bluuebunny/crossref_metadata_2025 huggingface dataset:
Cognate T Cell Help Is Sufficient to Trigger Anti-Nuclear Autoantibodies in Naive Mice
Authors: Keech Catherine L. , Farris A. Darise , Beroukas Dimitra , Gordon Tom P. , McCluskey James
Published: April 2014

The mechanisms involved in the initiation of anti-nuclear autoantibodies are unknown. In this study, we show that one factor allowing anti-nuclear autoantibodies to develop is the incomplete nature of immune tolerance to many of these proteins. Immune responses in mice toward the ubiquitous nuclear autoantigen La/SS-B are much weaker than responses to the xenoantigen, human La (hLa; 74% identical). However, in transgenic (Tg) mice expressing hLa, the Ab response to this neo-autoantigen was reduced to a level resembling the weak autoimmune response to mouse La. Partial tolerance to endogenous La autoantigen was restricted to the T compartment because transfer of CD4+ T cells specific for one or more hLa determinants into mice bearing the hLa transgene was sufficient to elicit production of anti-hLa autoantibodies. Notably, only hLa- specific T cells from non-Tg mice, and not T cells from hLa Tg mice, induced autoantibody production in hLa Tg mice. These findings confirm partial Th tolerance to endogenous La and indicate the existence in normal animals of autoreactive B cells continuously presenting La nuclear Ag. Therefore, the B cell compartment is constitutively set to respond to particular nuclear autoantigens, implicating limiting Th responses as a critical checkpoint in the development of anti-nuclear autoantibodies in normal individuals.
Anti-CD3ε F(ab′)2 Prevents Graft-Versus-Host Disease by Selectively Depleting Donor T Cells Activated by Recipient Alloantigens
Authors: Yu Xue-Zhong , Bidwell Sasha J. , Martin Paul J. , Anasetti Claudio
Published: April 2014

Transplantation tolerance is facilitated by activation-induced apoptosis of peripheral T cells triggered by specific Ag. Abs specific for the nonpolymorphic CD3 component of the TCR complex bind to APCs through Fc-FcR interactions, mimic MHC-peptide, and activate polyclonal T cells. In contrast, F(ab′)2 of anti-CD3ε Abs do not activate naive T cells but induce apoptosis of Ag-activated, cycling T cells. Here, we report that treatment with anti-CD3ε F(ab′)2 can selectively induce apoptosis of donor T cells that recognize a recipient alloantigen, thereby preventing graft-vs-host disease initiated by a TCR-transgenic T cell population. The selective elimination of Ag-activated T cells by non-FcR-binding anti-CD3ε Abs could serve as an ideal strategy to prevent graft-vs-host disease and allograft rejection or to treat autoimmune disorders.
Differential Regulation of Chemokine Production in Human Peritoneal Mesothelial Cells: IFN-γ Controls Neutrophil Migration Across the Mesothelium In Vitro and In Vivo
Authors: Robson Rachel L. , McLoughlin Rachel M. , Witowski Janusz , Loetscher Pius , Wilkinson Thomas S. , Jones Simon A. , Topley Nicholas
Published: April 2014

Leukocyte recruitment into the infected peritoneal cavity consists of an early, predominant polymorphonuclear leukocyte (PMN) influx and subsequent, prolonged mononuclear cell migration phase. Although chemokine secretion by resident peritoneal cells plays a primary role in mediating this migration, the mechanisms involved in controlling the switch in phenotype of cell infiltrate remain unclear. The present study investigates a potential role for the Th1-type cytokine IFN-γ in the process of leukocyte recruitment into the peritoneal cavity. Stimulation of cultured human peritoneal mesothelial cells with IFN-γ (1–100 U/ml) alone or in combination with IL-1β (100 pg/ml) or TNF-α (1000 pg/ml) resulted in significant up-regulation of monocyte chemoattractant protein-1 and RANTES protein secretion. In contrast, IFN-γ inhibited basal and IL-1β-, and TNF-α-induced production of IL-8. The modulating effects of IFN-γ on chemokine production occurred at the level of gene expression, and the degree of regulation observed was dependent on the doses of IL-1β and TNF-α used. Analysis of the functional effects of IFN-γ on IL-1β-induced transmesothelial PMN migration with an in vitro human transmigration system and an in vivo murine model of peritoneal inflammation demonstrated that IFN-γ was able to down-regulate PMN migration induced by optimal doses of IL-1β. These effects were mediated in vivo via down-regulation of CXC chemokine synthesis. These findings suggest that IFN-γ may play a role in controlling the phenotype of infiltrating leukocyte during the course of an inflammatory response, in part via regulation of resident cell chemokine synthesis.
Cutting Edge: IFN-Inducible ELR− CXC Chemokines Display Defensin-Like Antimicrobial Activity
Authors: Cole Alexander M. , Ganz Tomas , Liese Amy M. , Burdick Marie D. , Liu Lide , Strieter Robert M.
Published: April 2014

