bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Regulation of Vitamin D Receptor and Its Ligand in Human Monocyte-Derived Dendritic Cells
Authors: Hewison Martin , Freeman Lisa , Hughes Susan V. , Evans Katie N. , Bland Rosemary , Eliopoulos Aristides G. , Kilby Mark D. , Moss Paul A. H. , Chakraverty Ronjon
Published: April 2014

The functions of dendritic cells (DCs) are tightly regulated such that protective immune responses are elicited and unwanted immune responses are prevented. 1α25-dihydroxyvitamin D3 (1α25(OH)2D3) has been identified as a major factor that inhibits the differentiation and maturation of DCs, an effect dependent upon its binding to the nuclear vitamin D receptor (VDR). Physiological control of 1α25(OH)2D3 levels is critically dependent upon 25-hydroxyvitamin D3-1α-hydroxylase (1αOHase), a mitochondrial cytochrome P450 enzyme that catalyzes the conversion of inactive precursor 25-hydroxyvitamin D3 (25(OH)D3) to the active metabolite 1α25(OH)2D3. Using a human monocyte-derived DC (moDC) model, we have examined the relationship between DC VDR expression and the impact of exposure to its ligand, 1α25(OH)2D3. We show for the first time that moDCs are able to synthesize 1α25(OH)2D3 in vitro as a consequence of increased 1αOHase expression. Following terminal differentiation induced by a diverse set of maturation stimuli, there is marked transcriptional up-regulation of 1αOHase leading to increased 1αOHase enzyme activity. Consistent with this finding is the observation that the development and function of moDCs is inhibited at physiological concentrations of the inactive metabolite 25(OH)D3. In contrast to 1αOHase, VDR expression is down-regulated as monocytes differentiate into immature DCs. Addition of 1α25(OH)2D3 to moDC cultures at different time points indicates that its inhibitory effects are greater in monocyte precursors than in immature DCs. In conclusion, differential regulation of endogenous 1α25(OH)2D3 ligand and its nuclear receptor appear to be important regulators of DC biology and represent potential targets for the manipulation of DC function.
Memory Phenotype of CD8+ T Cells in MHC Class Ia-Deficient Mice
Authors: Kurepa Zoran , Su Jie , Forman James
Published: April 2014

B6.Kb−Db− mice are devoid of class Ia but express normal levels of class Ib molecules. They have low levels of CD8 T cells in both the thymus as well as peripheral T cell compartments. Although the percentage of splenic CD8αα T cells is increased in these animals, ∼90% of CD8 T cells are CD8αβ. In contrast to B6 animals, most of the CD8 T cells from these mice have a memory phenotype (CD44highCD122high CD62Llow) including both CD8αβ and CD8αα subsets. In the thymus of B6.Kb−Db− animals, there is a decrease in the percentage of SP CD8 T cells, although most are CD44low, similar to that seen in B6 mice. The spleens from day 1-old B6 and B6.Kb−Db− mice have a relatively high proportion of CD44highCD62Llow CD8 T cells. However, by day 28 most CD8 T cells in B6 mice have a naive phenotype while in B6.Kb−Db− mice the memory phenotype remains. Unlike CD44high cells that are found in B6 animals, most CD44high cells from B6.Kb−Db− mice do not secrete IFN-γ rapidly upon activation. The paucity of CD8 T cells in B6.Kb−Db− mice might be due in part to their inability to undergo homeostatic expansion. Consistent with this, we found that CD8 T cells from these animals expand poorly in X-irradiated syngeneic hosts compared with B6 CD8 T cells that respond to class Ia Ags. We examined homeostatic expansion of B6 CD8 T cells in single as well as double class Ia knockout mice and were able to estimate the fraction of cells reactive against class Ia vs class Ib molecules.
Genetic Dissection of Vα14Jα18 Natural T Cell Number and Function in Autoimmune-Prone Mice
Authors: Matsuki Naoto , Stanic Aleksandar K. , Embers Monica E. , Van Kaer Luc , Morel Laurence , Joyce Sebastian
Published: April 2014

Nonobese diabetic (NOD) mice, a model for type I diabetes (TID), have reduced numbers of invariant Vα14Jα18 TCR α-chain-positive natural T (iNKT) cells that do not release IL-4 in response to in vivo activation through their Ag receptor. The deficit in iNKT cell number and function is implicated in immune dysregulation and the etiology of TID. Therefore, we reasoned that the genetic determinant(s) that controls iNKT cell number and function might lie within Idd (insulin-dependent diabetes susceptibility locus) regions, which are known to contain TID resistance or susceptibility genes. A systematic analysis of iNKT cell number and function in Idd congenic mice revealed that neither iNKT cell number nor their inability to rapidly secrete IL-4 in response to acute in vivo activation by Ag underlies the mechanism of protection from diabetes in Idd congenic mice. Moreover, the regulation of iNKT cell number and function appears to be under the control of several genes. The most notable of these map to the Idd4, Idd5, Idd9.1, and Idd13 regions of the mouse genome. Together these findings provide a clue to the genetic mechanism(s) underlying iNKT cell deficiency in NOD mice.
IL-23 Production by Cosecretion of Endogenous p19 and Transgenic p40 in Keratin 14/p40 Transgenic Mice: Evidence for Enhanced Cutaneous Immunity
Authors: Kopp Tamara , Lenz Petra , Bello-Fernandez Concha , Kastelein Robert A. , Kupper Thomas S. , Stingl Georg
Published: April 2014

