bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-1β Induces IL-6 Expression in Human Orbital Fibroblasts: Identification of an Anatomic-Site Specific Phenotypic Attribute Relevant to Thyroid-Associated Ophthalmopathy
Authors: Chen Beiling , Tsui Shanli , Smith Terry J.
Published: April 2014

Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we report that these fibroblasts, when treated with IL-1β, express high levels of IL-6, a cytokine involved in B cell activation and the regulation of adipocyte metabolism. The magnitude of this induction is considerably greater than that in dermal fibroblasts and involves up-regulation of IL-6 mRNA levels. IL-1β activates both p38 and ERK 1/2 components of the MAPK pathways. Disrupting these could attenuate the IL-6 induction. The up-regulation involves enhanced IL-6 gene promoter activity and retardation of IL-6 mRNA decay by IL-1β. Dexamethasone completely blocked the effect of IL-1β on IL-6 expression. Orbital fibroblasts also express higher levels of IL-6R than do skin-derived cells. When treated with rIL-6 (10 ng/ml), STAT3 is transiently phosphorylated. Thus, the exaggerated capacity of orbital fibroblasts to express high levels of both IL-6 and its receptor in an anatomic site-selective manner could represent an important basis for immune responses localized to the orbit in Graves’ disease.
Genotype-Proteotype Linkage in the Wiskott-Aldrich Syndrome
Authors: Lutskiy Maxim I. , Rosen Fred S. , Remold-O’Donnell Eileen
Published: April 2014

Wiskott-Aldrich syndrome (WAS) is a platelet/immunodeficiency disease arising from mutations of WAS protein (WASP), a hemopoietic cytoskeletal protein. Clinical symptoms vary widely from mild (X-linked thrombocytopenia) to life threatening. In this study, we examined the molecular effects of individual mutations by quantifying WASP in peripheral lymphocytes of 44 patients and identifying the molecular variant (collectively called proteotype). Nonpredicted proteotypes were found for 14 genotypes. These include WASP-negative lymphocytes found for five missense genotypes and WASP-positive lymphocytes for two nonsense, five frameshift, and two splice site genotypes. Missense mutations in the Ena/VASP homology 1 (EVH1) domain lead to decreased/absent WASP but normal mRNA levels, indicating that proteolysis causes the protein deficit. Because several of the EVH1 missense mutations alter WIP binding sites, the findings suggest that abrogation of WIP binding induces proteolysis. Whereas platelets of most patients were previously shown to lack WASP, WASP-positive platelets were found for two atypical patients, both of whom have mutations outside the EVH1 domain. WASP variants with alternative splicing and intact C-terminal domains were characterized for eight nonsense and frameshift genotypes. One of these, a nonsense genotype in a mild patient, supports expression of WASP lacking half of the proline-rich region. With one notable exception, genotype and proteotype were linked, indicating that a genotype-proteotype registry could be assembled to aid in predicting disease course and planning therapy for newly diagnosed infants. Knowledge of the molecular effect of mutations would aid also in identifying disease-modifying genes.
Cutting Edge: IL-12 Induces CD4+CD25− T Cell Activation in the Presence of T Regulatory Cells
Authors: King Irah L. , Segal Benjamin M.
Published: April 2014

IL-12p40 cytokines have been implicated in the development of organ-specific autoimmune diseases as well as pathogen-specific adaptive immunity. In addition to inducing IFN-γ, IL-12 stimulates effector CD4+ T cells to express adhesion molecules and homing receptors that facilitate their migration to sites of inflammation. In this study, we expand upon those observations by demonstrating an alternative pathway by which IL-12 could promote Th1 inflammatory responses in mice, namely, by restoring proliferation and cytokine expression by effector T cells in the presence of CD4+CD25+ regulatory T cells (Treg). This effect of IL-12 was not replicated by IL-23 or IFN-γ and was dependent on signaling through the IL-12R expressed on CD25− responder cells, but not on Treg. Our studies suggest that IL-12 could act in concert with other proinflammatory factors to stimulate CD4+CD25− T cell activation in the presence of Treg.
Active Ca2+/Calmodulin-Dependent Protein Kinase IIγB Impairs Positive Selection of T Cells by Modulating TCR Signaling
Authors: McGargill Maureen A. , Sharp Leslie L. , Bui Jack D. , Hedrick Stephen M. , Calbo Sébastien
Published: April 2014

T cell development is regulated at two critical checkpoints that involve signaling events through the TCR. These signals are propagated by kinases of the Src and Syk families, which activate several adaptor molecules to trigger Ca2+ release and, in turn, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation. In this study, we show that a constitutively active form of CaMKII antagonizes TCR signaling and impairs positive selection of thymocytes in mice. Following TCR engagement, active CaMKII decreases TCR-mediated CD3ζ chain phosphorylation and ZAP70 recruitment, preventing further downstream events. Therefore, we propose that CaMKII belongs to a negative-feedback loop that modulates the strength of the TCR signal through the tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP-2).
Mechanisms of the Antitumor Responses and Host-versus-Graft Reactions Induced by Recipient Leukocyte Infusions in Mixed Chimeras Prepared with Nonmyeloablative Conditioning: A Critical Role for Recipient CD4+ T Cells and Recipient Leukocyte Infusion-Derived IFN-γ-Producing CD8+ T Cells
Authors: Rubio Marie-Therese , Saito Toshiki I. , Kattleman Kristin , Zhao Guiling , Buchli Jennifer , Sykes Megan
Published: April 2014

