effects and therefore used for treatment of autoimmune diseases. Given its anti-inflammatory effects, it has been under clinical trials to modify neurodegenerative processes. In this study, we examined whether chloroquine has an anti-inflammatory effect in the CNS by determining the in vitro effects of chloroquine on LPS-induced expression of cytokines by glial cells. We observed that (i) chloroquine augmented LPS-induced expression of pro-inflammatory cytokines such as lymphotoxin (LT)-beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta and IL-6 in human astroglial cells, while the same treatment suppressed LPS-induced expression of cytokines in monocytic and microglial cells; (ii) chloroquine alone induced expression of pro-inflammatory cytokines in a dose- and time-dependent manner in astroglial cells; (iii) other lysosomotropic agents such as ammonium chloride and bafilomycin A1 had minimal effects on cytokine expression; and (iv) chloroquine induced the activation of nuclear factor-kappa B in astroglial cells, which is a required component of chloroquine induction of cytokines. These results suggest that chloroquine may evoke either anti- or pro-inflammatory responses in the CNS depending on the cellular context.     

Noradrenergic depletion increases inflammatory responses in brain: effects on IkappaB and HSP70 expression

The inflammatory responses in many cell types are reduced by noradrenaline (NA) binding to beta-adrenergic receptors. We previously demonstrated that cortical inflammatory responses to aggregated amyloid beta (Abeta) are increased if NA levels were first depleted by lesioning locus ceruleus (LC) noradrenergic neurons, which replicates the loss of LC occurring in Alzheimer's disease. To examine the molecular basis for increased responses, we used the selective neurotoxin DSP4 to lesion the LC, and then examined levels of putative anti-inflammatory molecules. Inflammatory responses were achieved by injection of aggregated Abeta1-42 peptide and IL-1beta into frontal cortex, which induced neuronal inducible nitric oxide synthase (iNOS) and microglial IL-1beta expression. DSP4-treatment reduced basal levels of nuclear factor kappa B (NF-kappaB) inhibitory IkappaB proteins, and of heat shock protein (HSP)70. Inflammatory responses were prevented by co-injection (ibuprofen or ciglitzaone) or oral administration (pioglitazone) of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Treatment with PPARgamma agonists restored IkappaBalpha, IkappaBbeta, and HSP70 levels to values equal or above those observed in control animals, and reduced activation of cortical NF-kappaB. These results suggest that noradrenergic depletion reduces levels of anti-inflammatory molecules which normally limit cortical responses to Abeta, and that PPARgamma agonists can reverse that effect. These findings suggest one mechanism by which PPARgamma agonists could provide benefit in neurological diseases having an inflammatory component.     

Salvia miltiorrhiza inhibits intimal hyperplasia and monocyte chemotactic protein-1 expression after balloon injury in cholesterol-fed rabbits

Antioxidants that prevent low density lipoproteins (LDL) from oxidation may inhibit atherosclerosis and post-angioplasty restenosis. Salvia miltiorrhiza (SM) has been shown to inhibit LDL oxidation and reduce atherosclerosis in cholesterol-fed rabbits. The effects of SM on neointimal hyperplasia and monocyte chemotactic protein-1 (MCP-1) expression after balloon injury were studied. Male New Zealand white rabbits were fed a 2% cholesterol diet together with daily SM (4.8 gm/kg body wt.) treatment (SM; n=10) or without SM as a control (C; n=9) for 6 weeks. Probucol-treated (0.6 gm/kg body wt.) rabbits (P; n=9) were used as a positive control group. A balloon injury of the abdominal aorta was performed at the end of the third week. Aortas were harvested at the end of 6 weeks. The plasma cholesterol levels were lowered in SM group. The neointimal hyperplasia in abdominal aortas was significantly inhibited in SM group [neointima/media area ratio: 0.63+/-0.05 (SM) versus 0.78+/-0.05 (C); P < 0.05] and in P group [0.45+/-0.02 (P) versus 0.78+/-0.05 (C); P < 0.05] when compared with C group. SM treatment significantly reduced MCP-1 mRNA and protein expression in balloon-injured abdominal aorta. These inhibitory effects on intimal response after balloon injury might be attributed to antioxidant capacity and cholesterol lowering effect of SM. SM treatment may offer some protection against post-angioplasty restenosis.     