p40, the common subunit of the proinflammatory cytokines IL-12 and IL-23, is produced by resident skin cells. Whereas the in vivo effects of IL-12 are well established, little is known about the role of IL-23 in cutaneous immune responses. In this study we show that p40 transgenic (TG) mice constitutively produce IL-23 (p19/p40), but not IL-12 (p35/p40), in basal keratinocytes by cosecretion of TG p40 with endogenous p19. Repeated injections of rIL-23 in littermate (LM) mice result in an inflammatory skin disease similar to that of p40 TG mice, confirming the proinflammatory activity of IL-23. Furthermore, IL-23 secretion by p40 TG keratinocytes induces elevated numbers of Langerhans cells (LC) with a marked up-regulation of costimulatory molecules, indicating advanced maturation of keratin 14 (K14)/p40 LC when compared with LM LC. At the functional level, freshly isolated K14/p40 LC greatly exceeded LC from LM animals in their capacity to stimulate allogeneic T cell proliferation. To assess whether IL-23 regulates cutaneous immune responses in vivo, we used an allogeneic skin transplantation model. Full thickness skin grafts from K14/p40 donors (H-2q) transplanted across a MHC class I and class II barrier onto BALB/c (H-2d) recipients were rejected in a significantly accelerated fashion (mean survival time: 8.8 days) when compared with skin grafts from non-TG LM (H-2q) (mean survival time: 10.7 days, p < 0.01). Based on these results we propose that IL-23-induced changes of LC may be an important mechanism in directing the outcome of cutaneous immune responses.
Propagation and Control of T Cell Responses by Heparan Sulfate-Bound IL-2
Authors: Wrenshall Lucile E. , Platt Jeffrey L. , Stevens Elliot T. , Wight Thomas N. , Miller John D.
Published: April 2014

IL-2, a cytokine produced by T cells, is a key regulator of immune responses and T cell homeostasis. Controlling the availability of IL-2 is consequently of significant import to the immune system. Like other cytokines, IL-2 is thought to function as a soluble agonist, transiently present when secreted in response to appropriate stimuli. In this study, we show that the most salient properties of IL-2, propagation and control of T cell responses, are mediated in vivo by bound and not free cytokine and specifically by heparan sulfate-bound IL-2. These findings necessitate a new look at how IL-2 regulates immune responses and support the notion that the microenvironment plays a determining role in modulating the character of immune responses.
Isolated Lymphoid Follicle Formation Is Inducible and Dependent Upon Lymphotoxin-Sufficient B Lymphocytes, Lymphotoxin β Receptor, and TNF Receptor I Function
Authors: Lorenz Robin G. , Chaplin David D. , McDonald Keely G. , McDonough Jacquelyn S. , Newberry Rodney D.
Published: April 2014

The gastrointestinal mucosa contains a complex network of lymphoid compartments that have evolved to efficiently protect the host from invading pathogens. Recently, an additional lymphoid structure resembling Peyer’s patches (PP) in composition and architecture has been identified in the murine small intestine, the isolated lymphoid follicle (ILF). In this study we examine the nature and factors required for ILF formation. We observed a spectrum of structures fitting the previous descriptions of ILFs, ranging from clusters of B220+ cells (which we have termed immature ILFs) to well-organized lymphoid nodules (which we have termed mature ILFs). Here we demonstrate that that similar to PP formation, ILF formation requires lymphotoxin (LT)- and LTβ receptor-dependent events. However unlike PP formation, the LT- and LTβ receptor-dependent events required for ILF formation can occur in adulthood and require LT-sufficient B lymphocytes. We demonstrate that mature ILF formation occurs in response to lumenal stimuli, including normal bacterial flora, and requires TNF receptor I function. These findings suggest that ILFs are organized intestinal lymphoid structures whose formation can be induced and whose mass can be expanded in response to mucosal challenges.
IL-12 Administration Accelerates Autoimmune Diabetes in Both Wild-Type and IFN-γ-Deficient Nonobese Diabetic Mice, Revealing Pathogenic and Protective Effects of IL-12-Induced IFN-γ
Authors: Trembleau Sylvie , Penna Giuseppe , Gregori Silvia , Giarratana Nadia , Adorini Luciano
Published: April 2014

