bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological Role of Macrophage Inflammatory Protein-3α Induction during Maturation of Intestinal Macrophages
Authors: Hausmann Martin , Bataille Frauke , Spoettl Tanja , Schreiter Katja , Falk Werner , Schoelmerich Juergen , Herfarth Hans , Rogler Gerhard
Published: April 2014

Intestinal macrophages (IMAC) are a central component in the defense of the intestinal mucosa against luminal microbes. In normal mucosa, monocytes differentiate to immunologically tolerant IMAC with a typical phenotype lacking activation markers such as CD14 and TLRs 2 and 4. CD33+ IMAC were isolated from normal intestinal mucosa by immunomagnetic beads. A subtractive hybridization subtracting mRNA from normal IMAC from those of in vitro differentiated macrophages was performed. IMAC differentiation was studied in multicellular spheroids (MCS). Functional assays on migration of CD45R0+ T cells were performed in MCS coculture models. Of 76 clones, 3 obtained by subtractive mRNA hybridization showed >99% homology to mRNA of MIP-3α, indicating that this chemokine is induced in IMAC compared with in vitro differentiated macrophages. MIP-3α protein expression was confirmed in cryostat sections of normal intestinal mucosa by immunohistochemistry. IMAC in the lamina propria stained positive for MIP-3α. FACS of purified IMAC clearly indicated expression of MIP-3α in these cells. In the MCS-in vitro differentiation model for IMAC, MIP-3α protein expression was absent on day 1 but detectable on day 7 of coculture, demonstrating the induction of MIP-3α during differentiation of IMAC. IMAC attracted CD45R0+ T cells to migrate into an MCS coculture model. In human mucosa, a close contact between IMAC and CD45R0+ T cells could be demonstrated. MIP-3α is induced during the differentiation of monocytes into IMAC. Our data suggest that MIP-3α expression could be involved in the recruitment of CD45R0+ cells into the lamina propria.
The Colony-Stimulating Factor 1 Receptor Is Expressed on Dendritic Cells during Differentiation and Regulates Their Expansion
Authors: MacDonald Kelli P. A. , Rowe Vanessa , Bofinger Helen M. , Thomas Ranjeny , Sasmono Tedjo , Hume David A. , Hill Geoffrey R.
Published: April 2014

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11chigh DC subsets are reduced by 50–70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.
Tamoxifen Blocks Estrogen-Induced B Cell Maturation but Not Survival
Authors: Peeva Elena , Venkatesh Jeganathan , Diamond Betty
Published: April 2014

Estrogen treatment has been shown not only to exacerbate disease activity and accelerate death in spontaneous murine models of lupus but also to induce a lupus-like phenotype in nonspontaneously autoimmune mice. In mice transgenic for the H chain of an anti-DNA Ab, estrogen rescues naive autoreactive B cells that normally are deleted and causes them to mature to a marginal zone phenotype. Estrogen further leads to the activation of this population causing an elevation of serum anti-DNA Ab titers and renal disease. This study was designed to evaluate the therapeutic potential of tamoxifen, a selective estrogen receptor modulator, on estrogen-induced lupus. Mice treated with both estradiol and tamoxifen showed no elevation in anti-DNA Ab titers and consequently no glomerular IgG. The DNA-reactive B cell population that is rescued by estrogen was present in an anergic state in mice treated with both estradiol and tamoxifen. Estradiol enhances transitional B cell resistance to apoptosis and expands the population of marginal zone B cells; tamoxifen did not impede the enhanced resistance to apoptosis, but prevented the development of autoreactive cells as marginal zone B cells. Thus, estrogen-induced autoimmunity proceeds through two distinct molecular pathways, one affecting survival and the other maturation. Activation, but not survival, of autoreactive B cells can be abrogated by tamoxifen. Drugs that modulate even some of the effects of estrogen may be beneficial in patients with lupus. Eventually, understanding the pathways involved in survival and activation of autoreactive B cells will permit the development of therapeutics that target all relevant pathways.
Dynamic Differentiation of Activated Human Peripheral Blood CD8+ and CD4+ Effector Memory T Cells
Authors: Schwendemann Jochen , Choi Carmen , Schirrmacher Volker , Beckhove Philipp
Published: April 2014

