bluuebunny/crossref_metadata_2025 huggingface dataset:
Cbl-b Is a Negative Regulator of Inflammatory Cytokines Produced by IgE-Activated Mast Cells
Authors: Gustin Sonja E. , Thien Christine B. F. , Langdon Wallace Y.
Published: April 2014

c-Cbl and Cbl-b E3 ubiquitin ligases are abundantly expressed in hemopoietic cells where they negatively regulate the activity and levels of many cell surface receptors and associated signaling molecules. By comparing bone marrow-derived mast cells from c-Cbl and Cbl-b-deficient mice it has recently been shown that Cbl-b is the dominant family member for negatively regulating signaling responses from high-affinity IgE receptors. In this study, we suggest that a possible reason for the greater enhancement of IgE receptor signaling in Cbl-b-deficient mice is the relatively higher levels of Cbl-b protein over c-Cbl in mast cells compared with other hemopoietic cells. We also directly compare mast cells from c-Cbl and Cbl-b-deficient mice and find that loss of Cbl-b, but not c-Cbl, increases cell growth, retards receptor internalization, and causes the sustained tyrosine phosphorylation of Syk and its substrates. However, loss of Cbl-b does not enhance the activation of ERK or Akt, nor does it promote a greater calcium response. Furthermore, loss of Cbl-b or c-Cbl does not increase levels of the Syk or Lyn protein tyrosine kinases. Most notable, however, is the extremely large increase in the production of proinflammatory cytokines TNF-α, IL-6, and MCP-1 by Cbl-b−/− mast cells compared with levels produced by c-Cbl−/− or wild-type cells. This marked induction, which appears to be restricted to these three cytokines, is dependent on IgE receptor activation and correlates with enhanced IκB kinase phosphorylation. Thus, Cbl-b functions as a potent negative regulator of cytokines that promote allergic and inflammatory reactions.
Dendritic Cells Cross-Dressed with Peptide MHC Class I Complexes Prime CD8+ T Cells
Authors: Dolan Brian P. , Gibbs Kenneth D. , Ostrand-Rosenberg Suzanne
Published: April 2014

The activation of naive CD8+ T cells has been attributed to two mechanisms: cross-priming and direct priming. Cross-priming and direct priming differ in the source of Ag and in the cell that presents the Ag to the responding CD8+ T cells. In cross-priming, exogenous Ag is acquired by professional APCs, such as dendritic cells (DC), which process the Ag into peptides that are subsequently presented. In direct priming, the APCs, which may or may not be DC, synthesize and process the Ag and present it themselves to CD8+ T cells. In this study, we demonstrate that naive CD8+ T cells are activated by a third mechanism, called cross-dressing. In cross-dressing, DC directly acquire MHC class I-peptide complexes from dead, but not live, donor cells by a cell contact-mediated mechanism, and present the intact complexes to naive CD8+ T cells. Such DC are cross-dressed because they are wearing peptide-MHC complexes generated by other cells. CD8+ T cells activated by cross-dressing are restricted to the MHC class I genotype of the donor cells and are specific for peptides generated by the donor cells. In vivo studies demonstrate that optimal priming of CD8+ T cells requires both cross-priming and cross-dressing. Thus, cross-dressing may be an important mechanism by which DC prime naive CD8+ T cells and may explain how CD8+ T cells are primed to Ags that are inefficiently cross-presented.
Enhanced IgA Class Switching in Marginal Zone and B1 B Cells Relative to Follicular/B2 B Cells
Authors: Kaminski Denise A. , Stavnezer Janet
Published: April 2014

Mouse splenic marginal zone (MZ) B cells and B1 B cells enriched in the peritoneal cavity respond preferentially to T cell-independent Ags compared with follicular (FO)/B2 B cells. Despite the differential responses of B cell subsets to various stimuli, and despite the need for multiple stimuli to induce IgA class switching, the relative contribution of B cell subpopulations to IgA production is unknown. By culturing purified B cell populations, we find that MZ and peritoneal B1 cells switch more readily to IgA than do splenic FO or peritoneal B2 cells in BLyS/LPS/TGF-β. Addition of IL-4, IL-5, and anti-IgD dextran to the cultures enhances IgA switching in FO/B2 and MZ B cells to a similar frequency, but this treatment suppresses IgA class switching in B1 cells. Thus, IgA switching differs among purified B cell subsets, suggesting that individual B cell populations could contribute differentially to IgA expression in vivo, depending on available stimuli.
IL-10 Signaling Is Essential for 1,25-Dihydroxyvitamin D3-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis
Authors: Spach Karen M. , Nashold Faye E. , Dittel Bonnie N. , Hayes Colleen E.
Published: April 2014