IL-12 administration to nonobese diabetic (NOD) mice induces IFN-γ-secreting type 1 T cells and high circulating IFN-γ levels and accelerates insulin-dependent diabetes mellitus (IDDM). Here we show that IL-12-induced IFN-γ production is dispensable for diabetes acceleration, because exogenous IL-12 could enhance IDDM development in IFN-γ-deficient as well as in IFN-γ-sufficient NOD mice. Both in IFN-γ+/− and IFN-γ−/− NOD mice, IL-12 administration generates a massive and destructive insulitis characterized by T cells, macrophages, and CD11c+ dendritic cells, and increases the number of pancreatic CD4+ cells secreting IL-2 and TNF-α. Surprisingly, IL-12-induced IFN-γ hinders pancreatic B cell infiltration and inhibits the capacity of APCs to activate T cells. Although pancreatic CD4+ T cells from IL-12-treated IFN-γ−/− mice fail to up-regulate the P-selectin ligand, suggesting that their entry into the pancreas may be impaired, T cell expansion is favored in these mice compared with IL-12-treated IFN-γ+/− mice because IL-12 administration in the absence of IFN-γ leads to enhanced cell proliferation and reduced T cell apoptosis. NO, an effector molecule in β cell destruction, is produced ex vivo in high quantity by pancreas-infiltrating cells through a mechanism involving IL-12-induced IFN-γ. Conversely, in IL-12-treated IFN-γ-deficient mice, other pathways of β cell death appear to be increased, as indicated by the up-regulated expression of Fas ligand on Th1 cells in the absence of IFN-γ. These data demonstrate that IFN-γ has a dual role, pathogenic and protective, in IDDM development, and its deletion allows IL-12 to establish alternative pathways leading to diabetes acceleration.
Selective Induction of Dendritic Cells Using Granulocyte Macrophage-Colony Stimulating Factor, But Not fms-Like Tyrosine Kinase Receptor 3-Ligand, Activates Thyroglobulin-Specific CD4+/CD25+ T Cells and Suppresses Experimental Autoimmune Thyroiditis
Authors: Vasu Chenthamarakshan , Dogan Rukiye-Nazan E. , Holterman Mark J. , Prabhakar Bellur S.
Published: April 2014

fms-like tyrosine kinase receptor 3-ligand (Flt3-L) and GM-CSF cause expansion of different subsets of dendritic cells and skew the immune response toward predominantly Th1 and Th2 type, respectively. In the present study, we investigated their effects on experimental autoimmune thyroiditis in CBA/J mice. Relative to mouse thyroglobulin (mTg) immunized controls, mTg-immunized mice treated with Flt3-L showed more severe thyroiditis characterized by enhanced lymphocytic infiltration of the thyroid, and IFN-γ and IL-2 production. In contrast, mice treated with GM-CSF, either before or after immunization with mTg, showed suppressed T cell response to mTg and failed to develop thyroiditis. Lymphocytes from these mice, upon activation with mTg in vitro, produced higher levels of IL-4 and IL-10. Additionally, GM-CSF-treated mice showed an increase in the frequency of CD4+/CD25+ T cells, which suppressed the mTg-specific T cell response. Neutralization of IL-10, but not IL-4, or depletion of CD4+/CD25+ cells resulted in increased mTg-specific in vitro T cell proliferation suggesting that IL-10 produced by the Ag-specific CD4+/CD25+ regulatory T cells might be critical for disease suppression. These results indicate that skewing immune response toward Th2, through selective activation of dendritic cells using GM-CSF, may have therapeutic potential in Th1 dominant autoimmune diseases including Hashimoto’s thyroiditis.
Mast Cell Activation byMycobacterium tuberculosis: Mediator Release and Role of CD48
Authors: Muñoz Samira , Hernández-Pando Rogelio , Abraham Soman N. , Enciso Jose Antonio
Published: April 2014

Mast cells (MC) are abundant in the lung and other peripheral tissue, where they participate in inflammatory processes against bacterial infections. Like other effector cells of the innate immune system, MC interact directly with a wide variety of infectious agents. This interaction results in MC activation and inflammatory mediator release. We demonstrated that MC interact with Mycobacterium tuberculosis, triggering the release of several prestored reagents, such as histamine and β-hexosaminidase, and de novo synthesized cytokines, such as TNF-α and IL-6. A number of M. tuberculosis Ags, ESAT-6, MTSA-10, and MPT-63, have been implicated in MC activation and mediator release. A MC plasmalemmal protein, CD48, was implicated in interactions with mycobacteria because CD48 appeared to aggregate in the MC membrane at sites of bacterial binding and because Abs to CD48 inhibited the MC histamine response to mycobacteria. Cumulatively, these findings suggest that MC, even in the absence of opsonins, can directly recognize M. tuberculosis and its Ags and have the potential to play an active role in mediating the host’s innate response to M. tuberculosis infection.
Subversion and Utilization of the Host Cell Cyclic Adenosine 5′-Monophosphate/Protein Kinase A Pathway byBrucellaDuring Macrophage Infection
Authors: Gross Antoine , Bouaboula Monsif , Casellas Pierre , Liautard Jean-Pierre , Dornand Jacques
Published: April 2014

Brucella spp. are intramacrophage pathogens that induce chronic infections in a wide range of mammals, including domestic animals and humans. Therefore, the macrophage response to infection has important consequences for both the survival of phagocytosed bacteria and the further development of host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection and the virulence strategy that Brucella use to counteract these responses and secure their survival. In a previous study, we have shown that macrophages activated by SR141716A, a ligand of the cannabinoid receptor CB1, acquired the capacity to control Brucella and observed that the CB1 receptor-triggering engages the microbicidal activity of phagocytes. To analyze the perturbation of cell signaling pathway during macrophage infection by Brucella, we hypothesized that SR141716A provides cell signaling that interferes with the bacterial message leading to inhibition of macrophage functions. As CB1 receptor belongs to the family of G protein-linked receptors, we explored the cAMP signaling pathway. In this study, we show that the CB1 ligand inhibited the bacteria-induced cell signaling. Taking advantage of this result, we then demonstrated that Brucella infection elicited a rapid activation of the cAMP/protein kinase A pathway. This activation resulted in a prolonged phosphorylation of the transcription factor CREB. We finally demonstrate that the activation of the cAMP/protein kinase A pathway is crucial for the survival and establishment of Brucella within macrophages. For the first time in phagocytes, we thus characterized a primordial virulence strategy of Brucella involving the host signaling pathway, a novel point of immune intervention of this virulent pathogen.
Hepatitis C Virus Core and Nonstructural Protein 3 Proteins Induce Pro- and Anti-inflammatory Cytokines and Inhibit Dendritic Cell Differentiation
Authors: Dolganiuc Angela , Kodys Karen , Kopasz Andrea , Marshall Christopher , Do Twan , Romics Laszlo , Mandrekar Pranoti , Zapp Maria , Szabo Gyongyi
Published: April 2014

