bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipoic Acid Affects Cellular Migration into the Central Nervous System and Stabilizes Blood-Brain Barrier Integrity
Authors: Schreibelt Gerty , Musters René J. P. , Reijerkerk Arie , de Groot Lody R. , van der Pol Susanne M. A. , Hendrikx Esther M. L. , Döpp Ed D. , Dijkstra Christine D. , Drukarch Benjamin , de Vries Helga E.
Published: April 2014

Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis (MS) pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction of the blood-brain barrier (BBB). In this study, we describe the beneficial effect of the antioxidant α-lipoic acid (LA) on these phenomena. In vivo, LA dose-dependently prevented the development of clinical signs in a rat model for MS, acute experimental allergic encephalomyelitis (EAE). Clinical improvement was coupled to a decrease in leukocyte infiltration into the CNS, in particular monocytes. Monocytes isolated from the circulation of LA-treated rats revealed a reduced migratory capacity to cross a monolayer of rat brain endothelial cells in vitro compared with monocytes isolated from untreated EAE controls. Using live cell imaging techniques, we visualized and quantitatively assessed that ROS are produced within minutes upon the interaction of monocytes with brain endothelium. Monocyte adhesion to an in vitro model of the BBB subsequently induced enhanced permeability, which could be inhibited by LA. Moreover, administration of exogenous ROS to brain endothelial cells induced cytoskeletal rearrangements, which was inhibited by LA. In conclusion, we show that LA has a protective effect on EAE development not only by affecting the migratory capacity of monocytes, but also by stabilization of the BBB, making LA an attractive therapeutic agent for the treatment of MS.
Human Mast Cell-Derived Gelatinase B (Matrix Metalloproteinase-9) Is Regulated by Inflammatory Cytokines: Role in Cell Migration
Authors: Di Girolamo Nick , Indoh Ikuko , Jackson Nicole , Wakefield Denis , McNeil H. Patrick , Yan Weixing , Geczy Carolyn , Arm Jonathan P. , Tedla Nicodemus
Published: April 2014

Mast cells are key effectors in the pathogenesis of inflammatory and tissue destructive diseases such as rheumatoid arthritis (RA). These cells contain specialized secretory granules loaded with bioactive molecules including cytokines, growth factors, and proteases that are released upon activation. This study investigated the regulation of matrix metalloproteinase MMP-9 (gelatinase B) in human mast cells by cytokines that are known to be involved in the pathogenesis of RA. Immunohistochemical staining of synovial tissue showed abundant expression of MMP-9 by synovial tissue mast cells in patients with RA but not in normal controls. The expression, activity, and production of MMP-9 in mast cells was confirmed by RT-PCR, zymography, and Western blotting using cord blood-derived human mast cells (CB-HMC). Treatment of CB-HMC with TNF-α significantly increased the expression of MMP-9 mRNA and up-regulated the activity of MMP-9 in a time- and dose-dependent manner. By contrast, IFN-γ inhibited MMP-9 mRNA and protein expression. The cytokine-mediated regulation of MMP-9 was also apparent in the human mast cell line (HMC-1) and in mouse bone marrow-derived mast cells. Furthermore, TNF-α significantly increased the invasiveness of CB-HMC across Matrigel-coated membranes while the addition of IFN-γ, rTIMP-1, or pharmacological MMP inhibitors significantly reduced this process. These observations suggest that MMP-9 is not a stored product in mast cells but these cells are capable of producing this enzyme under inflammatory conditions that may facilitate the migration of mast cell progenitors to sites of inflammation and may also contribute to local tissue damage.
Monocyte-Derived Human Macrophages Mediate Anergy in Allogeneic T Cells and Induce Regulatory T Cells
Authors: Hoves Sabine , Krause Stefan W. , Schütz Christian , Halbritter Dagmar , Schölmerich Jürgen , Herfarth Hans , Fleck Martin
Published: April 2014

Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (Mφ) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and Mφ in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with Mφ despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with Mφ, whereas high amounts of IL-2 and IFN-γ were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with Mφ and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by Mφ. Functional analysis of T cells rescued from cocultures with Mφ demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by Mφ. These results demonstrate that Mφ drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that Mφ maintain peripheral tolerance in vivo.
The Antineoplastic Agent Bryostatin-1 Differentially Regulates IFN-γ Receptor Subunits in Monocytic Cells: Transcriptional and Posttranscriptional Control of IFN-γR2
Authors: Garcia Carmen S. , Curiel Rafael E. , Mwatibo James M. , Pestka Sidney , Li Huifen , Espinoza-Delgado Igor
Published: April 2014

Bryostatin-1 (Bryo-1) is a potent ligand and modulator of protein kinase C that exerts antineoplastic and immunomodulatory activities both in vitro and in vivo. We have previously reported that Bryo-1 synergized with IFN-γ to induce NO synthase and NO by macrophages. To determine whether this effect was associated with changes in levels of IFN-γR, we investigated the effects of Bryo-1 on the expression and regulation of IFN-γR chains in monocytic cells. Northern blot analysis revealed that Bryo-1 treatment of the human monocytic cell lines MonoMac6 and THP-1 and human monocytes enhanced the expression of IFN-γR2 mRNA but did not affect IFN-γR1 mRNA expression. Bryo-1 increased IFN-γR2 mRNA in a dose-dependent manner as early as 3 h posttreatment. Bryo-1-induced up-regulation of IFN-γR2 mRNA levels is not dependent on de novo protein synthesis as shown by cell treatment with the protein-synthesis inhibitor cycloheximide. Bryo-1 treatment increased the IFN-γR2 mRNA half-life by 2 h. EMSA analysis from Bryo-1-treated MonoMac6 cells showed an increased nuclear protein binding to the NF-κB motif present in the 5′ flanking region of the human IFN-γR2 promoter that was markedly decreased by pretreatment with the NF-κB inhibitor SN50. These results show for the first time that Bryo-1 up-regulates IFN-γR2 expression in monocytic cells. Given the pivotal role that IFN-γ exerts on monocyte activation and in the initiation and outcome of the immune response, the induction of IFN-γR2 by Bryo-1 has significant implications in immunomodulation and could overcome some of the immune defects observed in cancer patients.
Cutting Edge: Decreased Accumulation and Regulatory Function of CD4+CD25high T Cells in Human STAT5b Deficiency
Authors: Cohen Aileen C. , Nadeau Kari C. , Tu Wenwei , Hwa Vivian , Dionis Kira , Bezrodnik Liliana , Teper Alejandro , Gaillard Maria , Heinrich Juan , Krensky Alan M. , Rosenfeld Ron G. , Lewis David B.
Published: April 2014

We show that STAT5b is important for the in vivo accumulation of CD4+CD25high T cells with regulatory cell function. A patient homozygous for a missense A630P STAT5b mutation displayed immune dysregulation and decreased numbers of CD4+CD25high T cells. STAT5bA630P/A630P CD4+CD25high T cells had low expression of forkhead box P3 and an impaired ability to suppress the proliferation of or to kill CD4+CD25− T cells. Expression of CD25, a component of the high-affinity IL-2R, was also reduced in response to IL-2 or after in vitro propagation. The impact of the STAT5b mutation was selective in that IL-2-mediated up-regulation of the common γ-chain cytokine receptor and perforin, and activation-induced expressions of CD154 and IFN-γ were normal. These results indicate that STAT5b propagates an important IL-2-mediated signal for the in vivo accumulation of functional regulatory T cells.
Molecular and Functional Characterization of CD300b, a New Activating Immunoglobulin Receptor Able to Transduce Signals through Two Different Pathways
Authors: Martínez-Barriocanal Águeda , Sayós Joan
Published: April 2014