Multiple sclerosis (MS) results from an aberrant, neuroantigen-specific, T cell-mediated autoimmune response. Because MS prevalence and severity decrease sharply with increasing sunlight exposure, and sunlight supports vitamin D3 synthesis, we proposed that vitamin D3 and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) may protect against MS. In support of this hypothesis, 1,25-(OH)2D3 strongly inhibited experimental autoimmune encephalomyelitis (EAE). This inhibition required lymphocytes other than the encephalitogenic T cells. In this study, we tested the hypothesis that 1,25-(OH)2D3 might inhibit EAE through the action of IL-10-producing regulatory lymphocytes. We report that vitamin D3 and 1,25-(OH)2D3 strongly inhibited myelin oligodendrocyte peptide (MOG35–55)-induced EAE in C57BL/6 mice, but completely failed to inhibit EAE in mice with a disrupted IL-10 or IL-10R gene. Thus, a functional IL-10-IL-10R pathway was essential for 1,25-(OH)2D3 to inhibit EAE. The 1,25-(OH)2D3 also failed to inhibit EAE in reciprocal, mixed bone marrow chimeras constructed by transferring IL-10-deficient bone marrow into irradiated wild-type mice and vice versa. Thus, 1,25-(OH)2D3 may be enhancing an anti-inflammatory loop involving hemopoietic cell-produced IL-10 acting on brain parenchymal cells and vice versa. If this interpretation is correct, and humans have a similar bidirectional IL-10-dependent loop, then an IL-10-IL-10R pathway defect could abrogate the anti-inflammatory and neuro-protective functions of sunlight and vitamin D3. In this way, a genetic IL-10-IL-10R pathway defect could interact with an environmental risk factor, vitamin D3 insufficiency, to increase MS risk and severity.
CD28 Ligation Costimulates Cell Death but Not Maturation of Double-Positive Thymocytes due to Defective ERK MAPK Signaling
Authors: Graham Daniel B. , Bell Michael P. , Huntoon Catherine J. , Griffin Matthew D. , Tai Xuguang , Singer Alfred , McKean David J.
Published: April 2014

The differentiation of double-positive (DP) CD4+CD8+ thymocytes to single-positive CD4+ or CD8+ T cells is regulated by signals that are initiated by coengagement of the Ag (TCR) and costimulatory receptors. CD28 costimulatory receptors, which augment differentiation and antiapoptotic responses in mature T lymphocytes, have been reported to stimulate both differentiation and apoptotic responses in TCR-activated DP thymocytes. We have used artificial APCs that express ligands for TCR and CD28 to show that CD28 signals increase expression of CD69, Bim, and cell death in TCR-activated DP thymocytes but do not costimulate DP thymocytes to initiate the differentiation program. The lack of a differentiation response is not due to defects in CD28-initiated TCR proximal signaling events but by a selective defect in the activation of ERK MAPK. To characterize signals needed to initiate the death response, a mutational analysis was performed on the CD28 cytoplasmic domain. Although mutation of all of CD28 cytoplasmic domain signaling motifs blocks cell death, the presence of any single motif is able to signal a death response. Thus, there is functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. In contrast, immobilized Abs can initiate differentiation responses and cell death in DP thymocytes. However, because Ab-mediated differentiation occurs through CD28 receptors with no cytoplasmic domain, the response may be mediated by increased adhesion to immobilized anti-TCR Abs.
Plasmacytoid Dendritic Cells Do Not Migrate in Intestinal or Hepatic Lymph
Authors: Yrlid Ulf , Cerovic Vuk , Milling Simon , Jenkins Christopher D. , Zhang Jiquan , Crocker Paul R. , Klavinskis Linda S. , MacPherson G. Gordon
Published: April 2014

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRαβ or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.
Complex Interplay of Activating and Inhibitory Signals Received by Vγ9Vδ2 T Cells Revealed by Target Cell β2-Microglobulin Knockdown
Authors: Trichet Valérie , Benezech Cécile , Dousset Christelle , Gesnel Marie-Claude , Bonneville Marc , Breathnach Richard
Published: April 2014

Tumor cells often escape immunosurveillance by down-regulating MHC class I molecule expression. For human Vγ9Vδ2 T cells, a major peripheral blood T cell subset with broad antitumor reactivity, this down-regulation can affect signals transmitted by both the inhibitory and the activating MHC class I and Ib-specific NK receptors (NKRs) that these lymphocytes frequently express. To assess the overall impact of MHC down-regulation on Vγ9Vδ2 T cell activation, we used stable β2-microglobulin knockdown to generate tumor cells with a ∼10-fold down-modulation of all MHC class I molecules. This down-modulation had little effect on T cell proliferation or cytokine production, but modified tumor cell killing efficiency. Ab-blocking studies identified ILT2 as an important inhibitor of tumor cell killing by Vγ9Vδ2 T cells. Down-modulation of MHC class I and Ib molecules severely reduced ILT2 inhibitory signaling, but still allowed signaling by activating CD94-based receptors. It also unveiled a frequent enhancing effect of NKG2D on tumor killing by Vγ9Vδ2 T cells. Current models suggest that activating NKRs have less affinity for their MHC ligands than homologous inhibitory NKRs. Our results show that, despite this, activating NKRs recognizing MHC class I molecules play an important role in the increased killing by Vγ9Vδ2 T cells of tumor cells with down-regulated MHC class I molecule expression, and suggest that these T cells will best lyse tumor cells combining MHC class I molecule expression down-regulation with up-regulated NKG2D ligand expression.
Epigenetic Histone Modifications Do Not Control Igκ Locus Contraction and Intranuclear Localization in Cells with Dual B Cell-Macrophage Potential
Authors: Hodawadekar Suchita , Wei Fang , Yu Duonan , Thomas-Tikhonenko Andrei , Atchison Michael L.
Published: April 2014