Recent reports highlighted the chemotactic activities of antimicrobial peptide defensins whose structure, charge, and size resemble chemokines. By assaying representative members of the four known families of chemokines we explored the obverse: whether some chemokines exert antimicrobial activity. In a radial diffusion assay, only recombinant monokine induced by IFN-γ (MIG/CXCL9), IFN-γ-inducible protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T cell α chemoattractant (I-TAC/CXCL11), members of the IFN-γ-inducible tripeptide motif Glu-Leu-Arg (ELR)− CXC chemokines, were antimicrobial against Escherichia coli and Listeria monocytogenes. Similar to human defensins, antimicrobial activities of the chemokines were inhibited by 50 and 100 mM NaCl. The concentration of MIG/CXCL9 and IP-10/CXCL10 released from IFN-γ-stimulated PBMC in 24 h were, respectively, 35- and 28-fold higher than from unstimulated cells. Additionally, the amounts of chemokines released per monocyte suggest that, in tissues with mononuclear cell infiltration, IFN-γ-inducible chemokines may reach concentrations necessary for microbicidal activity. IFN-γ-inducible chemokines may directly inactivate microbes before attracting other host defense cells to the area of infection.
Cutting Edge: Protein Kinase Cβ Expression Is Critical for Export of IL-2 from T Cells
Authors: Long Aideen , Kelleher Dermot , Lynch Sara , Volkov Yuri
Published: April 2014

Protein kinase C (PKC) plays an integral part in T cell activation and IL-2 secretion. We investigated the role of a particular PKC isoform, PKCβ, in IL-2 production and secretion. The T cell lymphoma line HuT 78 secretes IL-2 in response to the phorbol ester PMA. A PKCβ-deficient clone of HuT 78, K-4, did not secrete IL-2 in response to PMA stimulation. As assessed by RT-PCR, K-4 expressed mRNA for IL-2 following PMA activation, and intracellular IL-2 protein was detected by immunofluorescence. An enhanced green fluorescent protein-linked PKCβ construct was microinjected into K-4 cells, which were then stimulated with PMA; those cells that expressed PKCβ could secrete IL-2, as determined by an in situ immunofluorescent assay. This study demonstrates that PKCβ is not necessary for transcription of the IL-2 gene or translation of mRNA to protein, but that expression of this PKC isoform is critical to the export of IL-2 molecules from T cells.
Antigen Presentation by Liver Cells Controls Intrahepatic T Cell Trapping, Whereas Bone Marrow-Derived Cells Preferentially Promote Intrahepatic T Cell Apoptosis
Authors: Mehal Wajahat Z. , Azzaroli Francesco , Crispe I. Nicholas
Published: April 2014

Systemic activation and proliferation of CD8+ T cells result in T cell accumulation in the liver, associated with T cell apoptosis and liver injury. However, the role of Ag and APC in such accumulation is not clear. Bone marrow chimeras were constructed to allow Ag presentation in all tissues or alternatively to restrict presentation to either bone marrow-derived or non-bone marrow-derived cells. OVA-specific CD8+ T cells were introduced by adoptive transfer and then activated using peptide, which resulted in clonal expansion followed by deletion. Ag presentation by liver non-bone marrow-derived cells was responsible for most of the accumulation of activated CD8+ T cells. In contrast, Ag presentation by bone marrow-derived cells resulted in less accumulation of T cells in the liver, but a higher frequency of apoptotic cells within the intrahepatic T cell population. In unmodified TCR-transgenic mice, Ag-induced T cell deletion and intrahepatic accumulation of CD8+ T cells result in hepatocyte damage, with the release of aminotransaminases. Our experiments show that such liver injury may occur in the absence of Ag presentation by the hepatocytes themselves, arguing for an indirect mechanism of liver damage.
B Cell Receptor Cross-Linking Triggers a Caspase-8- Dependent Apoptotic Pathway That Is Independent of the Death Effector Domain of Fas-Associated Death Domain Protein
Authors: Besnault Laurence , Schrantz Nicolas , Auffredou Marie Thérèse , Leca Gérald , Bourgeade Marie Françoise , Vazquez Aimé
Published: April 2014

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt’s lymphoma cells. Whereas soluble anti-μ Ab triggers caspase-independent mitochondrial activation, cross-linked anti-μ Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt’s lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.
The Dendritic Cell Populations of Mouse Lymph Nodes
Authors: Henri Sandrine , Vremec David , Kamath Arun , Waithman Jason , Williams Stuart , Benoist Christophe , Burnham Kim , Saeland Sem , Handman Emanuela , Shortman Ken
Published: April 2014