Two functionally different memory T cell subsets were originally defined based on their different CCR7 expression profile, but the lineage relationship between these subsets referred to as central memory T cells (TCM) and effector memory T cells (TEM), is not resolved. A prevalent model proposes a linear progressive differentiation from TCM to TEM. Our results demonstrate that on activation, human CCR7−CD62L− peripheral blood CD8+ and CD4+ TEM cells exhibit a dynamic differentiation, involving transient as well as stable changes to TCM phenotype and properties. Whereas the larger fraction of TEM cells increases expression of effector molecules, such as perforin or IFN-γ, a smaller fraction first acquires CCR7 expression. We demonstrate that this acquisition of lymph node homing potential is associated with strong proliferation similar to that of activated TCM cells. After proliferation, most of these cells lose CCR7 expression again and acquire effector functions (e.g., perforin production). A small proportion (∼6%), however, maintain phenotypic and functional TCM properties over a long time interval. These results suggest that TEM cells provide immediate effector function by a fraction of cells as well as self-renewal by others through up-regulation of CCR7 followed by either secondary peripheral effector function or long term maintenance of TCM-like properties.
Provision of Granulocyte-Macrophage Colony-Stimulating Factor Converts an Autoimmune Response to a Self-Antigen into an Antitumor Response
Authors: Ji Qingyong , Gondek David , Hurwitz Arthur A.
Published: April 2014

Many tumor Ags recognized by T cells are self-Ags. Because high avidity, self-reactive T cells are deleted in the thymus, any residual self-reactive T cells existing in the periphery are likely to be low avidity and nonresponsive due to peripheral tolerance mechanisms. Activation of these residual T cells is critical for targeting tumors for immunotherapy. In this study, we studied immune responses against the murine B16 melanoma using a tyrosinase-related protein 2 (TRP-2) peptide as a model tumor/self-Ag. Our results showed that TRP-2 peptide vaccination alone elicited a weak T cell response and modestly decreased B16 lung tumor nodules. The combination of peptide vaccination and treatment with an Ab directed against the inhibitory receptor CTLA-4 enhanced the immune response against TRP-2 peptide, inducing autoimmune depigmentation and further decreasing lung tumor nodules. However, both vaccination methods failed to protect against orthotopic (s.c.) B16 tumor challenge. The addition of an irradiated GM-CSF-expressing, amelanotic tumor cell vaccine significantly delayed s.c. B16 tumor growth. Subsequent studies revealed that provision of GM-CSF increased dendritic cell numbers in lymph nodes and spleen. Furthermore, addition of CTLA-4 blockade increased the frequency of TRP-2-specific, IFN-secreting T cells in spleen and lymph nodes. Overall, our results indicate that combining enhancement of Ag presentation with removal of CTLA-4-mediated inhibition can convert a “weaker” autoimmune response into a more potent antitumor immune response.
T Cell Tolerance Induced by Cross-Reactive TCR Ligands Can Be Broken by Superagonist Resulting in Anti-Inflammatory T Cell Cytokine Production
Authors: Illés Zsolt , Waldner Hanspeter , Reddy Jayagopala , Bettelli Estelle , Nicholson Lindsay B. , Kuchroo Vijay K.
Published: April 2014

Cross-reactive activation of potentially autoreactive T cells by high-affinity nonself ligands may be important in breaking self-tolerance in autoimmunity. In a mouse transgenic for a cross-reactive TCR, we have previously shown that a hyperstimulating altered peptide ligand, L144, induced unresponsiveness to the self peptide, proteolipid protein 139–151. In this study, we demonstrate that a superagonist ligand can break T cell tolerance induced by the lower affinity cognate Ag. T cells tolerant to the cognate ligand, Q144, responded to superagonist, L144, by proliferation and the production of mainly IL-4 and IL-10 in vitro. In contrast, T cells that were tolerized to the superagonist were unable to respond to any peptide that cross-reacted with the transgenic TCR. Low-dose immunization with the superagonist L144 was able to break tolerance to the cognate ligand in vivo and resulted in a blunted proliferative response with production of Th2 cytokines.
Adenovirus Infection Dramatically Augments Lipopolysaccharide-Induced TNF Production and Sensitizes to Lethal Shock
Authors: Fejér György , Szalay Katalin , Győry Ildikó , Fejes Mária , Kúsz Erzsébet , Nedieanu Saviana , Páli Tibor , Schmidt Tibor , Siklódi Botond , Lázár György , Lázár György , Duda Ernő
Published: April 2014

