bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneous Nuclear Ribonucleoprotein P2 Is an Autoantibody Target in Mice Deficient for Mer, Axl, and Tyro3 Receptor Tyrosine Kinases
Authors: Radic Marko Z. , Shah Kinjal , Zhang Wenguang , Lu Qingxian , Lemke Greg , Hilliard George M.
Published: April 2014

Deficiencies in clearance of apoptotic cells predispose to the development of autoimmune disease. This is evident in mice lacking the receptor tyrosine kinases Tyro3, Axl, and Mer. Deficient mice exhibit an increased abundance of apoptotic cells in tissues and manifest diverse autoimmune conditions. To test these mice for the presence of autoantibodies to apoptotic cells, we generated spontaneous splenic B cell hybridomas and used a novel microscopy screen to detect Ab binding to apoptotic Jurkat cells. From hybridomas secreting IgG Abs reactive with apoptotic cells, we selected one that recreated the major serum specificity for apoptotic cells. The Ab LHC7.15 bound to an Ag that is differentially distributed between the nucleus and the cytoplasm in live and apoptotic cells. In late apoptotic cells, the Ag coalesces into aggregates that bleb from the cell surface. Immunopurification of the Ag, followed by mass spectrometry, identified a protein of 69 kDa whose partial sequence matched heterogeneous nuclear ribonucleoprotein P2. This multifunctional protein binds DNA, RNA, and several known ribonucleoprotein autoantigens. Our observations indicate that a ribonucleoprotein complex, formed and translocated to the cell surface in apoptosis, represents a potent stimulus for breaking tolerance and inducing systemic autoimmunity in mice with defective clearance of cell remnants.
Cutting Edge: The AP-1 Subunit JunB Determines NK Cell-Mediated Target Cell Killing by Regulation of the NKG2D-Ligand RAE-1ε
Authors: Nausch Norman , Florin Lore , Hartenstein Bettina , Angel Peter , Schorpp-Kistner Marina , Cerwenka Adelheid
Published: April 2014

The activating receptor NKG2D and its ligands RAE-1 play an important role in the NK, γδ+, and CD8+ T cell-mediated immune response to tumors. Expression levels of RAE-1 on target cells have to be tightly controlled to allow immune cell activation against tumors but to avoid destruction of healthy tissues. In this study, we report that cell surface expression of RAE-1ε is greatly enhanced on cells lacking JunB, a subunit of the transcription complex AP-1. Furthermore, tissue-specific junB knockout mice respond to 12-O-tetradecanoyl-phorbol-13-acetate, a potent AP-1 activator, with markedly increased and sustained epidermal RAE-1ε expression. Accordingly, junB-deficient cells are efficiently killed via NKG2D by NK cells and induce IFN-γ production. Our data indicate that the transcription factor AP-1, which is involved in tumorigenesis and cellular stress responses, regulates RAE-1ε. Thus, up-regulated RAE-1ε expression due to low levels of JunB could alert immune cells to tumors and stressed cells.
Granzyme B, a New Player in Activation-Induced Cell Death, Is Down-Regulated by Vasoactive Intestinal Peptide in Th2 but Not Th1 Effectors
Authors: Sharma Vikas , Delgado Mario , Ganea Doina
Published: April 2014

Following antigenic stimulation and differentiation, Th1 and Th2 effector cells contribute differently to cellular and humoral immunity. Vasoactive intestinal peptide (VIP) induces Th2 responses by promoting Th2 differentiation and survival. In this study, we investigate the mechanisms for the protective effect of VIP against activation-induced cell death (AICD) of Th2 effectors. Surprisingly, microarray and protein data indicate that VIP prevents the up-regulation of granzyme B (GrB) in Th2 but not Th1 effectors. This is the first report of GrB expression in Th cells and of its involvement in activation-induced apoptosis. The enhanced responsiveness of Th2 cells to VIP is probably due to the higher expression of VIP receptors. The effect of VIP on Th2 survival and GrB expression is mediated through the VIP receptors 1 and 2 and cAMP signaling through exchange protein activated by cAMP and, to a lesser degree, protein kinase A. In addition to effects on GrB, VIP also down-regulates Fas ligand (FasL) and perforin (Pfr) expression. The extrinsic Fas/FasL pathway and the intrinsic GrB-dependent pathway act independently in inducing AICD. The mechanisms by which GrB induces cell death in Th1/Th2 effectors include both fratricide and suicide. Fratricide killing, prevalent in wild-type cells, is calcium and Pfr dependent, whereas the cell death of Pfr-deficient Th cells involves Fas and GrB but is calcium independent. This study identifies GrB as a new significant player in Th1/Th2 AICD and characterizes two mechanisms for the protective effect of VIP on Th2 survival, i.e., the down-regulation of GrB and FasL expression.
Hormonal Regulation of B Cell Development: 17β-Estradiol Impairs Negative Selection of High-Affinity DNA-Reactive B Cells at More Than One Developmental Checkpoint
Authors: Grimaldi Christine M. , Jeganathan Venkatesh , Diamond Betty
Published: April 2014