Somatic rearrangement of the Ig genes during B cell development is believed to be controlled, at least in part, by accessibility of the loci to the recombinational machinery. Accessibility is poorly understood, but appears to be controlled by a combination of histone posttranslational modifications, large scale Ig locus contractions, and changes in intranuclear localization of the loci. These changes are regulated by developmental stage-specific as well as tissue-specific mechanisms. We previously isolated a murine B cell lymphoma line, Myc5, that can oscillate between the B cell and macrophage lineages depending upon growth conditions. This line provides an opportunity to study tissue-specific regulation of epigenetic mechanisms operating on the Ig loci. We found that when Myc5 cells are induced to differentiate from B cells into macrophages, expression of macrophage-specific transcripts was induced (M-CSFR, F4/80, and CD14), whereas B cell-specific transcripts decreased dramatically (mb-1, E47, IRF4, Pax5, and Igκ). Loss of Igκ transcription was associated with reduced Igκ locus contraction, as well as increased association with heterochromatin protein-1 and association of the Igκ locus with the nuclear periphery. Surprisingly, however, we found that histone modifications at the Igκ locus remained largely unchanged whether the cells were grown in vivo as B cells, or in vitro as macrophages. These results mechanistically uncouple histone modifications at the Igκ locus from changes in locus contraction and intranuclear localization.
CD1 Antigen Presentation by Human Dendritic Cells as a Target for Herpes Simplex Virus Immune Evasion
Authors: Raftery Martin J. , Winau Florian , Kaufmann Stefan H. E. , Schaible Ulrich E. , Schönrich Günther
Published: April 2014

In contrast to MHC molecules, which present peptides, the CD1 molecules have been discovered to present lipid Ags to T cells. CD1-restricted T lymphocytes have been recently associated with resistance to virus infection. The mechanisms underlying activation of CD1-restricted T cells in the course of virus infection are not defined. In this study, we wanted to investigate the interaction of HSV with the antiviral CD1 Ag presentation system in human dendritic cells (DC). In response to low titers of HSV, the surface expression of CD1b and CD1d on human DC was up-regulated. These phenotypic changes enhanced the capacity of infected DC to stimulate proliferation of CD1-restricted T lymphocytes. High titers of HSV, however, lead to strong down-regulation of all surface CD1 molecules. This modulation of surface expression was associated with intracellular accumulation, colocalization with viral proteins, and disruption of the CD1 recycling machinery. Finally, even at low titers HSV interfered with the capacity of infected DC to stimulate the release of important cytokines by CD1d-restricted NKT cells. Thus, we demonstrate both the existence of a CD1 pathway allowing human DC to react to viral infection, as well as its blockage by a human herpesvirus.
Polymeric Immunoglobulin Receptor in Intestinal Immune Defense against the Lumen-Dwelling Protozoan ParasiteGiardia
Authors: Davids Barbara J. , Palm J. E. Daniel , Housley Michael P. , Smith Jennifer R. , Andersen Yolanda S. , Martin Martin G. , Hendrickson Barbara A. , Johansen Finn-Eirik , Svärd Staffan G. , Gillin Frances D. , Eckmann Lars
Published: April 2014

The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, α-enolase, and α- and β-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.
TLR3 Deletion Limits Mortality and Disease Severity due to Phlebovirus Infection
Authors: Gowen Brian B. , Hoopes Justin D. , Wong Min-Hui , Jung Kie-Hoon , Isakson Kevin C. , Alexopoulou Lena , Flavell Richard A. , Sidwell Robert W.
Published: April 2014

TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3−/− mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3−/− mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-γ, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3−/− mice.
Cooperative Activation of TLR2 and Bradykinin B2 Receptor Is Required for Induction of Type 1 Immunity in a Mouse Model of Subcutaneous Infection by Trypanosoma cruzi
Authors: Monteiro Ana Carolina , Schmitz Verônica , Svensjo Erik , Gazzinelli Ricardo T. , Almeida Igor C. , Todorov Alex , de Arruda Luciana B. , Torrecilhas Ana Cláudia T. , Pesquero João B. , Morrot Alexandre , Bouskela Eliete , Bonomo Adriana , Lima Ana Paula C. A. , Müller-Esterl Werner , Scharfstein Julio
Published: April 2014