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-α production in monocytes. Degradation of I-κBα suggested involvement of NF-κB activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-α and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients’ DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.
Dendritic Cell-Based Immunotherapy Combined with Antimony-Based Chemotherapy Cures Established Murine Visceral Leishmaniasis
Authors: Ghosh Monidipa , Pal Chiranjib , Ray Mitali , Maitra Sudipta , Mandal Labanya , Bandyopadhyay Santu
Published: April 2014

Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4+ T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.
Rac1 Deletion in Mouse Neutrophils Has Selective Effects on Neutrophil Functions
Authors: Glogauer Michael , Marchal Christophe C. , Zhu Fei , Worku Aelaf , Clausen Björn E. , Foerster Irmgard , Marks Peter , Downey Gregory P. , Dinauer Mary , Kwiatkowski David J.
Published: April 2014

Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To elucidate the specific role of Rac1 in neutrophils, we generated mice with a conditional Rac1 deficiency restricted to cells of the granulocyte/monocyte lineage. As observed in Rac2-deficient neutrophils, Rac1-deficient neutrophils demonstrated profound defects in inflammatory recruitment in vivo, migration to chemotactic stimuli, and chemoattractant-mediated actin assembly. In contrast, superoxide production is normal in Rac1-deficient neutrophils but markedly diminished in Rac2 null cells. These data demonstrate that although Rac1 and Rac2 are both required for actin-mediated functions, Rac2 is specifically required for activation of the neutrophil NADPH oxidase.
CC Chemokine Ligand 5/RANTES Chemokine Antagonists Aggravate Glomerulonephritis Despite Reduction of Glomerular Leukocyte Infiltration
Authors: Anders Hans-Joachim , Frink Michael , Linde Yvonne , Banas Bernard , Wörnle Markus , Cohen Clemens D. , Vielhauer Volker , Nelson Peter J. , Gröne Hermann-Josef , Schlöndorff Detlef
Published: April 2014

The chemokine CC chemokine ligand (CCL)5/RANTES as well as its respective receptor CCR5 mediate leukocyte infiltration during inflammation and are up-regulated early during the course of glomerulonephritis (GN). We tested the effects of the two CCL5/RANTES blocking analogs, Met-RANTES and amino-oxypentane-RANTES, on the course of horse apoferritin (HAF)-induced GN. HAF-injected control mice had proliferative GN with mesangial immune complex deposits of IgG and HAF. Daily i.p. injections of Met-RANTES or amino-oxypentane-RANTES markedly reduced glomerular cell proliferation and glomerular macrophage infiltration, which is usually associated with less glomerular injury and proteinuria in HAF-GN. Surprisingly, however, HAF-GN mice treated with both analogs showed worse disease with mesangiolysis, capillary obstruction, and nephrotic range albuminuria. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state, characterized by a distinct morphology and increased inducible NO synthetase expression in vitro and in vivo, but a reduced uptake of apoptotic cells in vivo. The humoral response and the Th1/Th2 balance in HAF-GN and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs. We conclude that, despite blocking local leukocyte recruitment, chemokine analogs can aggravate some specific disease models, most likely due to interactions with systemic immune reactions, including the removal of apoptotic cells and inducible NO synthetase expression.
IL-1α, Innate Immunity, and Skin Carcinogenesis: The Effect of Constitutive Expression of IL-1α in Epidermis on Chemical Carcinogenesis
Authors: Murphy Jo-Ellen , Morales Romeo E. , Scott Jordan , Kupper Thomas S.
Published: April 2014

Tumor promoters such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) are proinflammatory agents, and their mechanism of action in epithelial carcinogenesis has been linked to the release of IL-1α and the induction of chronic inflammation in skin. To test the role of IL-1α and inflammation in models of cutaneous carcinogenesis, we used our previously described FVB/N transgenic mice overexpressing 17-kDa IL-1α in the epidermis under the keratin 14 (K14) promoter. Strikingly, the K14/IL-1α mice were completely resistant to papilloma and carcinoma formation induced by a two-stage DMBA/TPA protocol, while littermate controls developed both tumor types. K14/IL-1α mice crossed with the highly sensitive TG.AC mice, constitutively expressing mutant Ha-Ras, also failed to develop papillomas or carcinomas. When the K14/IL-1α transgene was bred onto a recombinase-activating gene-2-deficient background, the resistance persisted, indicating that innate, but not acquired, mechanisms may be involved in the resistance to the initiation/promotion model. As an alternative approach, a complete carcinogenesis protocol using repetitive application of DMBA alone was applied. Surprisingly, although the IL-1α mice still did not develop papillomas, they did develop carcinomas de novo at an accelerated rate compared with controls. We conclude that constitutive IL-1α expression rendered FVB mice completely resistant to carcinomas that required evolution from prior papillomas, but facilitated carcinomas that did not evolve from papillomas, as in the complete carcinogenesis protocol. Thus, the role of IL-1α and, by extension that of other proinflammatory factors, in epithelial carcinogenesis are more complex than previously appreciated. These mice may provide a mechanism to investigate the validity of these models of human skin tumorigenesis.
Regulation of Lipopolysaccharide Sensitivity by IFN Regulatory Factor-2
Authors: Cuesta Natalia , Salkowski Cindy A. , Thomas Karen E. , Vogel Stefanie N.
Published: April 2014