There are increasing data suggesting that sex hormones, such as estrogen, have immunomodulatory effects and play a role in disease progression and pathogenesis in patients with the autoimmune disorder systemic lupus erythematosus. We have shown previously that treatment with 17β-estradiol (E2) induces a lupus phenotype in BALB/c mice that express a transgene-encoded H chain of an anti-DNA Ab. Because E2 treatment interferes with normal tolerance of naive DNA-reactive B cells, we elected to study the effects of hormonal modulation on the regulation of autoreactive B cells at early developmental checkpoints. Single-cell PCR was performed to study the repertoire of DNA-reactive B cell subsets. High-affinity DNA-reactive B cells were rescued at both the immature and transitional B cell stage in E2-treated mice. Interestingly, although low-affinity DNA-reactive B cells survive negative selection in control mice, the frequency of these cells was significantly reduced in the mature pool of E2-treated mice, suggesting that the high-affinity DNA-reactive cells that mature to immunocompetence out-compete the low-affinity population for survival as mature B cells. These data provide evidence that an elevation in serum levels of E2 facilitates the maturation of a pathogenic naive autoreactive B cell repertoire and hampers the maturation of a potentially protective autoreactive B cell repertoire. Furthermore, these data show that both positive and negative selection occur within the transitional B cell stage.
Dendritic Cell-Mediated Induction of Mucosal Cytotoxic Responses following Intravaginal Immunization with the Nontoxic B Subunit of Cholera Toxin
Authors: Luci Carmelo , Hervouet Catherine , Rousseau Déborah , Holmgren Jan , Czerkinsky Cecil , Anjuère Fabienne
Published: April 2014

The use of the nontoxic B subunit of cholera toxin (CTB) as mucosal adjuvant and carrier-delivery system for inducing secretory Ab responses has been documented previously with different soluble Ags. In this study, we have evaluated this approach for inducing CTL responses against a prototype Ag, OVA, in the female genital mucosa. We report here the ability of an immunogen comprised of CTB conjugated to OVA (CTB-OVA) given by intravaginal (ivag) route to induce genital OVA-specific CTLs in mice. Using adoptive transfer models, we demonstrate that ivag application of CTB-OVA activates OVA-specific IFN-γ-producing CD4 and CD8 T cells in draining lymph nodes (DLN). Moreover, ivag CTB induces an expansion of IFN-γ-secreting CD8+ T cells in DLN and genital mucosa and promotes Ab responses to OVA. In contrast, ivag administration of OVA alone or coadministered with CTB failed to induce such responses. Importantly, we demonstrate that ivag CTB-OVA generates OVA-specific CTLs in DLN and the genital mucosa. Furthermore, genital CD11b+CD11c+ dendritic cells (DCs), but not CD8+CD11c+ or CD11c− APCs, present MHC class I epitopes acquired after ivag CTB-OVA, suggesting a critical role of this DC subset in the priming of genital CTLs. Inhibition studies indicate that the presentation of OVA MHC class I epitopes by DCs conditioned with CTB-OVA involves a proteasome-dependent and chloroquine-sensitive mechanism. These results demonstrate that CTB is an efficient adjuvant-delivery system for DC-mediated induction of genital CTL responses and may have implications for the design of vaccines against sexually transmitted infections.
B Cell Induction of IL-13 Expression in NK Cells: Role of CD244 and SLAM-Associated Protein
Authors: Gao Ning , Schwartzberg Pamela , Wilder Julie A. , Blazar Bruce R. , Yuan Dorothy
Published: April 2014

NK cells are an important component of the innate immune system that can also interact with B cells in a mutually productive manner. We have previously shown that activated B cells can induce NK cells to up-regulate their secretion of IFN-γ. In this study, we show that B cells, and, particularly, marginal zone B cells, can, in addition, induce NK cells via direct cell-cell interactions to express mRNA encoding the Th2 cytokine IL-13. The induction of NK cell IL-13 mRNA expression requires the ligation of the CD244 receptor by the CD48 ligand on B cells via signaling pathways that depend upon expression of the X-linked lymphoproliferative disease gene product, SH2D1A/DSHP/SAP (SLAM-associated protein, or SAP) in NK cells. Thus, the positive signals attributed to the B cell activation of CD244 on murine NK cells appears to be more similar to the activity of CD244 on human cells. The induction of IL-13 mRNA by B cells may account for the effect of NK cells on the generation of Th2-type responses in the presence of some adjuvants.
Recombinant Ig-Like Transcript 3-Fc Modulates T Cell Responses via Induction of Th Anergy and Differentiation of CD8+ T Suppressor Cells
Authors: Kim-Schulze Seunghee , Scotto Luigi , Vlad George , Piazza Flavia , Lin Hana , Liu Zhuoru , Cortesini Raffaello , Suciu-Foca Nicole
Published: April 2014