We have previously reported that exogenous bradykinin activates immature dendritic cells (DCs) via the bradykinin B2 receptor (B2R), thereby stimulating adaptive immunity. In this study, we show that these premises are met in a model of s.c. infection by Trypanosoma cruzi, a protozoan that liberates kinins from kininogens through its major protease, cruzipain. Intensity of B2R-dependent paw edema evoked by trypomastigotes correlated with levels of IL-12 produced by CD11c+ dendritic cells isolated from draining lymph nodes. The IL-12 response induced by endogenously released kinins was vigorously increased in infected mice pretreated with inhibitors of angiotensin converting enzyme (ACE), a kinin-degrading metallopeptidase. Furthermore, these innate stimulatory effects were linked to B2R-dependent up-regulation of IFN-γ production by Ag-specific T cells. Strikingly, the trypomastigotes failed to up-regulate type 1 immunity in TLR2−/− mice, irrespective of ACE inhibitor treatment. Analysis of the dynamics of inflammation revealed that TLR2 triggering by glycosylphosphatidylinositol-anchored mucins induces plasma extravasation, thereby favoring peripheral accumulation of kininogens in sites of infection. Further downstream, the parasites generate high levels of innate kinin signals in peripheral tissues through the activity of cruzipain. The demonstration that the deficient type 1 immune responses of TLR2−/− mice are rescued upon s.c. injection of exogenous kininogens, along with trypomastigotes, supports the notion that generation of kinin “danger” signals is intensified through cooperative activation of TLR2 and B2R. In summary, we have described a s.c. infection model where type 1 immunity is vigorously up-regulated by bradykinin, an innate signal whose levels in peripheral tissues are controlled by an intricate interplay of TLR2, B2R, and ACE.
Disruption of JNK2 Decreases the Cytokine Response toPlasmodium falciparumGlycosylphosphatidylinositol In Vitro and Confers Protection in a Cerebral Malaria Model
Authors: Lu Ziyue , Serghides Lena , Patel Samir N. , Degousee Norbert , Rubin Barry B. , Krishnegowda Gowdahali , Gowda D. Channe , Karin Michael , Kain Kevin C.
Published: April 2014

Host inflammatory responses to Plasmodium falciparum GPI (pfGPI) anchors are believed to play an important role in the pathophysiology of severe malaria. However, relatively little is known about the signal transduction pathways involved in pfGPI-stimulated inflammatory response and its potential contribution to severe malaria syndromes. In this study, we investigated the role of MAPK activation in pfGPI-induced cytokine secretion and examined the role of selected MAPKs in a model of cerebral malaria in vivo. We demonstrate that ERK1/2, JNK, p38, c-Jun, and activating transcription factor-2 became phosphorylated in pfGPI-stimulated macrophages. A JNK inhibitor (1,9-pyrazoloanthrone) inhibited pfGPI-induced phosphorylation of JNK, c-Jun, and activating transcription factor-2 and significantly decreased pfGPI-induced TNF-α secretion. pfGPI-stimulated JNK and c-Jun phosphorylation was absent in Jnk2−/− macrophages but unchanged in Jnk1−/− and Jnk3−/− macrophages compared with wild-type macrophages. Jnk2−/− macrophages secreted significantly less TNF-α in response to pfGPI than macrophages from Jnk1−/−, Jnk3−/−, and wild-type counterparts. Furthermore, we demonstrate a role for JNK2 in mediating inflammatory responses and severe malaria in vivo. In contrast to wild-type or Jnk1−/− mice, Jnk2−/− mice had lower levels of TNF-α in vivo and exhibited significantly higher survival rates when challenged with Plasmodium berghei ANKA. These results provide direct evidence that pfGPI induces TNF-α secretion through activation of MAPK pathways, including JNK2. These results suggest that JNK2 is a potential target for therapeutic interventions in severe malaria.
VCAM-1 Signals Activate Endothelial Cell Protein Kinase Cα via Oxidation
Authors: Abdala-Valencia Hiam , Cook-Mills Joan M.
Published: April 2014

Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 μM H2O2. This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase Cα (PKCα) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm2 laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKCα or the PKCα inhibitors, Rö-32-0432 or Gö-6976. Phosphorylation of PKCαThr638, an autophosphorylation site indicating enzyme activity, was increased by Ab cross-linking of VCAM-1 on endothelial cells or by the exogenous addition of 1 μM H2O2. The anti-VCAM-1-stimulated phosphorylation of PKCαThr638 was blocked by scavenging of H2O2 and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKCα. Oxidized PKCα is a transiently active form of PKCα that is diacylglycerol independent. This oxidation was blocked by inhibition of NADPH oxidase. In summary, VCAM-1 activation of endothelial cell NADPH oxidase induces transient PKCα activation that is necessary for VCAM-1-dependent transendothelial cell migration.
Characterization of the Divergent Wound-Healing Responses Occurring in the Pathergy Reaction and Normal Healthy Volunteers
Authors: Melikoglu Melike , Uysal Serpil , Krueger James G. , Kaplan Gilla , Gogus Feride , Yazici Hasan , Oliver Stephen
Published: April 2014