Surprisingly, antitumor responses can occur in patients who reject donor grafts following nonmyeloablative hemopoietic cell transplantation. In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism, IFN-γ production, and antitumor responses against host-type tumors. However, the mechanisms behind this phenomenon remain to be determined. We now demonstrate that the effects of RLI are mediated by distinct and complex mechanisms. Donor marrow rejection is induced by RLI-derived alloactivated T cells, which activate non-RLI-derived, recipient IFN-γ-producing cells. RLI-derived CD8 T cells induce the production of IFN-γ by both RLI and non-RLI-derived recipient cells. The antitumor responses of RLI involve mainly RLI-derived IFN-γ-producing CD8 T cells and recipient-derived CD4 T cells and do not involve donor T cells. The pathways of donor marrow and tumor rejection lead to the development of tumor-specific cell-mediated cytotoxic responses that are not due to bystander killing by alloreactive T cells.
The Synthetic Peptide Trp-Lys-Tyr-Met-Val-D-Met Inhibits Human Monocyte-Derived Dendritic Cell Maturation via Formyl Peptide Receptor and Formyl Peptide Receptor-Like 2
Authors: Kang Hyun Kyu , Lee Ha-Young , Kim Mi-Kyoung , Park Kyoung Sun , Park Yeong Min , Kwak Jong-Young , Bae Yoe-Sik
Published: April 2014

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) has been reported to stimulate monocytes, neutrophils, and dendritic cells (DCs). However, although WKYMVm has been reported to function as a DC chemoattractant, its role on DC maturation has not been examined. In this study, we investigated the effects of WKYMVm on human DC maturation. The costimulation of DCs with WKYMVm and LPS dramatically inhibited LPS-induced IL-12 production, CD86 and HLA-DR surface expression, and DC-mediated T cell proliferation. However, DC phagocytic activity was increased by WKYMVm stimulation. These findings demonstrate that WKYMVm inhibits DC maturation by LPS. In terms of the mechanism underlying DC maturation inhibition by WKYMVm, we found that LPS-induced DC maturation was negatively regulated by WKYMVm-stimulated ERK activity. Moreover, the costimulation of DCs with WKYMVm and LPS dramatically inhibited the LPS-induced accumulations of IL-12 mRNA, thus suggesting that WKYMVm inhibits LPS-induced IL-12 production at the transcriptional level. We also found that DCs express two WKYMVm receptors, formyl peptide receptor (FPR) and FPR-like 2 (FPRL2). In addition, formyl-Met-Leu-Phe (a FPR ligand), Trp-Lys-Tyr-Met-Val-Met, Hp(2–20) peptide, and F2L (three FPRL2 ligands) inhibited LPS-induced IL-12 production in DCs. Taken together, our findings indicate that the activations of FPR and FPRL2 inhibit LPS-induced DC maturation, and suggest that these two receptors should be regarded as important potential therapeutic targets for the modulation of DC maturation.
In Vivo Hydrodynamic Delivery of cDNA Encoding IL-2: Rapid, Sustained Redistribution, Activation of Mouse NK Cells, and Therapeutic Potential in the Absence of NKT Cells
Authors: Ortaldo John R. , Winkler-Pickett Robin T. , Bere Earl W. , Watanabe Morihiro , Murphy William J. , Wiltrout Robert H.
Published: April 2014

In the present study, we have tested the ability of hydrodynamically delivered IL-2 cDNA to modulate the number and function of murine leukocyte subsets in different organs and in mice of different genetic backgrounds, and we have evaluated effects of this mode of gene delivery on established murine tumor metastases. Hydrodynamic administration of the IL-2 gene resulted in the rapid and transient production of up to 160 ng/ml IL-2 in the serum. The appearance of IL-2 was followed by transient production of IFN-γ and a dramatic and sustained increase in NK cell numbers and NK-mediated cytolytic activity in liver and spleen leukocytes. In addition, significant increases in other lymphocyte subpopulations (e.g., NKT, T, and B cells) that are known to be responsive to IL-2 were observed following IL-2 cDNA plasmid delivery. Finally, hydrodynamic delivery of only 4 μg of the IL-2 plasmid to mice bearing established lung and liver metastases was as effective in inhibiting progression of metastases as was the administration of large amounts (100,000 IU/twice daily) of IL-2 protein. Studies performed in mice bearing metastatic renal cell tumors demonstrated that the IL-2 cDNA plasmid was an effective treatment against liver metastasis and moderately effective against lung metastasis. Collectively, these results demonstrate that hydrodynamic delivery of relatively small amounts of IL-2 cDNA provides a simple and inexpensive method to increase the numbers of NK and NKT cells, to induce the biological effects of IL-2 in vivo for use in combination with other biological agents, and for studies of its antitumor activity.
Bordetella pertussis Adenylate Cyclase Toxin Modulates Innate and Adaptive Immune Responses: Distinct Roles for Acylation and Enzymatic Activity in Immunomodulation and Cell Death
Authors: Boyd Aoife P. , Ross Pádraig J. , Conroy Helen , Mahon Nicola , Lavelle Ed C. , Mills Kingston H. G.
Published: April 2014