The Ig-like transcript (ILT)3 is crucial to the tolerogenic activity acquired by dendritic cells exposed to allospecific T suppressor (Ts) cells. We have explored the immunomodulatory property of the extracellular region of ILT3 using a cytoplasmic deletion mutant of ILT3 (ILT3δ), expressed as membrane-bound ILT3 on KG1 cells, and a rILT3-Fc fusion protein. We found that both membrane-bound and soluble ILT3 inhibited T cell proliferation in primary and secondary MLC inducing anergy in CD4+ Th cells and suppressing the differentiation of IFN-γ-producing CD8+ CTL. Furthermore, membrane-bound and soluble ILT3 induced the differentiation of CD8+ FOXP3+ Ts cells in primary 7-day MLC. The suppressive activity of these CD8+ Ts cells is alloantigen specific and mediated by their capacity to induce the up-regulation of ILT3 and down-regulation of costimulatory molecules such as CD86 in APC from the stimulator used for priming, but not on control HLA-mismatched APC. Our finding that ILT3-Fc has potent immunosuppressive activity in vitro and that it acts on T cells only upon activation suggests the possibility that this agent may be of use for specific suppression of the immune response in autoimmunity or transplantation.
Dengue Virus-Reactive CD8+ T Cells Display Quantitative and Qualitative Differences in Their Response to Variant Epitopes of Heterologous Viral Serotypes
Authors: Bashyam Hema S. , Green Sharone , Rothman Alan L.
Published: April 2014

Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-γ, TNF-α, MIP-1β, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.
Quantitative Analysis of Phosphotyrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells
Authors: Kim Ji-Eun , White Forest M.
Published: April 2014

The mechanism by which stimulation of coreceptors such as CD28 contributes to full activation of TCR signaling pathways has been intensively studied, yet quantitative measurement of costimulation effects on functional TCR signaling networks has been lacking. In this study, phosphotyrosine networks triggered by CD3, CD28, or CD3 and CD28 costimulation were analyzed by site-specific quantitative phosphoproteomics, resulting in identification of 101 tyrosine and 3 threonine phosphorylation sites and quantification of 87 sites across four cell states. As expected, CD3 stimulation induced phosphorylation of CD3 chains and upstream components of TCR pathways such as Zap70, while CD28 stimulation induced phosphorylation of CD28, Vav-1, and other adaptor proteins including downstream of tyrosine kinase 1, Grb2-associated protein 2 (Grap2), and Wiskott-Aldrich syndrome protein. CD3 and CD28 costimulation induced a complex response including decreased threonine phosphorylation in the ERK1 and ERK2 activation loops and increased phosphorylation of selected tyrosine sites on ERK1/2, p38, phospholipase C-γ, Src homology 2 domain-containing transforming protein 1, Grap2, and Vav-1, potentiating T cell activation. Hierarchical clustering and self-organizing maps were used to identify modules of coregulated phosphorylation sites within the network. Quantitative information in our study suggests quantitative and qualitative contribution by costimulation of CD28 on CD3-stimulated TCR signaling networks via enhanced phosphorylation of phospholipase C-γ/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and their effects on downstream components including MAPKs.
Self-Limitation of Th1-Mediated Inflammation by IFN-γ
Authors: Feuerer Markus , Eulenburg Katharina , Loddenkemper Christoph , Hamann Alf , Huehn Jochen
Published: April 2014

IFN-γ is an effector cytokine of cell-mediated immunity that plays an essential role in both innate and adaptive phases of an immune response. Interestingly, in several Th1-dependent autoimmune models, lack of IFN-γ is associated with an acceleration of disease. To distinguish the influence of IFN-γ on the polarization of naive precursors from the influence on effector cells, we used an adoptive transfer model of differentiated Ag-specific Th1 cells. In this study, IFN-γ displayed a dual function in a Th1-dependent immune reaction. In the early phase, IFN-γ accelerated the inflammation, whereas in the late phase it mediated the process of self-limitation. We demonstrated that IFN-γ limits the number of Th1 effector cells after Ag challenge. Studies using IFN-γR−/− mice as recipients showed that IFN-γ acts indirectly via host cells to regulate the pool size of Th1 cells. NO was a downstream effector molecule. Transfer experiments of Th1 cells into IFN-γ−/− mice revealed that Th1 cells control both themselves and the corresponding inflammation by the release of IFN-γ. Thus, the proinflammatory cytokine IFN-γ can act as a negative feedback regulator to control Th1-mediated immune responses.
Resistance to Experimental Autoimmune Encephalomyelitis and Impaired IL-17 Production in Protein Kinase Cθ-Deficient Mice
Authors: Tan Seng-Lai , Zhao Jingyong , Bi Chen , Chen XinYi Cynthia , Hepburn Deena L. , Wang Jian , Sedgwick Jonathon D. , Chintalacharuvu Subba R. , Na Songqing
Published: April 2014