The dendritic cells (DC) of mouse lymph nodes (LN) were isolated, analyzed for surface markers, and compared with those of spleen. Low to moderate staining of LN DC for CD4 and low staining for CD8 was shown to be attributable to pickup of these markers from T cells. Excluding this artifact, five LN DC subsets could be delineated. They included the three populations found in spleen (CD4+8−DEC-205−, CD4−8−DEC-205−, CD4−8+DEC-205+), although the CD4-expressing DC were of low incidence. LN DC included two additional populations, characterized by relatively low expression of CD8 but moderate or high expression of DEC-205. Both appeared among the DC migrating out of skin into LN, but only one was restricted to skin-draining LN and was identified as the mature form of epidermal Langerhans cells (LC). The putative LC-derived DC displayed the following properties: large size; high levels of class II MHC, which persisted to some extent even in CIITA null mice; expression of very high levels of DEC-205 and of CD40; expression of many myeloid surface markers; and no expression of CD4 and only low to moderate expression of CD8. The putative LC-derived DC among skin emigrants and in LN also showed strong intracellular staining of langerin.
Unexpected Characteristics of the IFN-γ Reporters in Nontransformed T Cells
Authors: Zhu Hong , Yang Jianfei , Murphy Theresa L. , Ouyang Wenjun , Wagner Fred , Saparov Arman , Weaver Casey T. , Murphy Kenneth M.
Published: April 2014

Analysis of the IFN-γ promoter has primarily been conducted by transient expression of reporter constructs in transformed cells. However, the activity of cis elements may differ when expressed transiently compared with their activity within native chromatin. Furthermore, the transcription factors and signaling mechanisms in transformed cells may differ from those in normal T cells. To analyze IFN-γ promoter regulation in normal T cells, we developed a novel retroviral bottom-strand reporter system to allow the chromatin integration of promoter regions in primary developing T cells. As controls, both the IL-2 and IL-4 promoters were inducible in this system, with the IL-4 reporter having Th2-specific activity. Strikingly, the IFN-γ promoter exhibited constitutive activity in both Th1 and Th2 subsets, in contrast to the behavior of the endogenous IFN-γ gene, which is inducible only in Th1 cells. In mapping this activity, we found that the AP-1/GM-CSF site in the distal promoter element is the most critical element for the constitutive activity. Transgenic reporter lines for the IFN-γ promoter confirmed the constitutive behavior of the isolated IFN-γ promoter. This constitutive activity was resistant to inhibition by cyclosporin A and was independent of Stat4 and p38 mitogen-activated protein kinase. These results suggest that IFN-γ promoter regulation may require cis elements residing either downstream or >3.4 kb upstream of the transcriptional start site, involving repression of constitutive activity.
Functional CD4 T Cells after Intercellular Molecular Transfer of OX40 Ligand
Authors: Baba Eishi , Takahashi Yoshiaki , Lichtenfeld Juliane , Tanaka Reiko , Yoshida Atsushi , Sugamura Kazuo , Yamamoto Naoki , Tanaka Yuetsu
Published: April 2014

OX40/OX40 ligand (OX40L) proteins play critical roles in the T cell-B cell and T cell-dendritic cell interactions. Here we describe the intercellular transfer of OX40L molecules by a non-Ag specific manner. After 2-h coculture of activated CD4+ T cell (OX40L−, OX40+) with FLAG peptide-tagged OX40L (OX40L-flag) protein-expressing COS-1 cells, the OX40L-flag protein was detected on the cell surface of the CD4+ T cells by both anti-OX40L and anti-FLAG mAbs. The intercellular OX40L transfer was specifically abrogated by pretreatment of the COS-1 cells with anti-OX40L mAb, 5A8. The OX40L transfer to OX40-negative cells was also observed, indicating an OX40-independent pathway of OX40L transfer. HUVECs, allostimulated monocytes, and human T cell leukemia virus type I-infected T cells, which all express OX40L, can potentially act as the donor cells of OX40L. The entire molecule of OX40L was transferred and stabilized on the recipient cell membrane with discrete punctate formation. The transferred OX40L on normal CD4+ T cells was functionally active as they stimulated latent HIV-1-infected cells to produce viral proteins via OX40 signaling. Therefore, these findings suggest that the intercellular molecular transfer of functional OX40L may be involved in modifying the immune responses.
Oxidative Responses of Human and Murine Macrophages During Phagocytosis ofLeishmania chagasi
Authors: Gantt Kira R. , Goldman Todd L. , McCormick Michael L. , Miller Melissa A. , Jeronimo Selma M. B. , Nascimento Eliana T. , Britigan Bradley E. , Wilson Mary E.
Published: April 2014