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-α, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties.
Phospholipase C-γ2 Is a Critical Signaling Mediator for Murine NK Cell Activating Receptors
Authors: Tassi Ilaria , Presti Rachel , Kim Sungjin , Yokoyama Wayne M. , Gilfillan Susan , Colonna Marco
Published: April 2014

Phospholipase C-γ (PLCγ) is a key regulator of intracellular Ca2+ mobilization. Two isoforms of PLCγ have been identified, PLCγ1 and PLCγ2. Previously, in vitro studies indicated that activating NK cell receptors signal through both isoforms. However, PLCγ2 deficiency alone was sufficient to induce a substantial impairment of NK cell-mediated cytotoxicity in vitro. Why PLCγ2 is more important than PLCγ1 for NK cell activation and whether PLCγ2 is also critical for NK cell development, secretion of IFN-γ, and clearance of viral infections in vivo is not known. In this study, we report that PLCγ2 is the predominant isoform expressed in murine NK cells. PLCγ2 deficiency did not affect NK cell numbers in bone marrow and spleen, but acquisition of Ly49 receptors by NK cells was partially impaired. PLCγ2-deficient NK cells exhibited a dramatic impairment of cytolytic function and IFN-γ production upon ligation of activating receptors, whereas they did secrete IFN-γ in response to cytokines. Consequently, mice lacking PLCγ2 controlled murine CMV infection substantially less effectively than did wild-type animals, and this defect was most evident in the spleen, where viral clearance mostly depends on NK cell lytic function. These results demonstrate that PLCγ2 is crucial for development of the NK cell receptor repertoire and signaling of activating NK cell receptors, mediating optimal NK cell function in vivo.
IL-21 Sustains CD28 Expression on IL-15-Activated Human Naive CD8+ T Cells
Authors: Alves Nuno L. , Arosa Fernando A. , van Lier René A. W.
Published: April 2014

Human naive CD8+ T cells are able to respond in an Ag-independent manner to IL-7 and IL-15. Whereas IL-7 largely maintains CD8+ T cells in a naive phenotype, IL-15 drives these cells to an effector phenotype characterized, among other features, by down-regulation of the costimulatory molecule CD28. We evaluated the influence of the CD4+ Th cell-derived common γ-chain cytokine IL-21 on cytokine-induced naive CD8+ T cell activation. Stimulation with IL-21 did not induce division and only slightly increased IL-15-induced proliferation of naive CD8+ T cells. Strikingly, however, IL-15-induced down-modulation of CD28 was completely prevented by IL-21 at the protein and transcriptional level. Subsequent stimulation via combined TCR/CD3 and CD28 triggering led to a markedly higher production of IL-2 and IFN-γ in IL-15/IL-21-stimulated cells compared with IL-15-stimulated T cells. Our data show that IL-21 modulates the phenotype of naive CD8+ T cells that have undergone IL-15 induced homeostatic proliferation and preserves their responsiveness to CD28 ligands.
Mechanisms of Gene Therapy for Tolerance: B7 Signaling Is Required for Peptide-IgG Gene-Transferred Tolerance Induction
Authors: Litzinger Mary T. , Su Yan , Lei Tie Chi , Soukhareva Nadejda , Scott David W.
Published: April 2014

LPS-activated B cells, transduced with IgG fusion proteins, are highly tolerogenic APCs. To analyze the mechanisms for this B cell-delivered gene therapy, we first followed the fate of CFSE-labeled B cell blasts. These cells primarily localized to the spleen, where a small population persisted for at least 1 mo after injection. By day 7 after injection, ∼95% of the transduced cells had divided at least once, presumably an effect of the in vitro LPS activation into the cycle, because resting cells did not divide. B cells from gld donors were not tolerogenic, initially suggesting a role for Fas ligand (FasL) in tolerance. Because transduced normal B cells expressed only low levels of FasL and did not kill Fas-expressing Jurkat or A20 B lymphoma cells in vitro, these data suggest that gld B cells are not tolerogenic due to unique characteristics of these B cells rather than the lack of functional FasL expression. The transduced B cell blasts displayed significant up-regulation of both B7 costimulatory molecules, and B7.2 up-regulation was maintained through day 7 in vivo. When B cells from B7 knockout donors were transduced to express Ig fusion proteins, they were not tolerogenic in two different mouse strains and Ag models. Moreover, anti-B7 Ab blocked tolerance induction in this model, a result consistent with a role for B7 in tolerance induction. We propose that tolerance may be induced in this model by B7-driven negative regulatory signaling, but tolerance is maintained by a lack of signal 2, because expression of B7 is eventually lost in vivo.
IL-23 Compensates for the Absence of IL-12p70 and Is Essential for the IL-17 Response during Tuberculosis but Is Dispensable for Protection and Antigen-Specific IFN-γ Responses if IL-12p70 Is Available
Authors: Khader Shabaana A. , Pearl John E. , Sakamoto Kaori , Gilmartin Leigh , Bell Guy K. , Jelley-Gibbs Dawn M. , Ghilardi Nico , deSauvage Fred , Cooper Andrea M.
Published: April 2014