Behçet’s disease (BD) is a multisystem inflammatory disorder of unknown etiology characterized by recurrent oral and genital ulcerations and uveitis, with varying other manifestations associated with vascular inflammation. A unifying feature of BD inflammation is the skin pathergy reaction (SPR), a nonspecific tissue hyperreactivity to minor trauma involving epithelial disruption. This study compared skin responses to needle prick in BD patients and normal healthy volunteers. Two study groups, each consisting of 10 BD patients with SPR+ and 6 controls, were evaluated using either immunohistochemistry or quantitative real-time PCR to measure inflammatory cell and cytokine levels in biopsy specimens obtained serially from independent sites at 0, 8, and 48 h after needle prick. We found similar cellular infiltration patterns in response to needle prick in BD patients and controls between 0 and 8 h. Further development of this immune response was limited in skin of normal control subjects, with stable or decreased inflammatory mediators observed at 48 h. In contrast, in BD-derived skin specimens, increased influxes of mature dendritic cells, monocytes, and lymphocytes, including T regulatory cells, were noted by 48 h. Similarly, increases in cytokines (IFN-γ, IL-12 p40, IL-15), chemokines (MIP3-α, IP-10, Mig, and iTac), and adhesion molecules (ICAM-1, VCAM-1) were noted at 48 h in the skin of BD patients with SPR+ but not in the skin of normal controls. These results suggest that, in contrast to the self-limited inflammation associated with epithelial disruption of normal skin, BD patients experience marked cellular influxes into the injury site, leading to an exaggerated lymphoid Th1-type response.
Involvement of TNF-Like Weak Inducer of Apoptosis in the Pathogenesis of Collagen-Induced Arthritis
Authors: Kamata Koichi , Kamijo Seiji , Nakajima Atsuo , Koyanagi Akemi , Kurosawa Hisashi , Yagita Hideo , Okumura Ko
Published: April 2014

TNF-like weak inducer of apoptosis (TWEAK) is a type II membrane protein belonging to the TNF family that regulates apoptotic cell death, cellular proliferation, angiogenesis, and inflammation. However, the role of TWEAK in the pathogenesis of rheumatoid arthritis (RA) remains unclear. In this study, we have investigated the effect of neutralizing anti-TWEAK mAb on the development of collagen-induced arthritis (CIA), a well-established murine model of RA. Administration of anti-TWEAK mAb significantly ameliorated paw swelling, synovial hyperplasia, and infiltration of inflammatory cells. The levels of proinflammatory chemokines such as MCP-1 and MIP-2 in serum and knee joints were reduced by this treatment. Consistently, recombinant TWEAK enhanced the proliferation of MCP-1 and MIP-2 production by synovial cells from CIA mice in vitro. Histological examination also revealed that the treatment with anti-TWEAK mAb suppressed the development of small vessels in synovial tissues. These results indicated anti-inflammatory and antiangiogenic effects of the TWEAK blockade in CIA, which may be also beneficial for the treatment of RA.
Decreased Alveolar Macrophage Apoptosis Is Associated with Increased Pulmonary Inflammation in a Murine Model of Pneumococcal Pneumonia
Authors: Marriott Helen M. , Hellewell Paul G. , Cross Simon S. , Ince Paul G. , Whyte Moira K. B. , Dockrell David H.
Published: April 2014

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS−/− mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-α. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS−/− mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS−/− mice and decreased TNF-α mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.
Poly(ADP-ribose) Polymerase-1 Inhibition Prevents Eosinophil Recruitment by Modulating Th2 Cytokines in a Murine Model of Allergic Airway Inflammation: A Potential Specific Effect on IL-5
Authors: Mustapha Oumouna , Datta Rahul , Oumouna-Benachour Karine , Suzuki Yasuhiro , Hans Chetan , Matthews Kametra , Fallon Kenneth , Boulares Hamid
Published: April 2014

We recently used a murine model of allergic airway inflammation to show that poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in the pathogenesis of asthma-related lung inflammation. In this study, we show that PARP-1 inhibition, by a novel inhibitor (TIQ-A) or by gene deletion, prevented eosinophilic infiltration into the airways of OVA-challenged mice. Such impairment of eosinophil recruitment appeared to take place after IgE production. OVA challenge of wild-type mice resulted in a significant increase in IL-4, IL-5, IL-10, IL-13, and GM-CSF secretions. Although IL-4 production was moderately affected in OVA-challenged PARP-1−/− mice, the production of IL-5, IL-10, IL-13, and GM-CSF was completely inhibited in ex vivo OVA-challenged lung cells derived from these animals. A single TIQ-A injection before OVA challenge in wild-type mice mimicked the latter effects. The marked effect PARP-1 inhibition exerted on mucus production corroborated the effects observed on the Th2 response. Although PARP-1 inhibition by gene knockout increased the production of the Th1 cytokines IL-2 and IL-12, the inhibition by TIQ-A exerted no effect on these two cytokines. The failure of lung cells derived from OVA-challenged PARP-1−/− mice to synthesize GM-CSF, a key cytokine in eosinophil recruitment, was reestablished by replenishment of IL-5. Furthermore, intranasal administration of IL-5 restored the impairment of eosinophil recruitment and mucus production in OVA-challenged PARP-1−/− mice. The replenishment of either IL-4 or IgE, however, did not result in such phenotype reversals. Altogether, these results suggest that PARP-1 plays a critical role in eosinophil recruitment by specifically regulating the cascade leading to IL-5 production.
Adoptive Transfer of Vaccine-Induced Peripheral Blood Mononuclear Cells to Patients with Metastatic Melanoma following Lymphodepletion
Authors: Powell Daniel J. , Dudley Mark E. , Hogan Katherine A. , Wunderlich John R. , Rosenberg Steven A.
Published: April 2014