IFN regulatory factors (IRFs) are a family of transcription factors and include several members that regulate expression of pro- and anti-inflammatory genes. Mice with a targeted mutation in IRF-2 (IRF-2−/−) were studied after injection of LPS to evaluate the importance of IRF-2 in the regulation of endotoxicity. IRF-2−/− mice were highly refractory to LPS-induced lethality. Although hepatic TNF-α mRNA and circulating TNF-α were significantly elevated in LPS-challenged IRF-2−/− mice, levels of IL-1, IL-12, and IFN-γ mRNA and protein, as well as IL-6 protein, were significantly lower than levels seen in LPS-challenged IRF-2+/+ mice. IRF-2−/− mice were also more refractory to TNF-α challenge than were control mice, which was consistent with their diminished sensitivity to LPS, yet no significant difference in the mRNA expression of TNFRs was observed. IL-12Rβ2 mRNA levels from LPS-challenged IRF-2−/− mice were significantly different after 1, 6, and 8 h, suggesting that both diminished IL-12 and altered IL-12R expression contribute to the paucity of IFN-γ produced. IRF-2 knockout mice also failed to sustain LPS-inducible levels of IRF-1 and IFN consensus sequence binding protein mRNA expression, two transacting factors required for IL-12 transcription, perhaps as a result of diminished IL-1β, IL-6, and IFN-γ levels. Liver sections from IRF-2+/+ and IRF-2−/− mice were analyzed 6 h after a typically lethal injection of LPS. IRF-2−/− mice exhibited greater numbers of apoptotic Kupffer cells than did wild-type mice, suggesting a novel anti-apoptotic role for IRF-2. Collectively, these findings reveal a critical role for IRF-2 in endotoxicity, and point to a previously unappreciated role for IRF-2 in the regulation of apoptosis.
Expression of a Novel Murine Type I IFN in the Pancreatic Islets Induces Diabetes in Mice
Authors: Vassileva Galya , Chen Shu-Cheng , Zeng Ming , Abbondanzo Susan , Jensen Kristian , Gorman Daniel , Baroudy Bahige M. , Jiang Ying , Murgolo Nicholas , Lira Sergio A.
Published: April 2014

IFN-κ belongs to a recently identified subclass of type I IFNs. In this study, we report the cloning and preliminary characterization of the murine homologue of IFN-κ. The gene encodes a 200-aa protein which is 38.5% homologous to human IFN-κ. Murine IFN-κ contains four cysteines in analogous positions to those observed in the IFN-α and an additional fifth unique cysteine, C174. The murine gene is located on chromosome 4, where other type I murine IFN genes, IFN-α and IFN-β, are clustered. This region is syntenic with human chromosome 9 where the gene encoding IFN-κ and the type I IFN gene cluster are found. Mouse IFN-κ is expressed at low levels in peritoneal macrophages and its expression is up-regulated by dsRNA and IFN-γ. Similar to previously reported transgenic mice carrying type I and type II IFNs, transgenic mice overexpressing murine IFN-κ in the β cells of the pancreas develop overt diabetes with hyperglycemia. Histological characterization of pancreatic islets from these transgenic mice showed inflammatory infiltrates with corresponding destruction of β cells.
Complement C5a Receptor-Mediated Signaling May Be Involved in Neurodegeneration in Alzheimer’s Disease
Authors: Farkas Imre , Takahashi Mitsuo , Fukuda Atsuo , Yamamoto Naoki , Akatsu Hiroyasu , Baranyi Lajos , Tateyama Hisashi , Yamamoto Takayuki , Okada Noriko , Okada Hidechika
Published: April 2014

In our earlier results, we demonstrated that cells expressing the complement C5aR are vulnerable since abnormal activation of C5aR caused apoptosis of these cells. In this study, we demonstrate that activation of C5aR by antisense homology box (AHB) peptides synthesized in multiple antigenic peptide form and representing putative interaction sites of the C5a/C5aR evoked calcium influx in TGW neuroblastoma cells. Dose-dependent inhibition of the response was found when the cells were pretreated with C5a, suggesting that C5aR was involved in this process. In addition, pretreatment with monomeric forms of the AHB peptides resulted in attenuation of the calcium signals, supporting the idea of the role of C5aR in this process. Cells of a neuron-rich primary culture and pyramidal cells of rat brain slices also responded to the AHB peptide activation with an increase in the intracellular calcium level, showing that calcium metabolism might be affected in these cells. TUNEL staining demonstrated that C5aR-mediated apoptosis could be induced both in cells of the primary culture as well as in cortical pyramidal neurons of the rat brain. In addition, we investigated expression of C5aR in the hippocampal and cortical neurons of human brains of healthy and demented patients using two anti-human C5aR Abs. Pyramidal cells of the hippocampus and cortex and granular cells of the hippocampus were immunopositive on staining. Although staining was also positive in the vascular dementia brain, it disappeared in the brain with Alzheimer’s disease. These results provide further support that C5aR may be involved in neurodegeneration.
Impaired Secretion of IL-10 by T Cells from Patients with Common Variable Immunodeficiency–Involvement of Protein Kinase A Type I
Authors: Holm Are Martin , Aukrust Pål , Aandahl Einar Martin , Müller Fredrik , Taskén Kjetil , Frøland Stig S.
Published: April 2014