We observed a remarkable synergism of adenoviruses and LPS in triggering the production of TNF in intact animals. We found that in mice pre-exposed to adenoviruses, LPS injections generated extremely high levels of TNF with altered kinetics. The elevated TNF synthesis stemmed mostly from posttranscriptional up-regulation of TNF production, although transcription of the TNF gene was also induced. Adenoviruses and LPS exhibited a significant but less dramatic synergism in the induction of IL-6, IFN-γ, and NO. Only marginal changes were detected in the synthesis of a panel of other cytokines. Different serotypes of the virus showed practically identical effects. As deletion mutants lacking indispensable viral genes or UV inactivated virions exhibited similar activities as the infectious, wild-type virus, it seems unlikely that the viral genome plays any significant role in the phenomenon. Published data indicate that other viruses also show some kind of synergism with LPS, although by different cellular mechanisms. T cells and their IFN-γ production—crucial in the synergism of influenza viruses and LPS—were dispensable in our experiments. We suggest that the phenomenon is probably a general one: an overlap between different molecular mechanisms detecting bacterial and viral pathogens and inducing mediators of nonspecific cell-mediated host defense. The synergism of viruses and LPS (bacteria) could be a concern in medical practice as well as in gene therapy experiments with high doses of recombinant adenoviruses.
Regulation of Bim by TCR Signals in CD4/CD8 Double-Positive Thymocytes
Authors: Bunin Anna , Khwaja Fatima W. , Kersh Gilbert J.
Published: April 2014

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3ε injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that BimEL is rapidly phosphorylated in thymocytes in response to CD3ε cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-xL bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-xL in immature thymocytes.
α-Galactosylceramide-Induced Liver Injury in Mice Is Mediated by TNF-α but Independent of Kupffer Cells
Authors: Biburger Markus , Tiegs Gisa
Published: April 2014

NKT cells expressing phenotypic markers of both T and NK cells seem to be pivotal in murine models of immune-mediated liver injury, e.g., in Con A-induced hepatitis. Also α-galactosylceramide (α-GalCer), a specific ligand for invariant Vα14 NKT cells, induces hepatic injury. To improve the comprehension of NKT-cell mediated liver injury, we investigated concomitants and prerequisites of α-GalCer-induced hepatitis in mice. Liver injury induced by α-GalCer injection into C57BL/6 mice was accompanied by intrahepatic caspase-3 activity but appeared independent thereof. α-GalCer injection also induces pronounced cytokine responses, including TNF-α, IFN-γ, IL-2, IL-4, and IL-6. We provide a detailed time course for the expression of these cytokines, both in liver and plasma. Cytokine neutralization revealed that, unlike Con A-induced hepatitis, IFN-γ is not only dispensable for α-GalCer-induced hepatotoxicity but even appears to exert protective effects. In contrast, TNF-α was clearly identified as an important mediator for hepatic injury in this model that increased Fas ligand expression on NKT cells. Whereas intrahepatic Kupffer cells are known as a pivotal source for TNF-α in Con A-induced hepatitis, they were nonessential for α-GalCer-mediated hepatotoxicity. In α-GalCer-treated mice, TNF-α was produced by intrahepatic lymphocytes, in particular NKT cells. BALB/c mice were significantly less susceptible to α-GalCer-induced liver injury than C57BL/6 mice, in particular upon pretreatment with d-galactosamine, a hepatocyte-specific sensitizer to TNF-α-mediated injury. Finally, we demonstrate resemblance of murine α-GalCer-induced hepatitis to human autoimmune-like liver disorders. The particular features of this model compared with other immune-mediated hepatitis models may enhance comprehension of basic mechanisms in the etiopathogenesis of NKT cell-comprising liver disorders.
Human Tumor Antigen MUC1 Is Chemotactic for Immature Dendritic Cells and Elicits Maturation but Does Not Promote Th1 Type Immunity
Authors: Carlos Casey A. , Dong Hui Fang , Howard O. M. Zack , Oppenheim Joost J. , Hanisch Franz-Georg , Finn Olivera J.
Published: April 2014

The immunostimulatory outcome of the interactions of many pathogens with dendritic cells (DCs) has been well characterized. There are many fewer examples of similar interactions between DCs and self-molecules, especially the abnormal self-proteins such as many tumor Ags, and their effects on DC function and the immune response. We show that human epithelial cell Ag MUC1 mucin is recognized in its aberrantly glycosylated form on tumor cells by immature human myeloid DCs as both a chemoattractant (through its polypeptide core) and a maturation and activation signal (through its carbohydrate moieties). On encounter with MUC1, similar to the encounter with LPS, immature DCs increase cell surface expression of CD80, CD86, CD40, and CD83 molecules and the production of IL-6 and TNF-α cytokines but fail to make IL-12. When these DCs are cocultured with allogeneic CD4+ T cells, they induce production of IL-13 and IL-5 and lower levels of IL-2, thus failing to induce a type 1 response. Our data suggest that, in vivo in cancer patients, MUC1 attracts immature DCs to the tumor through chemotaxis and subverts their function by negatively affecting their ability to stimulate type 1 helper T cell responses important for tumor rejection.
NFκB-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation
Authors: Lu Li-Fan , Gondek David C. , Scott Zachary A. , Noelle Randolph J.
Published: April 2014