Human airway epithelial cells in culture for studying the molecular mechanisms of the inflammatory response triggered by diesel exhaust particles

Epidemiological studies have shown that particulate air pollution is linked to the increase of morbidity and mortality due to respiratory diseases. Diesel exhaust particles (DEPs), which are the most important part of PM2.5 in Western European and Japanese urban areas, have been suspected. The mechanisms of proinflammatory response induced by DEPS were elucidated using a human epithelial cell line (16-HBE). It has been shown that DEPs can be phagocytosed by HBE cells, inducing the release of cytokines. MAP kinase pathways (i.e., ERK1/2 and P38) were triggered as well as the activation of the nuclear factor NF-κB. Reactive oxygen species (ROS) were strongly incriminated in this response because DEPs induce the increase of intracellular hydroperoxides and antioxidants inhibit the release of DEP-induced cytokines, the activation of MAP kinases and NF-κB. Organic compounds adsorbed on DEPs seemed to be involved in the response and the production of ROS. Moreover, we have demonstrated that DEPs can activate CYP1A1 in HBE cells. These experimental results give biological plausibility to the epidemiological findings. This revised version was published online in August 2006 with corrections to the Cover Date.     

The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone,and not mimicked by rh-TNF-alpha,IL-1beta and IFN-gamma

It is becoming widely accepted that the inflammatory response is involved in neurodegenerative disease. In this context, we have developed an animal model of dopaminergic system degeneration by the intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation. To address the importance of the inflammatory response in the LPS-induced degeneration of nigral dopaminergic neurones, we carried out two different kinds of studies: (i) the possible protective effect of an anti-inflammatory compound, and (ii) the effect of the intranigral injection of inflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) on dopaminergic neurones viability. Present results show that dexamethasone, a potent anti-inflammatory drug that interferes with many of the features characterizing pro-inflammatory glial activation, prevented the loss of catecholamine content, Tyrosine hydroxylase (TH) activity and TH immunostaining induced by LPS-injection and also the bulk activation of microglia/macrophages. Surprisingly, injection of the pro-inflammatory cytokines failed to reproduce the LPS effect. Taken together, our results suggest that inflammatory response is implicated in LPS-induced neurodegeneration. This damage may be due, at least in part, to a cascade of events independent of that described for TNF-alpha/IL-1 beta/IFN-gamma.     

Potentiation of Fas- and TRAIL-mediated apoptosis by IFN-gamma in A549 lung epithelial cells: enhancement of caspase-8 expression through IFN-response element

Epithelial cell apoptosis triggered cooperatively by multiple cytokines contributes to the injury induced by inflammatory responses in the lung and elsewhere. Here we show that interferon-gamma (IFN-gamma) sensitizes A549 cells, human lung epithelial cells, to cytokine-mediated apoptosis by upregulating caspase-8 expression. Pretreating the cells with IFN-gamma potentiated Fas- and TNF-related apoptosis inducing ligand (TRAIL)-induced cell death, but other forms of apoptosis, not mediated via receptors, were unaffected. Western blotting and inhibitor assays showed that IFN-gamma selectively increased expression of caspases-7 and -8, but not caspases-2, -3, -9, or -10, as a necessary step leading to apoptosis. Assaying promoter activity using a luciferase reporter gene indicated that an IFN-gamma response element was located in the 5'-flanking region of the caspase-8 gene, spanning positions -227 to -219. Taken together, these findings suggest that IFN-gamma potentiates Fas- and TRAIL-mediated apoptosis by increasing caspase-8 expression via an IFN-gamma response element in A549 cells.