Common variable immunodeficiency (CVID) is a heterogeneous group of B cell deficiency syndromes. T cell abnormalities are present in a high proportion of patients with CVID, suggesting impaired T cell-mediated stimulation of B cells. Based on the importance of IL-10 for B cell function and the involvement of the cAMP/protein kinase A type I (PKAI) system in IL-10 synthesis, we examined IL-10 secretion in T cells from CVID patients and controls, particularly focusing on possible modulatory effects of the cAMP/PKAI system. Our main findings were: 1) anti-CD3 and anti-CD3/anti-CD28 activated T cells from CVID patients secreted less IL-10 than healthy controls. This defect was not related to varying proportions of T cell subsets (e.g., CD4+/CD8+, CD45RA+/RO+, or CD28− T cells); 2) PKAI activation through the cAMP agonist 8-CPT-cAMP markedly inhibited IL-10 secretion from T cells through CD3 and CD28 activation in both patients and controls, but the sensitivity for cAMP-dependent inhibition was increased in CVID; 3) selective PKAI inhibition by Rp-8-Br-cAMPS markedly increased IL-10 secretion in anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in both patients and controls. Even at the lowest concentrations of Rp-8-Br-cAMPS, IL-10 secretion in CVID patients reached levels comparable to those in controls. Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The involvement of the cAMP/PKAI system in this defect suggests a novel target for therapeutic immunomodulation in CVID.
Retinoic Acid Reduces Autoimmune Renal Injury and Increases Survival in NZB/W F1 Mice
Authors: Kinoshita Koji , Yoo Byun-Suk , Nozaki Yuji , Sugiyama Masafumi , Ikoma Shinya , Ohno Motoki , Funauchi Masanori , Kanamaru Akihisa
Published: April 2014

Retinoic acids, a group of natural and synthetic vitamin A derivatives, have potent antiproliferative and anti-inflammatory properties. Recently, retinoic acids were reported to inhibit Th1 cytokine production. We investigated the effects of retinoic acid on lupus nephritis in a model of NZB/NZW F1 (NZB/W F1) mice. Three-month-old NZB/W F1 mice were separated into two groups: one treated with all-trans-retinoic acid (ATRA; 0.5 mg i.p., three times weekly for 7 mo) and one with saline as a control. Compared with controls, ATRA-treated mice survived longer and exhibited a significant reduction of proteinuria, renal pathological findings including glomerular IgG deposits, and serum anti-DNA Abs. Splenomegaly was less marked in the treated mice than in controls. Transcripts encoding IFN-γ, IL-2, and IL-10 in splenic CD4+ T cells were significantly reduced in treated mice compared with controls. We conclude that treatment with ATRA in SLE-prone NZB/W F1 mice significantly alleviates autoimmune renal disorder and prolongs survival; this may thus represent a novel approach to the treatment of patients with lupus nephritis.
Proliferation and Survival of Activated B Cells Requires Sustained Antigen Receptor Engagement and Phosphoinositide 3-Kinase Activation
Authors: Donahue Amber C. , Fruman David A.
Published: April 2014

In this study, we investigate the extracellular and intracellular signals that drive cell cycle progression of activated B cells in the absence of T cell help. We find that brief engagement of the B cell receptor is sufficient to induce a single cell division in a fraction of cells, but that survival during successive cell divisions requires sustained receptor stimulation. In contrast, T cells have been shown previously to commit to multiple cell divisions following brief TCR engagement. Both early and late B cell receptor signals are blocked by inhibitors of phosphoinositide 3-kinase and mammalian target of rapamycin and are associated with S6 kinase activation and increased cell size. The requirement for ongoing Ag receptor signaling can be overcome by engagement of CD40 but only partially by IL-4. Proliferation driven by LPS also requires sustained exposure to the stimulus. These findings reveal checkpoints that may limit T-independent B cell responses when Ag exposure is transient.
Aod1Controlling Day 3 Thymectomy-Induced Autoimmune Ovarian Dysgenesis in Mice Encompasses Two Linked Quantitative Trait Loci with Opposing Allelic Effects on Disease Susceptibility
Authors: Roper Randall J. , McAllister Ryan D. , Biggins Julia E. , Michael Sandra D. , Min Soo Hong , Tung Kenneth S. K. , Call Stanford B. , Gao Jianfeng , Teuscher Cory
Published: April 2014