CD4+CD25+ regulatory T cells (Treg) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on Treg has been shown to be a key player of regulating Treg-mediated suppression. A recent study reports that NF-κB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of Treg but not for its suppression capacity. In this report, we have shown that Treg from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive Treg proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type Treg. Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62Llow Treg both in thymus and periphery than their wild-type littermates. This CD62low subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.
Human Follicular Dendritic Cells Express Prostacyclin Synthase: A Novel Mechanism to Control T Cell Numbers in the Germinal Center
Authors: Lee In Yong , Ko Eun-Mi , Kim Sang-Hyun , Jeoung Doo-Il , Choe Jongseon
Published: April 2014

Stromal cells in the lymphoid organs provide a microenvironment where lymphocytes undergo various biological processes such as development, homing, clonal expansion, and differentiation. Follicular dendritic cells (FDCs) in the primary and secondary follicles of the peripheral lymphoid tissues interact with lymphocytes by contacting directly or producing diffusible molecules. To understand the biological role of human FDC at the molecular level, we developed a mAb, 3C8, that recognizes FDC but not bone marrow-derived cells. Through expression cloning and proteome analysis, we identified the protein that is recognized by 3C8 mAb, which revealed that FDC expresses prostacyclin synthase. The 3C8 protein purified from FDC-like cells indeed displayed the enzymatic activity of prostacyclin synthase and converted PGH2 into prostacyclin. In addition, prostacyclin significantly inhibited proliferation of T cells but delayed their spontaneous apoptosis. These findings may help explain why T cells constitute only a minor population compared with B cells in the germinal center.
Molecular Basis of Peptide Recognition by the TCR: Affinity Differences Calculated Using Large Scale Computing
Authors: Wan Shunzhou , Coveney Peter V. , Flower Darren R.
Published: April 2014

Free energy calculations of the wild-type and the variant human T cell lymphotropic virus type 1 Tax peptide presented by the MHC to the TCR have been performed using large scale massively parallel molecular dynamics simulations. The computed free energy difference (−1.86 ± 0.44 kcal/mol) using alchemical mutation-based thermodynamic integration agrees well with experimental data (−2.9 ± 0.2 kcal/mol). Our simulations exploit state-of-the-art hardware and codes whose algorithms have been optimized for supercomputing platforms. This enables us to simulate larger, more realistic biological systems for longer durations without the imposition of artificial constraints.
Delineation of the Function of a Major γδ T Cell Subset during Infection
Authors: Andrew Elizabeth M. , Newton Darren J. , Dalton Jane E. , Egan Charlotte E. , Goodwin Stewart J. , Tramonti Daniela , Scott Philip , Carding Simon R.
Published: April 2014

γδ T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among γδ T cells. Using mice deficient in Vγ1+ T cells which are a major component of the γδ T cell response to microbial infection, a specific immunoregulatory role for Vγ1+ T cells in macrophage and γδ T cell homeostasis during infection has been established. By contrast, Vγ1+ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited Vγ1+ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of Vγ1+ T cells. These findings, therefore, identify distinct and nonoverlapping roles for γδ T cell subsets in infection and establish the complexity and adaptability of a single population of γδ T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.
Critical Role for IgM in Host Protection in Experimental Filarial Infection
Authors: Rajan Bhargavi , Ramalingam Thirumalai , Rajan Thiruchandurai V.
Published: April 2014

We have previously shown that B cells (in particular B1 cells) are important in host protection against brugian infections in a murine i.p. model. In this study, we show that mice deficient in circulating IgM (secIgM−/−), but otherwise normal in their humoral responses, manifest a significant impairment in worm elimination, suggesting that one critical B cell function is the production of Ag-specific IgM. Efficient elimination of larvae is IgM dependent for both primary and challenge infections. The ability to eliminate worms is restored in secIgM−/− mice by administering sera from primed mice. We corroborated these in vivo studies with in vitro observations which show that IgM is the only isotype that reacts strongly with the surface of Brugia L3. Furthermore, activated peritoneal exudate cells adhere to L3 only in the presence of filaria-specific sera or IgM purified from them. This attachment is not reduced by heat inactivation of the serum, suggesting complement independent activity. Peritoneal exudate cells from primed mice, especially activated macrophages, carry high levels of IgM on their surfaces. Our observations suggest that an IgM-mediated reaction initiates the formation of host-protective granulomas.
Constitutive Nuclear Expression of the IκB Kinase Complex and Its Activation in Human Neutrophils
Authors: Ear Thornin , Cloutier Alexandre , McDonald Patrick P.
Published: April 2014