IL-12p70 induced IFN-γ is required to control Mycobacterium tuberculosis growth; however, in the absence of IL-12p70, an IL-12p40-dependent pathway mediates induction of IFN-γ and initial bacteriostatic activity. IL-23 is an IL-12p40-dependent cytokine containing an IL-12p40 subunit covalently bound to a p19 subunit that is implicated in the induction of CD4 T cells associated with autoimmunity and inflammation. We show that in IL-23 p19-deficient mice, mycobacterial growth is controlled, and there is no diminution in either the number of IFN-γ-producing Ag-specific CD4 T cells or local IFN-γ mRNA expression. Conversely, there is an almost total loss of both IL-17-producing Ag-specific CD4 T cells and local production of IL-17 mRNA in these mice. The absence of IL-17 does not alter expression of the antimycobacterial genes, NO synthase 2 and LRG-47, and the absence of IL-23 or IL-17, both of which are implicated in mediating inflammation, fails to substantially affect the granulomatous response to M. tuberculosis infection of the lung. Despite this redundancy, IL-23 is required to provide a moderate level of protection in the absence of IL-12p70, and this protection correlates with a requirement for IL-23 in the IL-12p70-independent induction of Ag-specific, IFN-γ-producing CD4 T cells. We also show that IL-23 is required for the induction of an IL-17-producing Ag-specific phenotype in naive CD4 T cells in vitro and that absence of IL-12p70 promotes an increase in the number of IL-17-producing Ag-specific CD4 T cells both in vitro and in vivo.
Cross-Presentation of the Long-Lived Lymphocytic Choriomeningitis Virus Nucleoprotein Does Not Require Neosynthesis and Is Enhanced via Heat Shock Proteins
Authors: Basta Sameh , Stoessel Ricarda , Basler Michael , van den Broek Maries , Groettrup Marcus
Published: April 2014

Many viral proteins that contain MHC class I-restricted peptides are long-lived, and it is elusive how they can give rise to class I epitopes. Recently, we showed that direct presentation of an epitope of the long-lived lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) required neosynthesis in accordance with the defective ribosomal products hypothesis. In this study, we report that LCMV-NP can be cross-primed in mice using either LCMV-NP-transfected human HEK293 or BALB/c-derived B8 cells as Ag donor cells. In addition, we establish that contrary to direct presentation, cross-presentation required accumulation of the mature LCMV-NP and could not be sustained by the newly synthesized LCMV-NP protein, intermediate proteasomal degradation products, or the minimal NP396 epitope. Nevertheless, NP cross-presentation was enhanced by heat shock and was blunted by inhibitors of heat shock protein 90 and gp96. We propose that cross-presentation has evolved to sustain the presentation of stable viral proteins when their neosynthesis has ceased in infected donor cells.
Orally Tolerized T Cells Can Form Conjugates with APCs but Are Defective in Immunological Synapse Formation
Authors: Ise Wataru , Nakamura Kentaro , Shimizu Nobuko , Goto Hirofumi , Fujimoto Kenichiro , Kaminogawa Shuichi , Hachimura Satoshi
Published: April 2014

Oral tolerance is systemic immune hyporesponsiveness induced by the oral administration of soluble Ags. Hyporesponsiveness of Ag-specific CD4 T cells is responsible for this phenomenon. However, the molecular mechanisms underlying the hyporesponsive state of these T cells are not fully understood. In the present study, we investigated the ability of orally tolerized T cells to form conjugates with Ag-bearing APCs and to translocate TCR, protein kinase C-θ (PKC-θ), and lipid rafts into the interface between T cells and APCs. Orally tolerized T cells were prepared from the spleens of OVA-fed DO11.10 mice. Interestingly, the orally tolerized T cells did not show any impairment in the formation of conjugates with APCs. The conjugates were formed in a LFA-1-dependent manner. Upon antigenic stimulation, the tolerized T cells could indeed activate Rap1, which is critical for LFA-1 activation and thus cell adhesion. However, orally tolerized T cells showed defects in the translocation of TCR, PKC-θ, and lipid rafts into the interface between T cells and APCs. Translocation of TCR and PKC-θ to lipid raft fractions upon antigenic stimulation was also impaired in the tolerized T cells. Ag-induced activation of Vav, Rac1, and cdc42, which are essential for immunological synapse and raft aggregation, were down-regulated in orally tolerized T cells. These results demonstrate that orally tolerized T cells can respond to specific Ags in terms of conjugate formation but not with appropriate immunological synapse formation. This may account for the hyporesponsive state of orally tolerized T cells.
Enhanced Antibacterial Potential in UBP43-Deficient Mice against Salmonella typhimurium Infection by Up-Regulating Type I IFN Signaling
Authors: Kim Keun Il , Malakhova Oxana A. , Hoebe Kasper , Yan Ming , Beutler Bruce , Zhang Dong-Er
Published: April 2014

ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are dramatically induced upon viral or bacterial infection. We have generated a UBP43 knockout mouse model that is lacking an ISG15-specific isopeptidase to study the biological role of the protein ISGylation system. We report that UBP43-deficient mice are hypersensitive to LPS-induced lethality and that TIR domain-containing adapter inducing IFN-β → IFN regulatory factor 3 → type I IFN is the major axis to induce protein ISGylation and UBP43 expression in macrophages upon LPS treatment. In ubp43−/− macrophages, upon LPS treatment we detected increased expression of IFN-stimulated genes, including genes for several cytokines and chemokines involved in the innate immune response. The ubp43−/− mice were able to restrict the growth of Salmonella typhimurium more efficiently than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but a detriment to the body without strict control.
Antigen Presentation by an Immature Myeloid Dendritic Cell Line Does Not Cause CTL Deletion In Vivo, but Generates CD8+ Central Memory-Like T Cells That Can Be Rescued for Full Effector Function
Authors: Dumortier Hélène , van Mierlo Geertje J. D. , Egan Deirdre , van Ewijk Willem , Toes René E. M. , Offringa Rienk , Melief Cornelis J. M.
Published: April 2014

Immature dendritic cells (DC), in contrast to their mature counterparts, are incapable of mobilizing a CD8+ CTL response, and, instead, have been reported to induce CTL tolerance. We directly addressed the impact of immature vs mature DC on CTL responses by infusing adenovirus peptide-loaded DC (of the D1 cell line) into mice that had received adenovirus-specific naive TCR-transgenic CD8+ T cells. Whereas i.v. injection of mature DC triggered vigorous CTL expansion, immature DC elicited little proliferation involving only a minority of the TCR-transgenic CTL. Even though the latter CTL developed effector functions, including cytolytic activity and proinflammatory cytokine secretion, these cells differed significantly from CTL primed by mature DC in that they did not exhibit down-regulation of CD62L and CCR7, receptors involved in trapping of T cells in the lymphoid organs. Interestingly, adoptive transfer of CTL effector cells harvested after priming by either mature or immature DC into naive recipient mice, followed by exposure to adenovirus, yielded quantitatively and qualitatively indistinguishable CTL memory responses. Therefore, in vivo priming of naive CD8+ T cells by immature DC, although failing to induce a full-blown, systemic CTL response, resulted in the formation of central memory-like T cells that were able to expand and produce IFN-γ upon secondary antigenic stimulation.
CCR2 and CCR6, but Not Endothelial Selectins, Mediate the Accumulation of Immature Dendritic Cells within the Lungs of Mice in Response to Particulate Antigen
Authors: Osterholzer John J. , Ames Theresa , Polak Timothy , Sonstein Joanne , Moore Bethany B. , Chensue Stephen W. , Toews Galen B. , Curtis Jeffrey L.
Published: April 2014

Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II+CD11cmod cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.
Regulation of Follicular Dendritic Cell Networks by Activated T Cells: The Role of CD137 Signaling
Authors: Sun Yonglian , Blink Sarah E. , Chen Jonathan H. , Fu Yang-Xin
Published: April 2014

B cells, but not T cells, are considered to be important for the formation of follicular dendritic cell (FDC) clusters. Stimulation with agonist mAbs against CD137 (4-1BB), a TNFR family member primarily expressed on activated T cells, was effective in promoting T cell responses, but paradoxically suppressed T-dependent humoral immunity and autoantibody production in autoimmune disease models. Our present study shows that agonistic anti-CD137 treatment activates T cells, resulting in diminished FDC networks in B cell follicles, which are important components in T-dependent humoral immune responses both before and after the initiation of an immune response. Pretreatment with anti-CD137 before the secondary immunization inhibited memory Ab responses. Interestingly, CD137 costimulation-induced diminishment of FDC is T cell dependent. In addition, both CD4+ and CD8+ T cells are recruited into FDC area and are able to regulate FDCs by CD137 costimulation through a direct or indirect mechanism. These studies have revealed a previously unappreciated role of T cells in the regulation of FDC networks.
NKT Cells Are Critical for the Initiation of an Inflammatory Bowel Response againstToxoplasma gondii
Authors: Ronet Catherine , Darche Sylvie , de Moraes Maria Leite , Miyake Sachiko , Yamamura Takashi , Louis Jacques A. , Kasper Lloyd H. , Buzoni-Gatel Dominique
Published: April 2014