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2− during phagocytosis of opsonized promastigotes. Addition of the O2− scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2− enhances macrophage leishmanicidal activity. The importance of NO· produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO· in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor NG-monomethyl-l-arginine inhibited IFN-γ-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-γ. These data suggest that O2− and NO· each contribute to intracellular killing of L. chagasi in human and murine macrophages.
A Dominant Role for Fcγ Receptors in Antibody-Dependent Corneal Inflammation
Authors: Hall Laurie R. , Diaconu Eugenia , Pearlman Eric
Published: April 2014

Although production of specific Ab is a critical element of host defense, the presence of Ab in tissues leads to formation of immune complexes, which can trigger a type III Arthus reaction. Our studies on a mouse model of river blindness showed that Ab production is essential for recruitment of neutrophils and eosinophils to the cornea and for development of corneal opacification. In the current study, we determined the relative contribution of complement and FcγR interactions in triggering immune complex-mediated corneal disease. FcγR−/− mice, C3−/− mice, and immunocompetent control (B6/129Sj) mice were immunized s.c. and injected intrastromally with Onchocerca volvulus Ags. Slit lamp examination showed that control mice, C3−/− mice, and control mice injected with cobra venom factor developed pronounced corneal opacification, whereas corneas of FcγR−/− mice remained completely clear. Furthermore, recruitment of neutrophils and eosinophils to the corneal stroma was significantly impaired in FcγR−/− mice, but not in C3−/− mice or cobra venom factor-treated mice. We therefore conclude that FcγR-mediated cell activation, rather than complement activation, is the dominant pathway of immune complex disease in the cornea. These findings demonstrate a novel role for FcγR interactions in mediating ocular inflammation.
Regulation of Complement Classical Pathway by Association of C4b-Binding Protein to the Surfaces of SK-OV-3 and Caov-3 Ovarian Adenocarcinoma Cells
Authors: Holmberg Mikko T. , Blom Anna M. , Meri Seppo
Published: April 2014

The role of fluid-phase regulators of complement is to inhibit excessive complement activation and maintain homeostasis in blood. By binding to and inactivating complement components on cell surfaces, they can also protect autologous cells from complement-mediated cytotoxicity and phagocytosis. In this study, we wanted to find out whether C4b-binding protein (C4bp), a fluid-phase regulator of the classical complement pathway, could directly bind to cell surfaces in a functionally active form. After screening several malignant cell lines, we observed that the ovarian adenocarcinoma cell lines SK-OV-3, Caov-3, and SW626 were capable of binding C4bp. Binding tests with recombinant deletion mutants suggested that the primary binding site on C4bp is located on the α-chain complement control protein 4 domain. Functional tests showed that tumor cell-bound C4bp retained its cofactor activity for factor I-mediated inactivation of C4b, thus increasing the control of classical complement pathway activation on the surfaces of these cells. These results demonstrate a novel mechanism of complement regulation on cell surfaces, particularly on those of malignant ovarian tumor cells.
IL-12/IL-18-Dependent IFN-γ Release by Murine Dendritic Cells
Authors: Stober Detlef , Schirmbeck Reinhold , Reimann Jörg
Published: April 2014

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c+ myeloid DC from 7-day cultures stimulated with TNF-α, IFN-α, IFN-γ, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low “spontaneous” release of IL-18, and did not release IFN-γ. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-γ expression and release in 15–20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-γ release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8α+ and CD8α− DC (from immunocompetent and immunodeficient H-2d and H-2b mice) cultured with IL-12 and IL-18 released IFN-γ. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-γ release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-γ release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-γ generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.
Cutting Edge: The Mouse NK Cell-Associated Antigen Recognized by DX5 Moncoclonal Antibody is CD49b (α2 Integrin, Very Late Antigen-2)
Authors: Arase Hisashi , Saito Takashi , Phillips Joseph H. , Lanier Lewis L.
Published: April 2014

DX5 mAb is a useful reagent because it stains NK cells from all mouse strains examined. We have identified the molecule recognized by DX5 mAb by using a retrovirus-mediated expression cloning system. A 5-kb cDNA encoding a protein that is reactive with the DX5 mAb was isolated from a NK cell cDNA library, and this molecule was identical with CD49b (very late Ag-2, α2 integrin). The DX5 mAb reacted with transfectants expressing CD49b, and binding of DX5 to the NK cells and CD49b transfectants was blocked in the presence of other anti-CD49b mAbs. When NK1.1+ NK cells were cultured with IL-2, they progressively lost reactivity with DX5 mAb as a consequence of cellular proliferation. Cytotoxicity mediated by the DX5+ NK cells was dramatically higher as compared with DX5− NK cells. Therefore, DX5 mAb recognizes CD49b and can be used to define functionally distinct subsets of NK cells.
CD44 Is the Physiological Trigger of Fas Up-Regulation on Rheumatoid Synovial Cells
Authors: Fujii Koichi , Fujii Yuko , Hubscher Stefan , Tanaka Yoshiya
Published: April 2014