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-κB/Rel proteins and IκB-α in the nucleus. In this study, we show that in these cells, IκB kinase α (IKKα), IKKβ, and IKKγ also partially localize to the nucleus, whereas IKK-related kinases (IKKε, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-κB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKβ and IKKγ become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKα transiently vanishes from both compartments in what appears to be an IKKβ-dependent process. These responses are paralleled by the degradation of IκB-α, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IκB-α phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (α, β, γ) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-κB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IκB signaling cascade within the cell nucleus.
A Shared Epitope of the Interphotoreceptor Retinoid-Binding Protein Recognized by the CD4+ and CD8+ Autoreactive T Cells
Authors: Shao Hui , Peng Yong , Liao Tianjiang , Wang Meiqin , Song Ming , Kaplan Henry J. , Sun Deming
Published: April 2014

We previously demonstrated that cultures of rat uveitogenic T cells rapidly become dominated by CD4+ cells, but activation of CD8+ autoreactive T cells also occurred during the in vitro culture of in vivo-primed T cells. In the present study, we show that the commonly used uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP) 1–20, generated both CD4+ and CD8+ autoreactive T cells in the C57BL/6 (B6) mouse and that this 20-mer contains at least two distinct antigenic epitopes. To determine whether the CD8 response was Ag-specific and whether CD4+ and CD8+ IRBP1–20-specific T cells recognize distinct antigenic epitopes, we prepared highly purified CD4+ and CD8+ T cells from IRBP1–20-primed mice and tested their proliferative response to a large panel of truncated peptides derived from IRBP1–20. The results showed that both CD4+ and CD8+ T cells recognized the same spectrum of peptides. In addition, peptides P10–18 were found to bind effectively to CD8+ IRBP1–20-specific T cells when complexed with recombinant H-2Kb and also stimulate the proliferation and cytokine production of CD4+ IRBP1–20-specific T cells. Our results document for the first time that CD8+ and CD4+ autoreactive T cells display characteristic epitope recognition and they both recognize the same core epitope.
The Enhancement or Prevention of Airway Hyperresponsiveness during Reinfection with Respiratory Syncytial Virus Is Critically Dependent on the Age at First Infection and IL-13 Production
Authors: Dakhama Azzeddine , Park Jung-Won , Taube Christian , Joetham Anthony , Balhorn Annette , Miyahara Nobuaki , Takeda Katsuyuki , Gelfand Erwin W.
Published: April 2014

Respiratory syncytial virus (RSV) infection in early life is suspected to play a role in the development of postbronchiolitis wheezing and asthma. Reinfection is common at all ages, but factors that determine the development of altered airway function after reinfection are not well understood. This study was conducted in a mouse model to define the role of age in determining the consequences on airway function after reinfection. Mice were infected shortly after birth or at weaning and were reinfected 5 wk later, followed by assessment of airway function, airway inflammation, and lung histopathology. Infection of mice at weaning elicited a protective airway response upon reinfection. In this age group, reinfection resulted in increased airway inflammation, but without development of airway hyperresponsiveness (AHR) or eosinophilia and decreased IL-13 levels. By contrast, neonatal infection failed to protect the airways and resulted in enhanced AHR after reinfection. This secondary response was associated with the development of airway eosinophilia, increased IL-13 levels, and mucus hyperproduction. Both CD4- and CD8-positive T cells were a source of IL-13 in the lung, and inhibition of IL-13 abolished AHR and mucus production in these mice. Inoculation of UV-inactivated virus failed to elicit these divergent responses to reinfection, emphasizing the requirement for active lung infection during initial exposure. Thus, neonatal RSV infection predisposes to the development of airway eosinophilia and enhanced AHR via an IL-13-dependent mechanism during reinfection, whereas infection at a later age protects against the development of these altered airway responses after reinfection.
IL-4 and IL-13 Induce Protection of Porcine Endothelial Cells from Killing by Human Complement and from Apoptosis through Activation of a Phosphatidylinositide 3-Kinase/Akt Pathway
Authors: Grehan John F. , Levay-Young Brett K. , Fogelson Jeremy L. , François-Bongarçon Vanessa , Benson Barbara A. , Dalmasso Agustin P.
Published: April 2014

Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-α plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48–72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.
Neutrophil Dysfunction in Guanosine 3′,5′-Cyclic Monophosphate-Dependent Protein Kinase I-Deficient Mice
Authors: Werner Claudia G. , Godfrey Virginia , Arnold Roland R. , Featherstone Gerald L. , Bender Diane , Schlossmann Jens , Schiemann Matthias , Hofmann Franz , Pryzwansky Katherine B.
Published: April 2014