We demonstrated in this study the critical role of NKT cells in the lethal ileitis induced in C57BL/6 mice after infection with Toxoplasma gondii. This intestinal inflammation is caused by overproduction of IFN-γ in the lamina propria. The implication of NKT cells was confirmed by the observation that NKT cell-deficient mice (Jα281−/−) are more resistant than C57BL/6 mice to the development of lethal ileitis. Jα281−/− mice failed to overexpress IFN-γ in the intestine early after infection. This detrimental effect of NKT cells is blocked by treatment with α-galactosylceramide, which prevents death in C57BL/6, but not in Jα281−/−, mice. This protective effect is characterized by a shift in cytokine production by NKT cells toward a Th2 profile and correlates with an increased number of mesenteric Foxp3 lymphocytes. Using chimeric mice in which only NKT cells are deficient in the IL-10 gene and mice treated with anti-CD25 mAb, we identified regulatory T cells as the source of the IL-10 required for manifestation of the protective effect of α-galactosylceramide treatment. Our results highlight the participation of NKT cells in the parasite clearance by shifting the cytokine profile toward a Th1 pattern and simultaneously to immunopathological manifestation when this Th1 immune response remains uncontrolled.
In Vivo Assessment of the Relative Contributions of Deletion, Anergy, and Editing to B Cell Self-Tolerance
Authors: Hippen Keli L. , Schram Brian R. , Tze Lina E. , Pape Kathryn A. , Jenkins Marc K. , Behrens Timothy W.
Published: April 2014

In normal B cell development, a large percentage of newly formed cells bear receptors with high levels of self-reactivity that must be tolerized before entry into the mature B cell pool. We followed the fate of self-reactive B cells expressing high affinity anti-hen egg lysozyme (HEL) Ag receptors exposed in vivo to membrane HEL in a setting in which the anti-HEL L chain was “knocked-in” at the endogenous L chain locus. These mice demonstrated extensive and efficient L chain receptor editing responses and had B cell numbers comparable to those found in animals lacking membrane Ag. BrdU labeling indicated that the time required for editing in response to membrane HEL was ∼6 h. In mice transgenic for soluble HEL, anti-HEL B cells capable of editing showed evidence for both editing and anergy. These data identify receptor editing as a major physiologic mechanism by which highly self-reactive B cells are tolerized to membrane and soluble self-Ags.
Regulation of LFA-1 Activity through Cytoskeleton Remodeling and Signaling Components Modulates the Efficiency of HIV Type-1 Entry in Activated CD4+ T Lymphocytes
Authors: Tardif Mélanie R. , Tremblay Michel J.
Published: April 2014

Besides interactions between the viral envelope glycoproteins with cell surface receptors, interactions between cell-derived molecules incorporated onto virions and their ligand could also modulate HIV type-1 (HIV-1) entry inside CD4+ T lymphocytes. Although incorporation of host ICAM-1 within HIV-1 increases both virus attachment and fusion, the precise mechanism through which this phenomenon is occurring is still unclear. We demonstrate in this study that activation of primary human CD4+ T lymphocytes increases LFA-1 affinity and avidity states, two events promoting the early events of the HIV-1 replication cycle through interactions between virus-embedded host ICAM-1 and LFA-1 clusters. Confocal analyses suggest that HIV-1 is concentrated in microdomains rich in LFA-1 clusters that also contain CD4 and CXCR4 molecules. Experiments performed with specific inhibitors revealed that entry of HIV-1 in activated CD4+ T cells is regulated by LFA-1-dependent ZAP70, phospholipase Cγ1, and calpain enzymatic activities. By using laboratory and clinical strains of HIV-1 produced in primary human cells, we demonstrate the importance of the LFA-1 activation state and cluster formation in the initial step of the virus life cycle. Overall, these data provide new insights into the complex molecular events involved in HIV-1 binding and entry.
Genetic Dissection of the Murine Lupus Susceptibility LocusSle2: Contributions to Increased Peritoneal B-1a Cells and Lupus Nephritis Map to Different Loci
Authors: Xu Zhiwei , Duan Biyan , Croker Byron P. , Wakeland Edward K. , Morel Laurence
Published: April 2014