CD44 is a ubiquitous molecule known as a hyaluronan receptor. However, the relevance of CD44 to inflammatory processes, for example, rheumatoid synovitis, remains unclear. In this study, we propose a novel function for CD44 using synovial cells from rheumatoid arthritis (RA) patients and demonstrated that CD44 cross-linking augmented Fas expression and subsequent Fas-mediated apoptosis of the cells: 1) cross-linking of CD44 on RA synovial cells markedly augmented Fas expression and its mRNA transcription; 2) engagement of CD44 up-regulated Fas on the cells within 3 h, much more than IL-1β and TNF-α did; 3) the Fas-mediated early apoptotic change of the cells was amplified by CD44 cross-linking; and 4) hyaluronan, especially when fragmented, also augmented Fas-mediated early apoptosis of the cells. Based on these findings, we postulate a new concept: that interaction of CD44 on RA synovial cells with hyaluronan fragments present in the surrounding extracellular matrix augments Fas expression as well as Fas-mediated apoptosis of synovial cells. This may lead to spontaneous growth arrest through Fas-Fas ligand pathway observed in synovial cells of RA synovitis in vivo.
Cross-Linking of EphB6 Resulting in Signal Transduction and Apoptosis in Jurkat Cells
Authors: Luo Hongyu , Wan Xiaochun , Wu Yulian , Wu Jiangping
Published: April 2014

Eph kinases are the largest family of receptor tyrosine kinases (RTK), and their ligands are cell surface molecules. The known functions of Eph kinases are mainly pattern formation in the CNS. Although several Eph kinases are expressed at high levels in hemopoietic cells and in the thymus, we have no knowledge of the functions of any Eph kinase in the immune system. In this study, we have demonstrated that an Eph kinase, EphB6, was expressed at high levels in Jurkat leukemic T cells. Co-cross-linking of EphB6 and CD3 led to an altered profile of lymphokine secretion along with proliferation inhibition of Jurkat cells. The cells subsequently underwent Fas-mediated apoptosis. Although EphB6 has no intrinsic kinase activity, its cross-linking triggered general protein tyrosine phosphorylation in Jurkat cells. EphB6 was found to associate with a number of molecules in the signaling pathways, notably Cbl. EphB6 cross-linking resulted in Cbl dephosphorylation and dissociation from Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1). Our results show that EphB6 has important functions in T cells, and it can transduce signals into the cells via proteins it associates with.
Development and Maintenance of a B220− Memory B Cell Compartment
Authors: Driver David J. , McHeyzer-Williams Louise J. , Cool Melinda , Stetson Daniel B. , McHeyzer-Williams Michael G.
Published: April 2014

We have recently demonstrated that a novel somatically mutated B220− memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220+NP+ memory B cells produce large numbers of B220−NP+ B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220− memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220−NP+ B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220+ counterparts, the B220− B cells initially appear unmutated at days 5–7; however, the majority rapidly accumulate affinity increasing mutations by days 9–14 of the primary immune response. More extensive cell surface phenotype (GL7−BLA-1−CD24−CD43+) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220−CD11b+NP+ B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220− memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.
IL-12 Induction by a Th1-Inducing Adjuvant In Vivo: Dendritic Cell Subsets and Regulation by IL-10
Authors: Huang Li-Yun , Reis e Sousa Caetano , Itoh Yasushi , Inman John , Scott Dorothy E.
Published: April 2014

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8+ DC preferentially induce Th1 responses, whereas CD8− DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8− than CD8+ DC produced IL-12 after HKBA challenge, whereas only CD8+ DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11clow cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8+ and CD8− DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.
Both TCRα and TCRδ Chain Diversity Are Regulated During Thymic Ontogeny
Authors: Gallagher Maighréad , Obeïd Patricia , Marche Patrice N. , Jouvin-Marche Evelyne
Published: April 2014