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI−/−) mice. We demonstrate that murine neutrophils expressed cGKIα. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI−/− bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI−/− than in WT. Enhanced chemotaxis was also observed with cGKI−/− peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI−/− vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI−/− than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI−/− (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI−/− (∼60%) than in WT (∼90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIα down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIα on the secretagogue responses are complex, depending on the granule subtype.
Adenosine-Dependent Pulmonary Fibrosis in Adenosine Deaminase-Deficient Mice
Authors: Chunn Janci L. , Molina Jose G. , Mi Tiejuan , Xia Yang , Kellems Rodney E. , Blackburn Michael R.
Published: April 2014

Pulmonary fibrosis is a common feature of numerous lung disorders, including interstitial lung diseases, asthma, and chronic obstructive pulmonary disease. Despite the prevalence of pulmonary fibrosis, the molecular mechanisms governing inflammatory and fibroproliferative aspects of the disorder are not clear. Adenosine is a purine-signaling nucleoside that is generated in excess during cellular stress and damage. This signaling molecule has been implicated in the regulation of features of chronic lung disease; however, the impact of adenosine on pulmonary fibrosis is not well understood. The goal of this study was to explore the impact of endogenous adenosine elevations on pulmonary fibrosis. To accomplish this, adenosine deaminase (ADA)-deficient mice were treated with various levels of ADA enzyme replacement therapy to regulate endogenous adenosine levels in the lung. Maintaining ADA-deficient mice on low dosages of ADA enzyme therapy led to chronic elevations in lung adenosine levels that were associated with pulmonary inflammation, expression of profibrotic molecules, collagen deposition, and extreme alteration in airway structure. These features could be blocked by preventing elevations in lung adenosine. Furthermore, lowering lung adenosine levels after the establishment of pulmonary fibrosis resulted in a resolution of fibrosis. These findings demonstrate that chronic adenosine elevations are associated with pulmonary fibrosis in ADA-deficient mice and suggest that the adenosine functions as a profibrotic signal in the lung.
Muramyl Dipeptide Enhances Osteoclast Formation Induced by Lipopolysaccharide, IL-1α, and TNF-α through Nucleotide-Binding Oligomerization Domain 2-Mediated Signaling in Osteoblasts
Authors: Yang Shuhua , Takahashi Naoyuki , Yamashita Teruhito , Sato Nobuaki , Takahashi Masahiro , Mogi Makio , Uematsu Takashi , Kobayashi Yasuhiro , Nakamichi Yuko , Takeda Kiyoshi , Akira Shizuo , Takada Haruhiko , Udagawa Nobuyuki , Furusawa Kiyofumi
Published: April 2014

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and PGE2, LPS and IL-1α stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1α, or TNF-α but not 1α,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-κB ligand (RANKL) or TNF-α. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-α but not 1α,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1α, or TNF-α but not 1α,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-α in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-α-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1α, and TNF-α through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.
Antigen-Based Therapies Using Ignored Determinants of β Cell Antigens Can More Effectively Inhibit Late-Stage Autoimmune Disease in Diabetes-Prone Mice
Authors: Olcott Angelica P. , Tian Jide , Walker Valerie , Dang Hoa , Middleton Blake , Adorini Luciano , Washburn Lorraine , Kaufman Daniel L.
Published: April 2014

As organ-specific autoimmune diseases do not become manifest until well-advanced, interventive therapies must inhibit late-stage disease processes. Using a panel of immunogenic peptides from various β cell Ags, we evaluated the factors influencing the efficacy of Ag-based therapies in diabetes-prone NOD mice with advanced disease. The ability of the major β cell autoantigen target determinants (TDs) to prime Th2 responses declined sharply between 6 and 12 wk of age, whereas the ability of immunogenic ignored determinants (IDs) of β cell Ags to prime Th2 responses was unaffected by the disease process. The different patterns of TD and ID immunogenicity (even from the same β cell Ag) may be due to the exhaustion of uncommitted TD-reactive, but not ID-reactive, T cell pools by recruitment into the autoimmune cascade. Therapeutic efficacy was associated with a peptide’s immunogenicity and ability to promote Th2 spreading late in the disease process but not its affinity for I-Ag7 or its expression pattern (β cell specific/nonspecific or rare/abundant). Characterization of some IDs revealed them to be “absolute” cryptic determinants. Such determinants have little impact on T cell selection, leaving large precursor T cell pools available for priming by synthetic peptides. Traditional Ag-based therapeutics using whole autoantigens or their TDs cannot prime responses to such determinants. These findings suggest a new strategy for designing more efficacious Ag-based therapeutics for late-stage autoimmune diseases.
2B4/CD48-Mediated Regulation of Lymphocyte Activation and Function
Authors: Assarsson Erika , Kambayashi Taku , Persson Catrine M. , Chambers Benedict J. , Ljunggren Hans-Gustaf
Published: April 2014