Day 3 thymectomy (D3Tx) leads to a paucity of CD4+CD25+ suppressor T cells, a loss of peripheral tolerance, and the development of organ-specific autoimmune disease in adult mice. Importantly, D3Tx does not lead to autoimmune disease in all mouse strains, indicating that this process is genetically controlled. Previously, we reported linkage of D3Tx-induced autoimmune ovarian dysgenesis (AOD) and its intermediate phenotypes, antiovarian autoantibody responsiveness, oophoritis, and atrophy, to five quantitative trait loci (QTL), designated Aod1 through Aod5. We also showed interaction between these QTL and H2 as well as Gasa2, a QTL controlling susceptibility to D3Tx-induced autoimmune gastritis. To physically map Aod1, interval-specific bidirectional recombinant congenic strains of mice were generated and studied for susceptibility to D3Tx-induced AOD. Congenic mapping studies revealed that Aod1 controls susceptibility to oophoritis and comprises two linked QTL with opposing allelic effects. Aod1a resides between D16Mit211 (23.3 cM) and D16Mit51 (66.75 cM) on chromosome 16. Aod1b maps proximal of Aod1a between D16Mit89 (20.9 cM) and D16Mit211 (23.3 cM) and includes the candidate genes stefin A1, A2, and A3 (Stfa1-Stfa3), inhibitors of cathepsin S, a cysteine protease required for autoantigen presentation, and the development of autoimmune disease of the salivary and lacrimal glands following D3Tx. cDNA sequencing revealed the existence of structural polymorphisms for both Stfa1 and Stfa2. Given the roles of cathepsins in Ag processing and presentation, Stfa1 and Stfa2 alleles have the potential to control susceptibility to autoimmune disease at the level of both CD4+CD25+ suppressor and CD4+CD25− effector T cells.
B Cells Activated by Lipopolysaccharide, But Not By Anti-Ig and Anti-CD40 Antibody, Induce Anergy in CD8+ T Cells: Role of TGF-β1
Authors: Parekh Vrajesh V. , Prasad Durbaka V. R. , Banerjee Pinaki P. , Joshi Bimba N. , Kumar Anil , Mishra Gyan C.
Published: April 2014

B cells recognize Ag through their surface IgRs and present it in the context of MHC class II molecules to CD4+ T cells. Recent evidence indicates that B cells also present exogenous Ags in the context of MHC class I to CD8+ T cells and thus may play an important role in the modulation of CTL responses. However, in this regard, conflicting reports are available. One group of studies suggests that the interaction between B cells and CD8+ T cells leads to the activation of the T cells, whereas other studies propose that it induces T cell tolerance. For discerning this dichotomy, we used B cells that were activated with either LPS or anti-Ig plus anti-CD40 Ab, which mimic the T-independent and T-dependent modes of B cell activation, respectively, to provide accessory signals to resting CD8+ T cells. Our results show that, in comparison with anti-Ig plus anti-CD40 Ab-activated B cells, the LPS-activated B cells (LPS-B) failed to induce significant levels of proliferation, cytokine secretion, and cytotoxic ability of CD8+ T cells. This hyporesponsiveness of CD8+ T cells activated with LPS-B was significantly rescued by anti-TGF-β1 Ab. Moreover, it was found that such hyporesponsive CD8+ T cells activated with LPS-B had entered a state of anergy. Furthermore, LPS-B expresses a significantly higher level of TGF-β1 on the surface, which caused the observed hyporesponsiveness of CD8+ T cells. Therefore, this study, for the first time, provides a novel mechanism of B cell surface TGF-β1-mediated hyporesponsiveness leading to anergy of CD8+ T cells.
Pathogenesis of Autoimmunity After Xenogeneic Thymus Transplantation
Authors: Yan Yehong , Devos Timothy , Yu Lisong , Xia Guliang , Rutgeerts Omer , Goebels Jozef , Segers Constant , Lin Yuan , Vandeputte Michel , Waer Mark
Published: April 2014

Thymus transplantation is a promising strategy to induce xenotolerance, but may also induce an autoimmune syndrome (AIS). The pathogenesis of this AIS was explored using nude rats as recipients. Thymus grafts consisted of fetal hamster thymic tissue with or without mixing with fetal rat tissue such as thymus, thyroid, salivary gland, and heart. All hamster thymus recipients died of AIS within 2–3 mo. In most recipients of xenothymus mixed with rat tissues such as thymus, thyroid, and salivary gland, but not heart, AIS was prevented, indicating an insufficient presence of rat epithelial cell Ags within the xenothymus. AIS could be transferred to control nude rats by whole splenocytes or by splenocyte subpopulations such as CD3+, CD3−, and B lymphocytes, but not by non-T, non-B cells from AIS animals. This transfer could be suppressed by cotransferring either CD4+ or CD8+ lymphocytes from euthymic rats, but not by splenocytes from recipients of syngeneic or xenogeneic thymus mixed with rat tissue, indicating a defective generation of regulatory lymphocytes. As for CD4+ regulatory cells this defect was probably qualitative, because the percentages of CD4+CD25+ or CD4+CD45RClow populations were normal after xenothymus transplantation. As for the CD8+ regulatory cells, the defect was quantitative, as CD8+ cell levels always remained low. The latter was related to the nonvascularized nature of thymus grafts. In conclusion, AIS after xenothymus transplantation in nude rats is due to a combination of insufficient intrathymic presence of host-type epithelial cell Ags and a defective generation of regulatory T lymphocytes.
TCR Dynamics in Human Mature T Lymphocytes Lacking CD3γ
Authors: Torres Pilar S. , Alcover Andrés , Zapata David A. , Arnaud Jacques , Pacheco Alberto , Martín-Fernández José M. , Villasevil Eugenia M. , Sanal Ozden , Regueiro José R.
Published: April 2014