Lupus pathogenesis in the NZM2410 mouse model results from the expression of multiple interacting susceptibility loci. Sle2 on chromosome 4 was significantly linked to glomerulonephritis in a linkage analysis of a NZM2410 × B6 cross. Yet, Sle2 expression alone on a C57BL/6 background did not result in any clinical manifestation, but in an abnormal B cell development, including the accumulation of B-1a cells in the peritoneal cavity and spleen. Analysis of B6.Sle2 congenic recombinants showed that at least three independent loci, New Zealand White-derived Sle2a and Sle2b, and New Zealand Black-derived Sle2c, contribute to an elevated number of B-1a cells, with Sle2c contribution being the strongest of the three. To determine the contribution of these three Sle2 loci to lupus pathogenesis, we used a mapping by genetic interaction strategy, in which we bred them to B6.Sle1.Sle3 mice. We then compared the phenotypes of these triple congenic mice with that of previously characterized B6.Sle1.Sle2.Sle3, which express the entire Sle2 interval in combination with Sle1 and Sle3. Sle2a and Sle2b, but not Sle2c, contributed significantly to lupus pathogenesis in terms of survival rate, lymphocytic expansion, and kidney pathology. These results show that the Sle2 locus contains several loci affecting B cell development, with only the two NZW-derived loci having the least effect of B-1a cell accumulation significantly contributing to lupus pathogenesis.
Lipopolysaccharide Injection Induces Relapses of Experimental Autoimmune Encephalomyelitis in Nontransgenic Mice via Bystander Activation of Autoreactive CD4+ Cells
Authors: Nogai Axel , Siffrin Volker , Bonhagen Kerstin , Pfueller Caspar F. , Hohnstein Thordis , Volkmer-Engert Rudolf , Brück Wolfgang , Stadelmann Christine , Kamradt Thomas
Published: April 2014

Infections sometimes associate with exacerbations of autoimmune diseases through pathways that are poorly understood. Ag-specific mechanisms such as cross-reactivity between a microbial Ag and a self-Ag have received no direct support. In this study, we show that injection of LPS induces experimental autoimmune encephalomyelitis in TCR-transgenic mice and relapse of encephalomyelitis in normal mice. This form of treatment induces proliferation and cytokine production in a fraction of effector/memory Th lymphocytes in vitro via physical contact of Th cells with CD4− LPS-responsive cells. TCR-mediated signals are not necessary; rather what is required is ligation of costimulatory receptors on Th cells by costimulatory molecules on the CD4− cells. This form of bystander activation provides an Ag-independent link between infection and autoimmunity that might fit the clinical and epidemiological data on the connection between infection and autoimmunity better than the Ag-specific models.
Calnexin and ERp57 Facilitate the Assembly of the Neonatal Fc Receptor for IgG with β2-Microglobulin in the Endoplasmic Reticulum
Authors: Zhu Xiaoping , Peng Junmin , Chen Daohong , Liu Xindong , Ye Lilin , Iijima Hideki , Kadavil Kumar , Lencer Wayne I. , Blumberg Richard S.
Published: April 2014

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in nonconvalent association with β2-microglobulin (β2m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the β2m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and β2m, CNX bound to the FcRn H chain before the FcRn H chain association with β2m. However, calreticulin only bound to the FcRn H chain-β2m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for β2m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.
α-Lipoic Acid Inhibits Inflammatory Bone Resorption by Suppressing Prostaglandin E2 Synthesis
Authors: Ha Hyunil , Lee Jong-Ho , Kim Ha-Neui , Kim Hyun-Man , Kwak Han Bok , Lee Seungbok , Kim Hong-Hee , Lee Zang Hee
Published: April 2014

α-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-κB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.
B7+ Iris Pigment Epithelium Induce CD8+ T Regulatory Cells; Both Suppress CTLA-4+ T Cells
Authors: Sugita Sunao , Ng Tat Fong , Lucas Philip J. , Gress Ronald E. , Streilein J. Wayne
Published: April 2014

Ocular pigment epithelia contribute to immune privilege by suppressing T cell activation and converting T cells into regulatory T regulatory cells (Tregs) that inhibit bystander T cell activation. Iris pigment epithelium (IPE) does so through direct cell-cell contact with naive T cells, and this suppressive contact is via interactions between B7 expressed constitutively on IPE cells and CTLA-4 expressed on a subpopulation of CD8+ T cells. We have now examined whether TGFβ is required in this process. We report that IPE produces both soluble and membrane-bound active TGFβ, but that only the latter is actually delivered to CD8+ T cells. In turn, these T cells become IPE Tregs by up-regulating their own expression of B7-1/B7-2 and soluble and membrane-bound TGFβ. IPE Tregs through their expression of B7 are able to engage CTLA-4+ bystander T cells, and thus precisely, target delivery of membrane-bound TGFβ. We propose that this mechanism of suppression via TGFβ ensures that soluble active TGFβ is not released into the ocular microenvironment where it can have unregulated and deleterious effects, including elevation of intraocular pressure and development of glaucoma.
Differential Gene Expression in Endometrium, Endometrial Lymphocytes, and Trophoblasts during Successful and Abortive Embryo Implantation
Authors: Tayade Chandrakant , Black Gordon P. , Fang Yuan , Croy B. Anne
Published: April 2014