The protein kinase Cθ (PKCθ) serine/threonine kinase has been implicated in signaling of T cell activation, proliferation, and cytokine production. However, the in vivo consequences of ablation of PKCθ on T cell function in inflammatory autoimmune disease have not been thoroughly examined. In this study we used PKCθ-deficient mice to investigate the potential involvement of PKCθ in the development of experimental autoimmune encephalomyelitis, a prototypic T cell-mediated autoimmune disease model of the CNS. We found that PKCθ−/− mice immunized with the myelin oligodendrocyte glycoprotein (MOG) peptide MOG35–55 were completely resistant to the development of clinical experimental autoimmune encephalomyelitis compared with wild-type control mice. Flow cytometric and histopathological analysis of the CNS revealed profound reduction of both T cell and macrophage infiltration and demyelination. Ex vivo MOG35–55 stimulation of splenic T lymphocytes from immunized PKCθ−/− mice revealed significantly reduced production of the Th1 cytokine IFN-γ as well as the T cell effector cytokine IL-17 despite comparable levels of IL-2 and IL-4 and similar cell proliferative responses. Furthermore, IL-17 expression was dramatically reduced in the CNS of PKCθ−/− mice compared with wild-type mice during the disease course. In addition, PKCθ−/− T cells failed to up-regulate LFA-1 expression in response to TCR activation, and LFA-1 expression was also significantly reduced in the spleens of MOG35–55-immunized PKCθ−/− mice as well as in in vitro-stimulated CD4+ T cells compared with wild-type mice. These results underscore the importance of PKCθ in the regulation of multiple T cell functions necessary for the development of autoimmune disease.
Directed Differentiation of Human Embryonic Stem Cells into Functional Dendritic Cells through the Myeloid Pathway
Authors: Slukvin Igor I. , Vodyanik Maxim A. , Thomson James A. , Gumenyuk Maryna E. , Choi Kyung-Dal
Published: April 2014

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123lowHLA-DR− phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-α coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.
Unique Structural Determinants for Stat3 Recruitment and Activation by the Granulocyte Colony-Stimulating Factor Receptor at Phosphotyrosine Ligands 704 and 744
Authors: Shao Huang , Xu Xuejun , Jing Naijie , Tweardy David J.
Published: April 2014

G-CSFR cytoplasmic tyrosine (Y) residues (Y704, Y729, Y744, and Y764) become phosphorylated upon ligand binding and recruit specific Src homology 2 domain-containing proteins that link to distinct yet overlapping programs for myeloid cell survival, differentiation, proliferation, and activation. The structural basis for recruitment specificity is poorly understood but could be exploited to selectively target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation demonstrated in a subset of acute myeloid leukemia patients with poor prognosis. Recombinant Stat3 bound to G-CSFR phosphotyrosine peptide ligands pY704VLQ and pY744LRC with similar kinetics. Testing of three models for Stat3 Src homology 2-pY ligand binding in vitro and in vivo revealed unique determinants for Stat3 recruitment and activation by the G-CSFR, the side chain of Stat3 R609, which interacts with the pY ligand phosphate group, and the peptide amide hydrogen of E638, which bonds with oxygen/sulfur within the + 3 Q/C side chain of the pY ligand when it assumes a β turn. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of this interaction that can be exploited to target Stat3 activation for the treatment of a subset of acute myeloid leukemia patients.
Lack of Tyrosine 320 Impairs Spontaneous Endocytosis and Enhances Release of HLA-B27 Molecules
Authors: Santos Susana G. , Antoniou Antony N. , Sampaio Paula , Powis Simon J. , Arosa Fernando A.
Published: April 2014

Several lines of evidence suggest that endocytosis of MHC class I molecules requires conserved motifs within the cytoplasmic domain. In this study, we show, in the C58 rat thymoma cell line transfected with HLA-B27 molecules, that replacement of the highly conserved tyrosine (Tyr320) in the cytoplasmic domain of HLA-B27 does not hamper cell surface expression of β2-microglobulin H chain heterodimers or formation of misfolded molecules. However, Tyr320 replacement markedly impairs spontaneous endocytosis of HLA-B27. Although wild-type molecules are mostly internalized via endosomal compartments, Tyr320-mutated molecules remain at the plasma membrane in which partial colocalization with endogenous transferrin receptors can be observed, also impairing their endocytosis. Finally, we show that Tyr320 substitution enhances release of cleaved forms of HLA-B27 from the cell surface. These studies show for the first time that Tyr320 is most likely part of a cytoplasmic sorting motif involved in spontaneous endocytosis and shedding of MHC class I molecules.
Evidence That Complement Protein C1q Interacts with C-Reactive Protein through Its Globular Head Region
Authors: McGrath Fabian D. G. , Brouwer Mieke C. , Arlaud Gérard J. , Daha Mohamed R. , Hack C. Erik , Roos Anja
Published: April 2014