2B4 (CD244) is a member of the CD2 subset of the Ig superfamily. This molecule is expressed on innate immune cells, including NK cells, and on subsets of T cells. The 2B4 molecule interacts with CD48, which is widely expressed on hemopoietic cells. Although earlier reports demonstrated a role for 2B4 as an activating receptor in both mice and humans, recent studies of 2B4-deficient mice have suggested that 2B4 functions predominantly as an inhibitory receptor in mice. In addition, 2B4 may also act as a costimulatory ligand for cells expressing CD48. Thus, the 2B4 molecule is more multifunctional than previously understood. In this study, we delineate the current view of 2B4-CD48 interactions among lymphocytes and other cells.
Cutting Edge: A Single MHC Class Ia Is Sufficient for CD8 Memory T Cell Differentiation
Authors: Williams Matthew A. , Bevan Michael J.
Published: April 2014

Recent studies have suggested a role for MHC class Ib molecules in providing signals for memory T cell differentiation during the early phases of acute infection. To test this hypothesis, we assessed the development of effector and memory CD8 T cells in transgenic mice expressing a single chain H-2Dd/β2-microglobulin (β2M) fusion protein on a β2M-deficient background. These mice thus express a single MHC class Ia in the absence of all other β2M-dependent class Ia and Ib molecules. Following infection with a recombinant vaccinia virus expressing a known Dd-restricted epitope from HIV-1 gp160, the development of effector and memory cells CD8 T cells was comparable to control mice. Furthermore, these memory cells responded rapidly and robustly to antigenic restimulation. Therefore, we conclude that full CD8 memory differentiation requires only a single MHC class Ia chain, ruling out a requirement for MHC class Ib molecules in this process.
Both Dendritic Cells and Macrophages Can Stimulate Naive CD8 T Cells In Vivo to Proliferate, Develop Effector Function, and Differentiate into Memory Cells
Authors: Pozzi Lu-Ann M. , Maciaszek Joseph W. , Rock Kenneth L.
Published: April 2014

The generation of T cell immunity requires the acquisition and presentation of Ag on bone marrow-derived APCs. Dendritic cells (DC) are believed to be the most potent bone marrow-derived APCs, and the only ones that can stimulate naive T cells to productively respond to Ags. Because macrophages (MΦ) are bone marrow-derived APCs that are also found in tissues and lymphoid organs, can acquire and present Ag, and can express costimulatory molecules, we have investigated their potential to stimulate primary T cell responses in vivo. We find that both injected MΦ and DCs can migrate from peripheral tissues or blood into lymphoid organs. Moreover, injection of peptide-pulsed MΦ or DCs into mice stimulates CD8 T cells to proliferate, express effector functions including cytokine production and cytolysis, and differentiate into long-lived memory cells. MΦ and DCs stimulate T cells directly without requiring cross-presentation of Ag on host APCs. Therefore, more than one type of bone marrow-derived APC has the potential to prime T cell immunity. In contrast, another bone marrow-derived cell, the T lymphocyte, although capable of presenting Ag and homing to the T cell areas of lymphoid organs, is unable to stimulate primary responses. Because MΦ can be very abundant cells, especially at sites of infection and inflammation, they have the potential to play an important role in immune surveillance and the initiation of T cell immunity.
Invariant NKT Cells Exacerbate Type 1 Diabetes Induced by CD8 T Cells
Authors: Griseri Thibault , Beaudoin Lucie , Novak Jan , Mars Lennart T. , Lepault Françoise , Liblau Roland , Lehuen Agnès
Published: April 2014

Invariant NKT (iNKT) cells have been implicated in the regulation of autoimmune diseases. In several models of type 1 diabetes, increasing the number of iNKT cells prevents the development of disease. Because CD8 T cells play a crucial role in the pathogenesis of diabetes, we have investigated the influence of iNKT cells on diabetogenic CD8 T cells. In the present study, type 1 diabetes was induced by the transfer of CD8 T cells specific for the influenza virus hemagglutinin into recipient mice expressing the hemagglutinin Ag specifically in their β pancreatic cells. In contrast to previous reports, high frequency of iNKT cells promoted severe insulitis and exacerbated diabetes. Analysis of diabetogenic CD8 T cells showed that iNKT cells enhance their activation, their expansion, and their differentiation into effector cells producing IFN-γ. This first analysis of the influence of iNKT cells on diabetogenic CD8 T cells reveals that iNKT cells not only fail to regulate but in fact exacerbate the development of diabetes. Thus, iNKT cells can induce opposing effects dependent on the model of type 1 diabetes that is being studied. This prodiabetogenic capacity of iNKT cells should be taken into consideration when developing therapeutic approaches based on iNKT cell manipulation.
Opposing Roles for RelB and Bcl-3 in Regulation of T-Box Expressed in T Cells, GATA-3, and Th Effector Differentiation
Authors: Corn Radiah A. , Hunter Chris , Liou Hsiou-Chi , Siebenlist Ulrich , Boothby Mark R.
Published: April 2014