Prenatal mortality reaching 30% occurs during the first weeks of gestation in commercial swine. Mechanisms for this are unknown although poor uterine blood supply has been postulated. In other species, vascular endothelial growth factor, hypoxia-inducible factor 1-α, and IFN-γ regulate gestational endometrial angiogenesis. Vascular endothelial growth factor and hypoxia-inducible factor 1-α are also important for placental angiogenesis while trophoblastic expression of Fas ligand is thought to protect conceptuses against immune-mediated pregnancy loss. In this study, we document dynamic, peri-implantation differences in transcription of genes for angiogenesis, cytokine production, and apoptosis regulation in the endometrium, and laser capture microdissected endometrial lymphocytes and trophoblasts associated with healthy or viable but arresting porcine fetuses. In healthy implantation sites, endometrial gene expression levels differed between anatomic subregions and endometrial lymphocytes showed much greater transcription of angiogenic genes than trophoblasts. In arresting fetal sites, uterine lymphocytes had no angiogenic gene transcription and showed rapid elevation in transcription of proinflammatory cytokines Fas and Fas ligand while trophoblasts showed elevated transcription of IFN-γ and Fas. This model of experimentally accessible spontaneous fetal loss, involving blocked maternal angiogenesis, should prove valuable for further investigations of peri-implantation failure of normally conceived and surgically transferred embryos in many species, including the human.
A Key Regulatory Role for Histamine in Experimental Autoimmune Encephalomyelitis: Disease Exacerbation in Histidine Decarboxylase-Deficient Mice
Authors: Musio Silvia , Gallo Barbara , Scabeni Stefano , Lapilla Marilena , Poliani Pietro L. , Matarese Giuseppe , Ohtsu Hiroshi , Galli Stephen J. , Mantegazza Renato , Steinman Lawrence , Pedotti Rosetta
Published: April 2014

Histamine can modulate the cytokine network and influence Th1 and Th2 balance and Ab-isotype switching. Thus, pharmacological blockade or genetic deletion of specific histamine receptors has been shown to reduce the severity of experimental autoimmune encephalomyelitis (EAE), a prototypic Th1-mediated disease with similarities to human multiple sclerosis. To study the comprehensive contribution of endogenous histamine to the expression of EAE, we attempted to induce EAE in histidine decarboxylase-deficient mice, which are genetically unable to make histamine. In this study, we show that EAE is significantly more severe in HDC−/−, histamine-deficient mice, with diffuse inflammatory infiltrates, including a prevalent granulocytic component, in the brain and cerebellum. Unlike splenocytes from wild-type mice, splenocytes from HDC−/− mice do not produce histamine in response to the myelin Ag, whereas production of IFN-γ, TNF, and leptin are increased in HDC−/− splenocytes in comparison to those from wild-type mice. Endogenous histamine thus appears to regulate importantly the autoimmune response against myelin and the expression of EAE, in this model, and to limit immune damage to the CNS. Understanding which receptor(s) for histamine is/are involved in regulating autoimmunity against the CNS might help in the development of new strategies of treatment for EAE and multiple sclerosis.
The Glycosaminoglycan-Binding Domain of Decoy Receptor 3 Is Essential for Induction of Monocyte Adhesion
Authors: Chang Yung-Chi , Chan Yueh-Hsuan , Jackson David G. , Hsieh Shie-Liang
Published: April 2014

Decoy receptor 3 (DcR3), a soluble receptor for Fas ligand, LIGHT (homologous to lymphotoxins shows inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes), and TNF-like molecule 1A, is highly expressed in cancer cells and in tissues affected by autoimmune disease. DcR3.Fc has been shown to stimulate cell adhesion and to modulate cell activation and differentiation by triggering multiple signaling cascades that are independent of its three known ligands. In this study we found that DcR3.Fc-induced cell adhesion was inhibited by heparin and heparan sulfate, and that DcR3.Fc was unable to bind Chinese hamster ovary K1 mutants defective in glycosaminoglycan (GAG) synthesis. Furthermore, the negatively charged, sulfated GAGs of cell surface proteoglycans, but not their core proteins, were identified as the binding sites for DcR3.Fc. A potential GAG-binding site was found in the C-terminal region of DcR3, and the mutation of three basic residues, i.e., K256, R258, and R259, to alanines abolished its ability to trigger cell adhesion. Moreover, a fusion protein comprising the GAG-binding region of DcR3 with an Fc fragment (DcR3_HBD.Fc) has the same effect as DcR3.Fc in activating protein kinase C and inducing cell adhesion. Compared with wild-type THP-1 cells, cell adhesion induced by DcR3.Fc was significantly reduced in both CD44v3 and syndecan-2 knockdown THP-1 cells. Therefore, we propose a model in which DcR3.Fc may bind to and cross-link proteoglycans to induce monocyte adhesion.
Gap Junction-Mediated Intercellular Communication between Dendritic Cells (DCs) Is Required for Effective Activation of DCs
Authors: Matsue Hiroyuki , Yao Jian , Matsue Keiko , Nagasaka Akiko , Sugiyama Hideaki , Aoki Rui , Kitamura Masanori , Shimada Shinji
Published: April 2014

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-γ or TNF-α plus IFN-γ. LPS- plus IFN-γ-induced dye-coupling was mediated by DC-derived TNF-α. In addition, CpG plus IFN-γ induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-α. LPS- plus IFN-γ-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-γ-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.