TCRα and TCRδ chains are coded by a common genetic locus using a single set of V gene segments (ADV segments). This article addresses the question of regulation of the use of the ADV segments by the TCRα and TCRδ chains. Using both qualitative and quantitative analyses we have studied the use of 23 ADV gene families as part of TCRα and TCRδ transcripts. A number of previously undetected rearrangement and transcription events are described, indicating that the intrathymic TCRδ repertoire is much more diverse than previously supposed. Repertoire analysis at several developmental time points allowed the description of regulated waves of ADV gene use, not only for TCRδ chains, but also for TCRα chains, during thymic ontogeny. Control of these waves appears to be linked directly to the ADV segments and their local chromatin environment, which may change over the course of T cell differentiation.
Functional Roles of TAP and Tapasin in the Assembly of M3-N-Formylated Peptide Complexes
Authors: Chun Taehoon , Grandea Andreas G. , Lybarger Lonnie , Forman James , Van Kaer Luc , Wang Chyung-Ru
Published: April 2014

H2-M3 is a MHC class Ib molecule with a high propensity to bind N-formylated peptides. Due to the paucity of endogenous Ag, the majority of M3 is retained in the endoplasmic reticulum (ER). Upon addition of exogenous N-formylated peptides, M3 trafficks rapidly to the cell surface. To understand the mechanism underlying Ag presentation by M3, we examined the role of molecular chaperones in M3 assembly, particularly TAP and tapasin. M3-specific CTLs fail to recognize cells isolated from both TAP-deficient (TAPo) and tapasin-deficient mice, suggesting that TAP and tapasin are required for M3-restricted Ag presentation. Impaired M3 expression in TAPo mice is due to instability of the intracellular pool of M3. Addition of N-formylated peptides to TAPo cells stabilizes M3 in the ER and partially restores surface expression. Surprisingly, significant amounts of M3 are retained in the ER in tapasin-deficient mice, even in the presence of N-formylated peptides. Our results define the role of TAP and tapasin in the assembly of M3-peptide complexes. TAP is essential for stabilization of M3 in the ER, whereas tapasin is critical for loading of N-formylated peptides onto the intracellular pool of M3. However, neither TAP nor tapasin is required for ER retention of empty M3.
Differential Regulation of Mouse Germline Ig γ1 and ε Promoters by IL-4 and CD40
Authors: Mao Chun-sheng , Stavnezer Janet
Published: April 2014

Before Ig class switching, RNA transcription through the specific S regions undergoing recombination is induced by cytokines and other activators that induce and direct switching. The resulting germline (GL) transcripts are essential for switch recombination. To understand the differential regulation of mouse IgG1 and IgE, we compared the promoters for GL γ1 and ε transcripts. We addressed the question of why the promoter that regulates GL ε transcription is more responsive to IL-4 than the γ1 promoter and also why GL ε transcription is more dependent on IL-4 than is γ1 transcription. We found that the IL-4-responsive region of the GL ε promoter is more inducible than that of the γ1 promoter, although each promoter contains a binding site for the IL-4-inducible transcription factor Stat6, located immediately adjacent to a binding site for a basic region leucine zipper (bZip) family protein. However, the arrangement and sequences of the sites differ between the ε and γ1 promoters. The GL ε promoter binds Stat6 with a 10-fold higher affinity than does the γ1 promoter. Furthermore, the bZip elements of the two promoters bind different transcription factors, as the GL ε promoter binds and is activated by AP-1, whereas the γ1 promoter binds and is activated by activating transcription factor 2. C/EBPβ and C/EBPγ also bind the γ1 bZip element, although they inhibit rather than activate transcription. However, inhibition of promoter activity by C/EBPβ does not require the bZip element and may instead occur via inhibiting the activity of NF-κB.
Targeted Mutation of TNF Receptor I Rescues the RelA-Deficient Mouse and Reveals a Critical Role for NF-κB in Leukocyte Recruitment
Authors: Alcamo Elizabeth , Mizgerd Joseph P. , Horwitz Bruce H. , Bronson Rod , Beg Amer A. , Scott Martin , Doerschuk Claire M. , Hynes Richard O. , Baltimore David
Published: April 2014