The contribution of CD3γ to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3γ is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3γ-deficient (γ−) individuals. In contrast to γ− mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural γ− T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3γ. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3γ-dependent, as it was restored by retroviral transduction of CD3γ. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3γ contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.
Alterations in Fas Expression Are Characteristic of, But Not Solely Responsible for, Enhanced Metastatic Competence
Authors: Liu Kebin , Abrams Scott I.
Published: April 2014

Dysregulation of the Fas pathway has been implicated in tumor progression; however, how alterations in Fas expression influence metastatic behavior remains unresolved. In this study, we investigated the link between Fas expression and metastatic capacity in two mouse tumor models: one was a sarcoma, which was used to analyze the consequences of loss of Fas function in experimental pulmonary metastases, and the other was a mammary carcinoma, where Fas expression was examined in matched pairs of primary and metastatic cell lines as well as by immunohistochemistry of tissues taken from primary and metastatic sites of spontaneous tumor development. In the sarcoma model, a Fas-resistant/refractory subline was produced in vitro from the parental line by biologic selection against Fas-responsive cells, and it was then compared with the poorly metastatic parental line and to an in vivo-derived subline that was highly metastatic for growth in the lungs. In both tumor models, an inverse correlation was demonstrated between Fas expression and metastatic phenotype. Subsequent studies in the sarcoma model revealed that although the Fas-resistant/refractory subline displayed significant metastatic ability, the parental line from which it was derived exhibited little to no additional metastatic activity if experimentally rendered Fas-resistant by molecular-based strategies or transplanted into a Fas ligand-deficient host. Therefore, these findings suggested that down-regulation of Fas was associated with the metastatic phenotype, but alterations in Fas expression alone were insufficient for acquisition of full metastatic potential. Rather, the ability of such Fas-resistant neoplastic subpopulations to achieve metastatic competence apparently required co-possession of additional malignant characteristics.
IL-1β Scavenging by the Type II IL-1 Decoy Receptor in Human Neutrophils
Authors: Bourke Emer , Cassetti Arianna , Villa Antonello , Fadlon Emma , Colotta Francesco , Mantovani Alberto
Published: April 2014

IL-1 elicits its cellular effects by binding a heterodimeric receptor consisting of IL-1RI and the accessory protein, IL-1RAcPr. In addition, it binds to IL-1RII, which lacking signaling function has been ascribed a decoy role. The fate of the ligand following interaction with the decoy receptor was examined in human polymorphonuclear cells (PMN), which express predominantly (>90%) IL-1RII. Incubation of PMN with IL-1β results in a rapid decrease in cell surface-associated ligand accompanied by a concomitant increase in internalized IL-1 with 50–60% of IL-1β located intracellularly within 1 h at 37°C. The use of blocking Abs revealed that IL-1 internalization is mediated exclusively by the decoy receptor. The results of inhibitor analysis demonstrate that internalization requires ATP synthesis and involves clathrin-mediated endocytosis. Following removal of the ligand, the receptor was rapidly re-expressed on the cell surface. Cyclohexamide, a protein synthesis inhibitor, had no effect upon the process, suggesting that the re-expressed receptor was recycled. In addition, human keratinocytes stably transfected with IL-1RII (HaCAT 811) also internalized the IL-1RII with 43% cell surface receptor internalized after 90 min. Immunofluorescence microscopy revealed colocalization of the internalized receptor with wheat germ agglutinin-labeled internalized glycoproteins and early endosome Ag-1, a protein associated with the early endosome compartments, indicative of cellular uptake of IL-1RII by endocytosis. In contrast, little or no internalization was observed in other cells of immune origin. These results suggest that the decoy receptor IL-1RII can act as a scavenger of IL-1, representing a novel autoregulatory mechanism of the IL-1 system.
Mitochondrial Membrane Hyperpolarization Hijacks Activated T Lymphocytes Toward the Apoptotic-Prone Phenotype: Homeostatic Mechanisms of HIV Protease Inhibitors
Authors: Matarrese Paola , Gambardella Lucrezia , Cassone Antonio , Vella Stefano , Cauda Roberto , Malorni Walter
Published: April 2014

A decrease of mitochondrial membrane potential has been hypothesized to be a marker of apoptotic cells, including activated T lymphocytes. It was recently demonstrated that HIV protease inhibitors, independently from any viral infection, can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis. To analyze the mechanisms underlying these effects, a specific study was undertaken in both resting and activated human PBL exposed to either receptor (e.g., anti-Fas)- or nonreceptor (e.g., radiation)-mediated apoptotic stimuli. T cell activation was found to be accompanied by a significant increase in mitochondrial membrane potential, or hyperpolarization, which was undetectable in resting cells. We also detected apoptotic hindering by HIV protease inhibitors only in activated T lymphocytes. This was apparently due to the ability of these drugs to block activation-associated mitochondria hyperpolarization, which, in turn, was paralleled by an impairment of cell cycle progression. Remarkably, protease inhibitors also prevented zidovudine-mediated mitochondrial toxicity. Finally, HIV-infected cells from naive patients behaved identically to activated T cells, displaying hyperpolarized mitochondria, while lymphocytes from patients under highly active antiretroviral therapy (which included HIV protease inhibitors) seemed to react as resting cells. Altogether these results clearly indicate that the hyperpolarization state of mitochondria may represent a prerequisite for the sensitization of lymphocytes to the so-called activation-induced cell death. They also suggest that HIV protease inhibitors, by interfering with induction of the mitochondrial hyperpolarization state, can result in cell survival even independent of any viral infection.