Cancer vaccines can induce the in vivo generation of tumor Ag-specific T cells in patients with metastatic melanoma yet seldom elicit objective clinical responses. Alternatively, adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL) can mediate tumor regression in 50% of lymphodepleted patients, but are logistically and technically difficult to generate. In this study, we evaluated the capability of vaccine-induced PBMC to mediate tumor regression after transfer to patients receiving the same chemotherapy-induced lymphodepletion used for TIL transfer therapy. Autologous PBMC from nine gp100-vaccinated patients with metastatic melanoma were stimulated ex vivo with the gp100:209–217(210M) peptide and transferred in combination with high-dose IL-2 and cancer vaccine. Transferred PBMC contained highly avid, gp100:209–217 peptide-reactive CD8+ T cells. One week after transfer, lymphocyte counts peaked (median of 14.3 × 103 cells/μl; range of 0.9–59.7 × 103 cells/μl), with 56% of patients experiencing a lymphocytosis. gp100:209–217 peptide-specific CD8+ T cells persisted at high levels in the blood of all patients and demonstrated significant tumor-specific IFN-γ secretion in vitro. Melanocyte-directed autoimmunity was noted in two patients; however, no patient experienced an objective clinical response. These studies demonstrate the feasibility and safety of using vaccine-induced PBMC for cell transfer, but suggests that they are not as effective as TIL in adoptive immunotherapy even when transferred into lymphodepleted hosts.
Regulation of Reactive Oxygen Species by Atm Is Essential for Proper Response to DNA Double-Strand Breaks in Lymphocytes
Authors: Ito Keisuke , Takubo Keiyo , Arai Fumio , Satoh Hitoshi , Matsuoka Sahoko , Ohmura Masako , Naka Kazuhito , Azuma Masaki , Miyamoto Kana , Hosokawa Kentaro , Ikeda Yasuo , Mak Tak W. , Suda Toshio , Hirao Atsushi
Published: April 2014

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm−/− mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm−/− mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm−/− mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm−/− mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm−/− mice. Impairment of Ig class switch recombination seen in Atm−/− mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm−/− mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.
Activation of Type B T Cells after Protein Immunization Reveals Novel Pathways of In Vivo Presentation of Peptides
Authors: Lovitch Scott B. , Esparza Thomas J. , Schweitzer George , Herzog Jeremy , Unanue Emil R.
Published: April 2014

Type B T cells recognize a peptide-MHC conformer generated in recycling endosomes and eliminated by H2-DM in late endosomes; as a result, they recognize exogenous peptide, but fail to respond to the identical epitope generated from the native protein. To investigate the behavior of these cells in vivo, we generated mice transgenic for a type B TCR recognizing the 48-62 epitope of hen egg white lysozyme (HEL) presented by I-Ak. Type B T cells responded only to peptide ex vivo, but responded in vivo to immunization with either protein or peptide in the presence of Freund’s adjuvant or LPS. Presentation of the type B conformer was MyD88-independent, evident within 24 h after HEL immunization, and restricted to the CD11b/c+ APC subset. Immunization with listeriolysin O, a potent inducer of cell death, also primed type B T cells in vivo, and transfer of HEL-bearing allogeneic dendritic cells activated type B T cells. We conclude that a number of conditions in vivo, some of which induce inflammation and cell death, lead to peptide presentation through mechanisms distinct from the classical pathways involving H-2DM molecules.
1,25-Dihydroxyvitamin D3 Selectively Modulates Tolerogenic Properties in Myeloid but Not Plasmacytoid Dendritic Cells
Authors: Penna Giuseppe , Amuchastegui Susana , Giarratana Nadia , Daniel Kenn C. , Vulcano Marisa , Sozzani Silvano , Adorini Luciano
Published: April 2014