NF-κB binding sites are present in the promoter regions of many acute phase and inflammatory response genes, suggesting that NF-κB plays an important role in the initiation of innate immune responses. However, targeted mutations of the various NF-κB family members have yet to identify members responsible for this critical role. RelA-deficient mice die on embryonic day 15 from TNF-α-induced liver degeneration. To investigate the importance of RelA in innate immunity, we genetically suppressed this embryonic lethality by breeding the RelA deficiency onto a TNFR type 1 (TNFR1)-deficient background. TNFR1/RelA-deficient mice were born healthy, but were susceptible to bacterial infections and bacteremia and died within a few weeks after birth. Hemopoiesis was intact in TNFR1/RelA-deficient newborns, but neutrophil emigration to alveoli during LPS-induced pneumonia was severely reduced relative to that in wild-type or TNFR1-deficient mice. In contrast, radiation chimeras reconstituted with RelA or TNFR1/RelA-deficient hemopoietic cells were healthy and demonstrated no defect in neutrophil emigration during LPS-induced pneumonia. Analysis of RNA harvested from the lungs of mice 4 h after LPS insufflation revealed that the induction of several genes important for neutrophil recruitment to the lung was significantly reduced in TNFR1/RelA-deficient mice relative to that in wild-type or TNFR1-deficient mice. These results suggest that TNFR1-independent activation of RelA is essential in cells of nonhemopoietic origin during the initiation of an innate immune response.
Elevated Levels of Cyclooxygenase-2 in Antigen-Stimulated Mast Cells Is Associated with Minimal Activation of p38 Mitogen-Activated Protein Kinase
Authors: Hundley Thomas R. , Prasad Anjana R. , Beaven Michael A.
Published: April 2014

We have investigated possible factors that underlie changes in the production of eicosanoids after prolonged exposure of mast cells to Ag. Ag stimulation of cultured RBL-2H3 mast cells resulted in increased expression of cyclooxygenase (COX-2) protein and message. Other eicosanoid-related enzymes, namely COX-1, 5-lipoxygenase, and cytosolic phospholipase A2 were not induced. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein (MAP) kinase preceded the induction of COX-2, whereas phosphatidylinositol 3′ kinase and its substrate, Akt, were constitutively activated in RBL-2H3 cells. Studies with pharmacologic inhibitors indicated that of these kinases, only p38 MAP kinase regulated expression of COX-2. The induction of COX-2 was blocked by the p38 MAP kinase inhibitor SB202190, even when added 12–16 h after stimulation with Ag when p38 MAP kinase activity had returned to near basal, but still minimally elevated, levels. Interestingly, expression of COX-2 as well as cytosolic phospholipase A2 and 5-lipoxygenase were markedly reduced by SB202190 in unstimulated cells. Collectively, the results imply that p38 MAP kinase regulates expression of eicosanoid-related enzymes, passively or actively, at very low levels of activity in RBL-2H3 cells. Also, comparison with published data suggest that different MAP kinases regulate induction of COX-2 in inflammatory cells of different and even similar phenotype and suggest caution in extrapolating results from one type of cell to another.
RANTES-Induced Chemokine Cascade in Dendritic Cells
Authors: Fischer Falko R. , Luo Yi , Luo Moli , Santambrogio Laura , Dorf Martin E.
Published: April 2014

Dendritic cells (DC) are the most potent APCs and the principal activators of naive T cells. We now report that chemokines can serve as activating agents for immature DC. Murine bone marrow-derived DC respond to the CC chemokine RANTES (10–100 ng/ml) by production of proinflammatory mediators. RANTES induces rapid expression of transcripts for the CXC chemokines KC and macrophage inflammatory protein (MIP)-2, the CC chemokines MIP-1β and MIP-1α, and the cytokines TNF-α and IL-6. Synthesis of KC, IL-6, and TNF-α proteins were also demonstrated. After 4 h, autoinduction of RANTES transcripts was observed. These responses are chemokine specific. Although DC demonstrated weak responses to eotaxin, DC failed to respond to other chemokines including KC, MIP-2, stromal-derived factor-1α, MIP-1β, MIP-1α, monocyte chemoattractant protein-1, T cell activation gene 3, or thymus-derived chemotactic agent 4. In addition, RANTES treatment up-regulated expression of an orphan chemokine receptor termed Eo1. Chemokine induction was also observed after treatment of splenic DC and neonatal microglia with RANTES, but not after treatment of thymocytes or splenocytes depleted of adherent cells. TNF-α-treated DC lose responsiveness to RANTES. DC from mice deficient for CCR1, CCR3, and CCR5 respond to RANTES, indicating that none of these receptors are exclusively used to initiate the chemokine cascade. RANTES-mediated chemokine amplification in DC may prolong inflammatory responses and shape the microenvironment, potentially enhancing acquired and innate immune responses.
IL-13 Induces Airways Hyperreactivity Independently of the IL-4Rα Chain in the Allergic Lung
Authors: Mattes Joerg , Yang Ming , Siqueira Ana , Clark Kris , MacKenzie Jason , McKenzie Andrew N. J. , Webb Dianne C. , Matthaei Klaus I. , Foster Paul S.
Published: April 2014