C1q acts as the recognition unit of the first complement component, C1, and binds to immunoglobulins IgG and IgM, as well as to non-Ig ligands, such as C-reactive protein (CRP). IgG and IgM are recognized via the globular head regions of C1q (C1qGR), whereas CRP has been postulated to interact with the collagen-like region (C1qCLR). In the present study, we used a series of nine mAbs to C1q, five directed against C1qGR and four against C1qCLR, to inhibit the interaction of C1q with CRP. The F(ab′)2 of each of the five mAbs directed against C1qGR inhibited binding of C1q to polymerized IgG. These five mAbs also successfully inhibited the interaction of C1q with CRP. Moreover, these five mAbs inhibited C1 activation by CRP as well as by polymerized IgG in vitro. In contrast, none of the four mAbs against C1qCLR inhibited C1q interaction with CRP or IgG, or could reduce activation of complement by CRP or polymerized IgG. These results provide the first evidence that the interaction of C1q with CRP or IgG involves sites located in the C1qGR, whereas sites in the CLR do not seem to be involved in the physiological interaction of C1q with CRP.
Amino-Terminal Flanking Residues Determine the Conformation of a Peptide–Class II MHC Complex
Authors: Lovitch Scott B. , Pu Zheng , Unanue Emil R.
Published: April 2014

The peptide spanning residues 48–62 of hen egg white lysozyme presented by I-Ak molecules gives rise to two T cell populations, types A and B, that recognize distinct conformers of the complex generated in late and recycling endosomes. The class II–like accessory molecule H2-DM functions as a conformational editor, eliminating the type B conformer in late endosomes. Here, we show that the conformation of the complex, and its susceptibility to editing by H2-DM, are determined by peptide amino-terminal flanking residues. Elimination of these residues abolished editing, permitting formation of the type B conformer in late endosomes. Substitutions at P(−2) affected the stability of the type B conformer, preventing its formation and/or editing, without hindering peptide binding or formation of the type A conformer of the complex. We conclude that interactions involving amino-terminal flanking residues stabilize peptide-MHC conformers and confer resistance to editing by H2-DM, influencing the nature of the T cell repertoire.
Pituitary Adenylate Cyclase-Activating Polypeptide 27 Is a Functional Ligand for Formyl Peptide Receptor-Like 1
Authors: Kim Youndong , Lee Byoung Dae , Kim Oekyung , Bae Yoe-Sik , Lee Taehoon , Suh Pann-Ghill , Ryu Sung Ho
Published: April 2014

Although the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the regulation of several immune responses, its target receptors and signaling mechanisms have yet to be fully elucidated in immune cells. In this study, we found that PACAP27, but not PACAP38, specifically stimulated intracellular calcium mobilization and ERK phosphorylation in human neutrophils. Moreover, formyl peptide receptor-like 1 (FPRL1) was identified as a PACAP27 receptor, and PACAP27 was found to selectively stimulate intracellular calcium increase in FPRL1-transfected rat basophile leukocytes-2H3 cell lines. In addition, PACAP27-induced calcium increase and ERK phosphorylation were specifically inhibited by an FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp-Trp (WRW4), thus supporting the notion that PACAP27 acts on FPRL1. In terms of the functional role of PACAP27, we found that the peptide stimulated CD11b surface up-regulation and neutrophil chemotactic migration, and that these responses were completely inhibited by WRW4. The interaction between PACAP27 and FPRL1 was analyzed further using truncated PACAPs and chimeric PACAPs using vasoactive intestinal peptide, and the C-terminal region of PACAP27 was found to perform a vital function in the activation of FPRL1. Taken together, our study suggests that PACAP27 activates phagocytes via FPRL1 activation, and that this results in proinflammatory behavior, involving chemotaxis and the up-regulation of CD11b.
Molecular Genetic Analysis of the Idd4 Locus Implicates the IFN Response in Type 1 Diabetes Susceptibility in Nonobese Diabetic Mice
Authors: Ivakine Evgueni A. , Gulban Omid M. , Mortin-Toth Steven M. , Wankiewicz Ellen , Scott Christopher , Spurrell David , Canty Angelo , Danska Jayne S.
Published: April 2014