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is an immunomodulatory agent inducing dendritic cells (DCs) to become tolerogenic. To further understand its mechanisms of action, we have examined the effects of 1,25(OH)2D3 on tolerogenic properties of blood myeloid (M-DCs) and plasmacytoid (P-DCs) human DC subsets. Exposure of M-DCs to 1,25(OH)2D3 up-regulated production of CCL22, a chemokine attracting regulatory T cells, whereas production of CCL17, the other CCR4 ligand, was reduced. 1,25(OH)2D3 also decreased IL-12p75 production by M-DCs, as expected, and inhibited CCR7 expression. 1,25(OH)2D3 treatment markedly increased CD4+ suppressor T cell activity while decreasing the capacity of M-DCs to induce Th1 cell development. Surprisingly, 1,25(OH)2D3 did not exert any discernible effect on tolerogenic properties of P-DCs, and even their high production of IFN-α was not modulated. In particular, the intrinsically high capacity of P-DCs to induce CD4+ suppressor T cells was unaffected by 1,25(OH)2D3. Both DC subsets expressed similar levels of the vitamin D receptor, and its ligation by 1,25(OH)2D3 similarly activated the primary response gene cyp24. Interestingly, 1,25(OH)2D3 inhibited NF-κB p65 phosphorylation and nuclear translocation in M-DCs but not P-DCs, suggesting a mechanism for the inability of 1,25(OH)2D3 to modulate tolerogenic properties in P-DCs.
Essential Roles of c-Rel in TLR-Induced IL-23 p19 Gene Expression in Dendritic Cells
Authors: Carmody Ruaidhrí J. , Ruan Qingguo , Liou Hsiou-Chi , Chen Youhai H.
Published: April 2014

IL-23 plays crucial roles in both immunity against pathogens and autoimmunity against self. Although it is well recognized that IL-23 expression is restricted to the myeloid lineage and is tightly regulated at the transcriptional level, the nature of transcription factors required for IL-23 expression is poorly understood. We report, in this study, that murine dendritic cells deficient in c-Rel, a member of the NF-κB family, are severely compromised in their ability to transcribe the p19 gene, one of the two genes that encode the IL-23 protein. The p19 gene promoter contains three putative NF-κB binding sites, two of which can effectively bind c-Rel as determined by chromatin immunoprecipitation and EMSA. Unexpectedly, mutation of either of these two c-Rel binding sites completely abolished the p19 promoter activity induced by five TLRs (2, 3, 4, 6, and 9) and four members of the NF-κB family (c-Rel, p65, p100, and p105). Based on these observations, we conclude that c-Rel controls IL-23 p19 gene expression through two κB sites in the p19 promoter, and propose a c-Rel-dependent enhanceosome model for p19 gene activation.
Osteoprotegerin Reduces the Serum Level of Receptor Activator of NF-κB Ligand Derived from Osteoblasts
Authors: Nakamichi Yuko , Udagawa Nobuyuki , Kobayashi Yasuhiro , Nakamura Midrori , Yamamoto Yohei , Yamashita Teruhito , Mizoguchi Toshihide , Sato Masahiro , Mogi Makio , Penninger Josef M. , Takahashi Naoyuki
Published: April 2014

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-κB ligand (RANKL). We previously reported that OPG deficiency elevated the circulating level of RANKL in mice. Using OPG−/− mice, we investigated whether OPG is involved in the shedding of RANKL by cells expressing RANKL. Osteoblasts and activated T cells in culture released a large amount of RANKL in the absence of OPG. OPG or a soluble form of receptor activator of NF-κB (the receptor of RANKL) suppressed the release of RANKL from those cells. OPG- and T cell-double-deficient mice showed an elevated serum RANKL level equivalent to that of OPG−/− mice, indicating that circulating RANKL is mainly derived from bone. The serum level of RANKL in OPG−/− mice was increased by ovariectomy or administration of 1α,25-dihydroxyvitamin D3. Expression of RANKL mRNA in bone, but not thymus or spleen, was increased in wild-type and OPG−/− mice by 1α,25-dihydroxyvitamin D3. These results suggest that OPG suppresses the shedding of RANKL from osteoblasts and that the serum RANKL in OPG−/− mice exactly reflects the state of bone resorption.
Comparative Roles of IL-4, IL-13, and IL-4Rα in Dendritic Cell Maturation and CD4+ Th2 Cell Function
Authors: Webb Dianne C. , Cai Yeping , Matthaei Klaus I. , Foster Paul S.
Published: April 2014

IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. Although the function of these cytokines is partially linked through their shared use of IL-4Rα for signaling, the interplay between these cytokines in the development of memory Th2 responses is not well delineated. In this investigation, we show that both IL-4 and IL-13 influence the maturation of dendritic cells (DC) in the lung and their ability to regulate secretion of IFN-γ and Th2 cytokines by memory CD4+ T cells. Cocultures of wild-type T cells with pulmonary DC from allergic, cytokine-deficient mice demonstrated that IL-4 enhanced the capacity of DC to stimulate T cell secretion of Th2 cytokines, whereas IL-13 enhanced the capacity of DC to suppress T cell secretion of IFN-γ. Because IL-4Rα is critical for IL-4 and IL-13 signaling, we also determined how variants of IL-4Rα influenced immune cell function. T cells derived from allergic mice expressing a high-affinity IL-4Rα variant produced higher levels of IL-5 and IL-13 compared with T cells derived from allergic mice expressing a low-affinity IL-4Rα variant. Although DC expressing different IL-4Rα variants did not differ in their capacity to influence Th2 cytokine production, they varied in their capacity to inhibit IFN-γ production by T cells. Thus, IL-4 and IL-13 differentially regulate DC function and the way these cells regulate T cells. The affinity of IL-4Rα also appears to be a determinant in the balance between Th2 and IFN-γ responses and thus the severity of allergic disease.
Role of the Intracellular Domain of IL-7 Receptor in T Cell Development
Authors: Jiang Qiong , Huang Jiaqiang , Li Wen Qing , Cavinato Tiziana , Keller Jonathan R. , Durum Scott K.
Published: April 2014