The potent spasmogenic properties of IL-13 have identified this molecule as a potential regulator of airways hyperreactivity (AHR) in asthma. Although IL-13 is thought to primarily signal through the IL-13Rα1-IL-4Rα complex, the cellular and molecular components employed by this cytokine to induce AHR in the allergic lung have not been identified. By transferring OVA-specific CD4+ T cells that were wild type (IL-13+/+ T cells) or deficient in IL-13 (IL-13−/− T cells) to nonsensitized mice that were then challenged with OVA aerosol, we show that T cell-derived IL-13 plays a key role in regulating AHR, mucus hypersecretion, eotaxin production, and eosinophilia in the allergic lung. Moreover, IL-13+/+ T cells induce these features (except mucus production) of allergic disease independently of the IL-4Rα chain. By contrast, IL-13+/+ T cells did not induce disease in STAT6-deficient mice. This shows that IL-13 employs a novel component of the IL-13 receptor signaling system that involves STAT6, independently of the IL-4Rα chain, to modulate pathogenesis. We show that this novel pathway for IL-13 signaling is dependent on T cell activation in the lung and is critically linked to downstream effector pathways regulated by eotaxin and STAT6.
Adenoviral Augmentation of Elafin Protects the Lung Against Acute Injury Mediated by Activated Neutrophils and Bacterial Infection
Authors: Simpson A. John , Wallace William A. H. , Marsden Mark E. , Govan John R. W. , Porteous David J. , Haslett Chris , Sallenave Jean-Michel
Published: April 2014

During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils.
Cutting Edge: The Nucleotide Receptor P2X7 Contains Multiple Protein- and Lipid-Interaction Motifs Including a Potential Binding Site for Bacterial Lipopolysaccharide
Authors: Denlinger Loren C. , Fisette Philip L. , Sommer Julie A. , Watters Jyoti J. , Prabhu Usha , Dubyak George R. , Proctor Richard A. , Bertics Paul J.
Published: April 2014

The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of κB-α isoform (IκB-α) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.
Somatic Hypermutation and Selection of B Cells in Thymic Germinal Centers Responding to Acetylcholine Receptor in Myasthenia Gravis
Authors: Sims Gary P. , Shiono Hiroyuki , Willcox Nick , Stott David I.
Published: April 2014

The muscle weakness in myasthenia gravis (MG) is mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Production of these pathogenic autoantibodies is believed to be associated with germinal centers (GC) and anti-AChR-secreting plasma cells in the hyperplastic thymus of patients with early onset MG (EOMG). Here, we describe the repertoire of rearranged heavy chain V genes and their clonal origins in GC from a typical EOMG patient. Three hundred fifteen rearranged Ig VH genes were amplified, cloned, and sequenced from sections of four thymic GC containing AChR-specific B cells. We found that thymic GC contain a remarkably heterogeneous population of B cells. Both naive and circulating memory B cells undergo Ag-driven clonal proliferation, somatic hypermutation, and selection. Numerous B cell clones were present, with no individual clone dominating the response. Comparisons of B cell clonal sequences from different GC and known anti-AChR Abs from other patients showed convergent mutations in the complementarity determining regions. These results are consistent with AChR driving an ongoing GC response in the thymus of EOMG patients. This is the first detailed analysis of B cell clones in human GC responding to a defined protein Ag, and the response we observed may reflect the effects of chronic stimulation by autoantigen.
Signaling Lymphocytic Activation Molecule Is Expressed on Mature CD83+ Dendritic Cells and Is Up-Regulated by IL-1β
Authors: Kruse Monika , Meinl Edgar , Henning Golo , Kuhnt Christine , Berchtold Susanne , Berger Thomas , Schuler Gerold , Steinkasserer Alexander
Published: April 2014

Signaling lymphocyte activation molecule (SLAM), a 70-kDa costimulatory molecule that mediates CD28-independent proliferation of T cells and IFN-γ production, has been identified on human T cells, immature thymocytes, and a subset of B cells. We have found that SLAM is expressed on mature but not immature dendritic cells (DC). However, the SLAM-associated protein, is missing in DC. SLAM surface expression is strongly up-regulated by IL-1β. Addition of IL-1β to the DC maturation mixture also increases the stimulatory properties of DC. These findings provide a new marker for DC maturation and help to explain two areas of DC biology. First, SLAM is a receptor for the measles virus, previously shown to infect DC. Second, SLAM could possibly contribute to the enhanced immunostimulatory functions of DC that are observed following the addition of IL-1.
Inducible Costimulator Regulates Th2-Mediated Inflammation, but Not Th2 Differentiation, in a Model of Allergic Airway Disease
Authors: Tesciuba Amanda G. , Subudhi Sumit , Rother Russell P. , Faas Susan J. , Frantz Aric M. , Elliot David , Weinstock Joel , Matis Louis A. , Bluestone Jeffrey A. , Sperling Anne I.
Published: April 2014

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.