High-resolution mapping and identification of the genes responsible for type 1 diabetes (T1D) has proved difficult because of the multigenic etiology and low penetrance of the disease phenotype in linkage studies. Mouse congenic strains have been useful in refining Idd susceptibility loci in the NOD mouse model and providing a framework for identification of genes underlying complex autoimmune syndromes. Previously, we used NOD and a nonobese diabetes-resistant strain to map the susceptibility to T1D to the Idd4 locus on chromosome 11. Here, we report high-resolution mapping of this locus to 1.4 megabases. The NOD Idd4 locus was fully sequenced, permitting a detailed comparison with C57BL/6 and DBA/2J strains, the progenitors of T1D resistance alleles found in the nonobese diabetes-resistant strain. Gene expression arrays and quantitative real-time PCR were used to prioritize Idd4 candidate genes by comparing macrophages/dendritic cells from congenic strains where allelic variation was confined to the Idd4 interval. The differentially expressed genes either were mapped to Idd4 or were components of the IFN response pathway regulated in trans by Idd4. Reflecting central roles of Idd4 genes in Ag presentation, arachidonic acid metabolism and inflammation, phagocytosis, and lymphocyte trafficking, our combined analyses identified Alox15, Alox12e, Psmb6, Pld2, and Cxcl16 as excellent candidate genes for the effects of the Idd4 locus.
Expression of B7-H1 on Gastric Epithelial Cells: Its Potential Role in Regulating T Cells during Helicobacter pylori Infection
Authors: Das Soumita , Suarez Giovanni , Beswick Ellen J. , Sierra Johanna C. , Graham David Y. , Reyes Victor E.
Published: April 2014

Helicobacter pylori infection is associated with gastritis, ulcers, and gastric cancer. The infection becomes chronic as the host response is unable to clear it. Gastric epithelial cells (GEC) play an important role during the host response, and their expression of class II MHC and costimulatory molecules such as CD80 and CD86 suggests their role in local Ag presentation. Although T cells are recruited to the infected gastric mucosa, they have been reported to be hyporesponsive. In this study, we detected the expression of B7-H1 (programmed death-1 ligand 1), a member of B7 family of proteins associated with T cell inhibition on GEC. Quantitative real-time RT-PCR revealed that B7-H1 expression increased significantly on GEC after H. pylori infection. Western blot analysis showed that B7-H1 expression was induced by various H. pylori strains and was independent of H. pylori virulence factors such as Cag, VacA, and Urease. The functional role of B7-H1 in the cross talk between GEC and T cells was assessed by coculturing GEC or H. pylori-infected GEC with CD4+ T cells isolated from peripheral blood. Using blocking Abs to B7-H1 revealed that B7-H1 was involved in the suppression of T cell proliferation and IL-2 synthesis, and thus suggested a role for B7-H1 on the epithelium as a contributor in the chronicity of H. pylori infection.
Potent Inhibition of Macrophage Responses to IFN-γ by Live VirulentMycobacterium tuberculosisIs Independent of Mature Mycobacterial Lipoproteins but Dependent on TLR2
Authors: Banaiee Niaz , Kincaid Eleanor Z. , Buchwald Ulrike , Jacobs William R. , Ernst Joel D.
Published: April 2014

Mycobacterium tuberculosis is a highly successful pathogen that can persist and cause disease despite an immune response. One potential mechanism for resisting elimination is by inhibiting the action of IFN-γ. We have previously shown that live M. tuberculosis inhibits selected macrophage responses to IFN-γ, and that purified M. tuberculosis 19-kDa lipoprotein inhibits induction of selected IFN-γ-responsive genes through a TLR2-dependent pathway, whereas peptidoglycan inhibits responses to IFN-γ by a TLR2-independent pathway. To determine the relative contribution of lipoproteins to the inhibition of responses to IFN-γ, we deleted the M. tuberculosis gene (lspA) that encodes lipoprotein signal peptidase. This revealed that M. tuberculosis lipoprotein processing is indispensable for stimulation of TLR2 reporter cells, but that the lspA mutant inhibits macrophage responses to IFN-γ to the same extent as wild-type bacteria. Macrophages lacking TLR2 are more resistant to inhibition by either strain of M. tuberculosis, suggesting that nonlipoprotein TLR2 agonists contribute to inhibition. Indeed, we found that phosphatidylinositol mannan from M. tuberculosis inhibits macrophage responses to IFN-γ. M. tuberculosis inhibition of responses to IFN-γ requires new protein synthesis, indicating that a late effect of innate immune stimulation is the inhibition of responses to IFN-γ. These results establish that M. tuberculosis possesses multiple mechanisms of inhibiting responses to IFN-γ.
Cryptococcus neoformans Glycoantigens Are Captured by Multiple Lectin Receptors and Presented by Dendritic Cells
Authors: Mansour Michael K. , Latz Eicke , Levitz Stuart M.
Published: April 2014