Signals from the IL-7R are uniquely required for T cell development and maintenance, despite the resemblance of IL-7R to other cytokine receptors and the apparent sharing of common signaling pathways. This unique requirement could either reflect unique expression of IL-7R or IL-7, or it could indicate that the IL-7R delivers unique signals. To determine whether the IL-7R provided unique signals, we exchanged its intracellular domain with that of other cytokine receptors: IL-4R, IL-9R, and prolactin receptor (PRLR). Chimeric receptors were used to reconstitute development of IL-7R−/− hemopoietic progenitors by transducing the receptors in retroviral vectors. Whereas IL-7R−/− thymocytes are arrested at the double-negative stage, IL-4R, IL-9R, or PRLR all imparted some progression to the double-positive stage. IL-4R and PRLR gave only small numbers of thymocytes, whereas IL-9R gave robust αβ T cell development and reconstitution of peripheral CD4 and CD8 cells, indicating that it can duplicate many of the functions of IL-7R. However, IL-9R failed to reconstitute rearrangement of the TCRγ locus or development of γδ T cells. Thus, the IL-7R signals required in the αβ T cell lineage (such as survival and proliferation) are not unique to this receptor, whereas rearrangement of the TCRγ locus may require a signal that is not shared by other receptors.
Functional Polymorphism of the KIR3DL1/S1 Receptor on Human NK Cells
Authors: O’Connor Geraldine M. , Guinan Kieran J. , Cunningham Rodat T. , Middleton Derek , Parham Peter , Gardiner Clair M.
Published: April 2014

NK cells express both inhibitory and activatory receptors that allow them to recognize target cells through HLA class I Ag expression. KIR3DL1 is a receptor that recognizes the HLA-Bw4 public epitope of HLA-B alleles. We demonstrate that polymorphism within the KIR3DL1 receptor has functional consequences in terms of NK cell recognition of target. Inhibitory alleles of KIR3DL1 differ in their ability to recognize HLA-Bw4 ligand, and a consistent hierarchy of ligand reactivity can be defined. KIR3DS1, which segregates as an allele of KIR3DL1, has a short cytoplasmic tail characteristic of activatory receptors. Because it is very similar to KIR3DL1 in the extracellular domains, it has been assumed that KIR3DS1 will recognize a HLA-Bw4 ligand. In this study, we demonstrate that KIR3DS1 is expressed as a protein at the cell surface of NK cells, where it is recognized by the Z27 Ab. Using this Ab, we found that KIR3DS1 is expressed on a higher percentage of NK cells in KIR3DS1 homozygous compared with heterozygous donors. In contrast to the inhibitory KIR3DL1 allotypes, KIR3DS1 did not recognize HLA-Bw4 on EBV-transformed cell lines.
Cytokine-Dependent Blimp-1 Expression in Activated T Cells Inhibits IL-2 Production
Authors: Gong Dapeng , Malek Thomas R.
Published: April 2014

After Ag activation of naive T cells in vitro, extensive growth and differentiation into effector cells depend upon IL-2. DNA microarray analysis was used to identify IL-2-dependent molecules regulating this process. In this study, we show that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) is expressed by a cytokine-dependent pathway in activated T lymphocytes. IL-2 production by activated CD4+ and CD8+ T cells inversely correlated with Blimp-1 levels as higher IL-2 production was associated with lower Blimp-1 expression. Furthermore, ectopic expression of Blimp-1 by activated T cells inhibited IL-2 production but enhanced granzyme B and CD25 expression. Collectively, these findings indicate that there is a negative feedback regulatory loop in activated T cells such that IL-2 inhibits its own production through induction of Blimp-1 while promoting an effector cell phenotype.
Roundtrip Ticket for Secretory IgA: Role in Mucosal Homeostasis?
Authors: Corthésy Blaise
Published: April 2014

An important activity of mucosal surfaces is the production of Ab referred to as secretory IgA (SIgA). SIgA serves as the first line of defense against microorganisms through a mechanism called immune exclusion. In addition, SIgA adheres selectively to M cells in intestinal Peyer’s patches, thus mediating the transepithelial transport of the Ab molecule from the intestinal lumen to underlying gut-associated organized lymphoid tissue. In Peyer’s patches, SIgA binds and is internalized by dendritic cells in the subepithelial dome region. When used as carrier for Ags in oral immunization, SIgA induces mucosal and systemic responses associated with production of anti-inflammatory cytokines and limits activation of dendritic cells. In terms of humoral immunity at mucosal surfaces, SIgA appears thus to combine properties of a neutralizing agent (immune exclusion) and of a mucosal immunopotentiator inducing effector immune responses in a noninflammatory context favorable to preserve local homeostasis of the gastrointestinal tract.