CD4+ T cells with a block in the NF-κB signaling pathway exhibit decreases in Th1 responses and diminished nuclear levels of multiple transactivating NF-κB/Rel/IκB proteins. To determine the lineage-intrinsic contributions of these transactivators to Th differentiation, T cells from mice deficient in specific subunits were cultured in exogenous cytokines promoting either Th1 or Th2 differentiation. RelB-deficient cells exhibited dramatic defects in Th1 differentiation and IFN-γ production, whereas no consistent defect in either Th1 or Th2 responses was observed with c-Rel-deficient cells. In sharp contrast, Bcl-3-null T cells displayed no defect in IFN-γ production, but their Th2 differentiation and IL-4, IL-5, and IL-13 production were significantly impaired. The absence of RelB led to a dramatic decrease in the expression of T-box expressed in T cells and Stat4. In contrast, Bcl-3-deficient cells exhibited decreased GATA-3, consistent with evidence that Bcl-3 can transactivate a gata3 promoter. These data indicate that Bcl-3 and RelB exert distinct and opposing effects on the expression of subset-determining transcription factors, suggesting that the characteristics of Th cell responses may be regulated by titrating the stoichiometry of transactivating NF-κB/Rel/IκB complexes in the nuclei of developing helper effector cells.
Dendritic Cells in Human Thymus and Periphery Display a Proinsulin Epitope in a Transcription-Dependent, Capture-Independent Fashion
Authors: Garcia Carlos A. , Prabakar Kamalaveni R. , Diez Juan , Cao Zhu Alexander , Allende Gloria , Zeller Markus , Dogra Rajpreet , Mendez Armando , Rosenkranz Eliot , Dahl Ulf , Ricordi Camillo , Hanahan Douglas , Pugliese Alberto
Published: April 2014

The natural expression of tissue-specific genes in the thymus, e.g., insulin, is critical for self-tolerance. The transcription of tissue-specific genes is ascribed to peripheral Ag-expressing (PAE) cells, which discordant studies identified as thymic epithelial cells (TEC) or CD11c+ dendritic cells (DC). We hypothesized that, consistent with APC function, PAE-DC should constitutively display multiple self-epitopes on their surface. If recognized by Abs, such epitopes could help identify PAE cells to further define their distribution, nature, and function. We report that selected Abs reacted with self-epitopes, including a proinsulin epitope, on the surface of CD11c+ cells. We find that Proins+CD11c+ PAE cells exist in human thymus, spleen, and also circulate in blood. Human thymic Proins+ cells appear as mature DC but express CD8α, CD20, CD123, and CD14; peripheral Proins+ cells appear as immature DC. However, DC derived in vitro from human peripheral blood monocytes include Proins+ cells that uniquely differentiate and mature into thymic-like PAE-DC. Critically, we demonstrate that human Proins+CD11c+ cells transcribe the insulin gene in thymus, spleen, and blood. Likewise, we show that mouse thymic and peripheral CD11c+ cells transcribe the insulin gene and display the proinsulin epitope; moreover, by using knockout mice, we show that the display of this epitope depends upon insulin gene transcription and is independent of Ag capturing. Thus, we propose that PAE cells include functionally distinct DC displaying self-epitopes through a novel, transcription-dependent mechanism. These cells might play a role in promoting self-tolerance, not only in the thymus but also in the periphery.
Transmembrane Sequences Are Determinants of Immunoreceptor Signaling
Authors: Gosse Julie A. , Wagenknecht-Wiesner Alice , Holowka David , Baird Barbara
Published: April 2014

To investigate structural features critical for signal initiation by Ag-stimulated immunoreceptors, we constructed a series of single-chain chimeric receptors that incorporate extracellular human FcεRIα for IgE binding, a variable transmembrane (TM) segment, and the ITAM-containing cytoplasmic tail of the TCR ζ-chain. We find that functional responses mediated by these receptors are strongly dependent on their TM sequences, and these responses are highly correlated to cross-link-dependent association with detergent-resistant lipid rafts. For one chimera designated αFζ, mutation of a TM cysteine abolishes robust signaling and lipid raft association. In addition, TM disulfide-mediated oligomerization of another chimeric receptor, αζζ, enhances signaling. These results demonstrate an important role for TM segments in immunoreceptor signaling and a strong correspondence between strength of signaling and cross-link-dependent partitioning into ordered membrane domains.