Cell-mediated immune responses to glycoantigens have been largely uncharacterized. Protective T cell responses to the pathogenic yeast Cryptococcus neoformans are dependent on heavily mannosylated Ags termed mannoproteins. In the work presented, the innate immune response to mannoprotein was determined. Purified murine splenic dendritic cells (DC), B cells, and macrophages were used to stimulate mannoprotein-specific T cells. Only DC were capable of any measurable stimulation. Depletion of DC resulted in the abrogation of the T cell response. Human and murine DC rapidly captured fluorescent-labeled mannoprotein by a mannose receptor-mediated process. Using transfected cell lines, the type II C-type lectin receptor DC-specific ICAM-3-grabbing nonintegrin (CD209) was determined to have affinity for mannoprotein. Taken together with prior work demonstrating that mannoprotein was captured by the macrophage mannose receptor (CD206), these data suggest that multiple mannose receptors on DC recognize mannoprotein. Pulsing experiments demonstrated that DC captured sufficient mannoprotein over 2 h to account for 50% of total stimulation. Capture appeared dependent on mannose receptors, as competitive mannosylated inhibitors and calcium chelators each interfered with T cell stimulation. By confocal microscopy, intracellular mannoprotein trafficked to an endo-lysosomal compartment in DC, and at later time points extended into tubules in a similar fashion to the degradation marker DQ-OVA. Mannoprotein colocalized intracellularly with CD206 and CD209. These data suggest that DC provide the crucial link between innate and adaptive immune responses to C. neoformans via a process that is dependent upon the efficient uptake of mannoprotein by mannose receptors.
Enterocyte TLR4 Mediates Phagocytosis and Translocation of Bacteria Across the Intestinal Barrier
Authors: Neal Matthew D. , Leaphart Cynthia , Levy Ryan , Prince Jose , Billiar Timothy R. , Watkins Simon , Li Jun , Cetin Selma , Ford Henri , Schreiber Alan , Hackam David J.
Published: April 2014

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcγRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.
Mycobacterium bovis Bacillus Calmette-Guérin Induces TLR2-Mediated Formation of Lipid Bodies: Intracellular Domains for Eicosanoid Synthesis In Vivo
Authors: D’Avila Heloisa , Melo Rossana C. N. , Parreira Gleydes G. , Werneck-Barroso Eduardo , Castro-Faria-Neto Hugo C. , Bozza Patrícia T.
Published: April 2014

Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.
Vascular Endothelial Growth Factor-Induced Signaling Pathways in Endothelial Cells That Mediate Overexpression of the Chemokine IFN-γ-Inducible Protein of 10 kDa In Vitro and In Vivo
Authors: Boulday Gwénola , Haskova Zdenka , Reinders Marlies E. J. , Pal Soumitro , Briscoe David M.
Published: April 2014

Vascular endothelial growth factor (VEGF), an angiogenesis factor, has recently been found to have potent proinflammatory properties in vivo. However, the mechanism by which it mediates inflammation is poorly understood. In this study, we have evaluated the function of VEGF on the induced expression and function of the T cell chemoattractant chemokine IFN-γ-inducible protein of 10 kDa (IP-10). In vitro, we find that VEGF augments the effect of IFN-γ on the induction of IP-10 mRNA and protein expression in endothelial cells. Moreover, we show that VEGF and IFN-γ regulate the activation of the IP-10 promoter, and that the kinases PI3K, phosphoinositide-dependent kinase 1, and Akt act as intermediary signaling molecules for cytokine-inducible IP-10 transcriptional activation in endothelial cells. To examine whether VEGF is functional for IP-10 expression in vivo, Chinese hamster ovary cells that were designed to secrete VEGF were injected s.c. into the skin of nude mice and were found to mediate a time-dependent increase in IP-10 mRNA. This response was reduced in animals treated systemically with the PI3K inhibitor wortmannin. When the Chinese hamster ovary cells expressing VEGF plasmid were injected s.c. into C57BL/6 wild-type or CXCR3−/− mice, they elicited an inflammatory reaction in wild-type but not in CXCR3−/− mice. Collectively, these findings indicate that VEGF-induced augmentation of IP-10 expression is a major mechanism underlying its proinflammatory function.
Renal Ischemia-Reperfusion Injury and Adenosine 2A Receptor-Mediated Tissue Protection: The Role of CD4+ T Cells and IFN-γ
Authors: Day Yuan-Ji , Huang Liping , Ye Hong , Li Li , Linden Joel , Okusa Mark D.
Published: April 2014

A2A adenosine receptor (A2AR)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A2A agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A2AR expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A2A agonist, did not confer additional protection. IFN-γ is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-γ is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-γ KO mice (IFN-γ KO→WT chimera). We observed marked reduction in IRI in comparison to WT→WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A2AR in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+→Rag-1 KO) or A2A KO CD4+ cells (A2A KO CD4+→Rag-1 KO). ATL146e reduced injury in WT CD4+→Rag-1 KO mice but not in A2A KO CD4+→Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-γ KO mice (IFN-γ CD4+→Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-γ contributes to IRI and that A2A agonists mediate protection from IRI through action on CD4+ cells.
Exacerbation of Experimental Autoimmune Encephalomyelitis in P2X7R−/− Mice: Evidence for Loss of Apoptotic Activity in Lymphocytes
Authors: Chen Lanfen , Brosnan Celia F.
Published: April 2014

The purinergic receptor P2X7R is a nucleotide-gated ion channel that has been proposed to function as a major regulator of inflammation. In this study we examined the role of this receptor in regulating inflammation in the CNS by determining the effects of the loss of this receptor (P2X7R−/−) on experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We show here that P2X7R−/− mice developed more severe clinical and pathological expression of EAE than wild type (WT) controls and that spleen and lymph node cells from P2X7R−/− mice proliferated more vigorously to Ag in vitro. Bone marrow (BM) radiation chimeras revealed that enhanced susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R−/− BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. Coculture of P2X7R−/− macrophages with WT lymphocytes and vice versa showed that enhanced proliferative activity resided within the P2X7R−/− lymphocyte population and correlated with reduced levels of IFN-γ and NO and apoptosis of lymphocytes. mRNA and protein for IFN-γ were also significantly reduced in the CNS of P2X7R−/− mice with EAE. FACS analysis of cells isolated from the CNS showed significantly fewer annexin V/propidium iodide-positive lymphocytes in the CNS of P2X7R−/− mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. From these data we conclude that enhanced susceptibility of P2X7R−/− mice to EAE reflects a loss of apoptotic activity in lymphocytes, supporting an important role for this receptor in lymphocyte homeostasis.
The Human Specific CCR1 Antagonist CP-481,715 Inhibits Cell Infiltration and Inflammatory Responses in Human CCR1 Transgenic Mice
Authors: Gladue Ronald P. , Cole Susan H. , Roach Marsha L. , Tylaska Laurie A. , Nelson Robin T. , Shepard Richard M. , McNeish John D. , Ogborne Kevin T. , Neote Kuldeep S.
Published: April 2014

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715 inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED50 of 0.2 mg/kg. CP-481,715 did not inhibit cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hypersensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-γ and IL-2 produced by isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease.
The B12 Anti-Tryptase Monoclonal Antibody Disrupts the Tetrameric Structure of Heparin-Stabilized β-Tryptase to Form Monomers That Are Inactive at Neutral pH and Active at Acidic pH
Authors: Fukuoka Yoshihiro , Schwartz Lawrence B.
Published: April 2014

The novel tetrameric structure of human β-tryptase faces each active site into the central pore, thereby restricting access of most biologic protease inhibitors. The mechanism by which the anti-tryptase mAb B12 inhibits human β-tryptase peptidase and proteolytic activities at neutral pH, but augments proteolytic activity at acidic pH, was examined. At neutral pH, B12-β-tryptase complexes are inactive. At acidic pH, B12 (intact and Fab) minimally affects peptidase activity when added to β-tryptase tetramers, but does induce susceptibility to inhibition by soybean trypsin inhibitor and antithrombin III. Surprisingly, B12 Fab-β-tryptase complexes formed at both neutral and acidic pH exhibit the apparent molecular mass of a complex with 1 β-tryptase monomer and 1 Fab by gel filtration. B12 does not compete with heparin for binding to tryptase at either neutral or acidic pH. Thus, B12 directly disrupts β-tryptase tetramers to monomers that are inactive at neutral pH, whereas at acidic pH, are active and more accessible to protein inhibitors and substrates.
c-Jun Kinase Is a Critical Signaling Molecule in a Neonatal Model of Group B Streptococcal Sepsis
Authors: Kenzel Sybille , Mancuso Guiseppe , Malley Richard , Teti Guiseppe , Golenbock Douglas T. , Henneke Philipp
Published: April 2014

Group B streptococcus (GBS) is the major cause of sepsis in newborn infants. In vitro, inactivated GBS stimulates macrophages to produce inflammatory proteins via the TLR adapter protein MyD88. Furthermore, inflammatory cytokine release in response to GBS greatly exceeds that following stimulation with pneumococci. In this study, we attempted to unravel signaling events that are involved in GBS-, but not Streptococcus pneumoniae-stimulated phagocytes to identify molecular targets for adjunctive sepsis therapy. We found that inactivated GBS and S. pneumoniae differed in the activation of the MAPK JNK, but not IκB kinase. Furthermore, JNK was essential for the transcriptional activation of inflammatory cytokine genes in response to GBS. Inhibition of JNK by the anthrapyrazolone SP600125 abrogated GBS-induced cytokine formation via an AP-1- and NF-κB-dependent mechanism without impairing antibacterial properties such as phagocytosis of GBS and the formation of intracellular oxidative species. In contrast, inhibition of the MAPK p38 impaired both antibacterial processes. In a neonatal mouse model of GBS sepsis SP600125 inhibited the inflammatory response and improved survival. In conclusion, JNK plays a major role in the inflammatory, but not in the direct antibacterial response to inactivated GBS, and may thus serve as a rational target for an adjunctive GBS sepsis therapy.