In this study, we describe the characterization of human CD300b, a novel member of the CMRF-35/immune receptor expressed by myeloid cell (IREM) multigene family of immune receptors. Immune receptor expressed by myeloid cell-3 cDNA was cloned from a PHA-activated PBMC library and RT-PCR revealed the gene to be expressed preferentially in cells of myeloid origin. The CD300b cDNA open reading frame encodes a 201-aa type I protein composed of a single extracellular Ig V-type domain followed by a transmembrane region containing a positively charged residue (lysine) which is a common feature among receptors that associate with activating adaptor proteins. Indeed, CD300b was able to associate with DNAX-activating protein of 12 kDa (DAP-12) and deliver different activating signals through this ITAM-based adaptor. Unusually for an activating receptor, the 29-aa cytoplasmic tail of CD300b contains a tyrosine-based motif that, upon c-Fyn phosphorylation, became a docking site for the intracellular signaling mediator growth factor receptor-bound protein 2. Moreover, in the absence of DAP-12, CD300b was able to activate NFAT/AP-1-dependent transcriptional activity in RBL-2H3 cells. This activity could be abolished only by mutating both the cytoplasmic tyrosine and the transmembrane lysine. Our data suggest the existence of an unidentified molecule capable of interacting with CD300b through a charged residue of the transmembrane region and allowing receptor signaling independent of DAP-12. Therefore, CD300b defines a nonclassical Ig receptor able to trigger signals by coupling distinct mediators and thus initiating different signaling pathways.
P2X7 Receptor-Dependent Cell Death Is Modulated during Murine T Cell Maturation and Mediated by Dual Signaling Pathways
Authors: Tsukimoto Mitsutoshi , Maehata Machiko , Harada Hitoshi , Ikari Akira , Takagi Kuniaki , Degawa Masakuni
Published: April 2014

Extracellular ATP causes apoptosis and/or necrosis of the hemopoietic lineage through the activation of P2X7 receptors. In this study, we investigated P2X7 receptor-mediated cell death during murine T cell maturation. The expression level and activity of P2X7 receptors, as measured by induction of cell death and pore formation, were higher in splenocytes than thymocytes. Flow cytometric analysis revealed that cell shrinkage was induced by activation of the P2X7 receptor in murine lymphocytes and the responding cells were T cells. Splenic T cells were more responsive than their thymic counterpart. These observations indicate that the system of P2X7 receptor-mediated cell death in T cells could be modulated during T cell maturation. Furthermore, decreased extracellular Cl− suppressed ATP-induced cell shrinkage in splenocytes without inhibiting ERK1/2 phosphorylation, which is reported to mediate necrotic cell death. Treatment with U0126 (a MEK inhibitor) suppressed ATP-induced ERK1/2 phosphorylation without inhibiting cell shrinkage. Moreover, decreased extracellular Cl− and treatment with U0126 suppressed ATP-induced cell death. These observations indicate that the activation of P2X7 receptor leads to T cell death by two independent pathways, one of which is cell shrinkage dependent and the other of which involves the phosphorylation of ERK1/2. In conclusion, we demonstrate increasing P2X7 receptor activity during T cell maturation and the existence of two essential pathways in P2X7 receptor-mediated T cell death. Our findings suggest that ATP-induced cell death of peripheral T lymphocytes is important in P2X7 receptor-regulated immune responses.
T Helper Lymphocytes Rescue CTL from Activation-Induced Cell Death
Authors: Kennedy Richard , Celis Esteban
Published: April 2014

T cell activation is characterized by a vast expansion of Ag-specific T cells followed by an equally extensive reduction in T cell numbers. This decline is due, in part, to activation-induced apoptosis of the responding T cells during repeated encounter with Ag. In the current study, we used solid-phase MHC class I/peptide monomers to cause activation-induced cell death (AICD) of previously activated CD8 T cells in an Ag-specific manner. AICD occurred rapidly and was mediated primarily by Fas–FasL interactions. Most interestingly, we observed that Th cells could provide survival signals to CTL significantly reducing the level of AICD. Both Th1 and Th2 subsets were capable of protecting CTL from AICD, and a major role for soluble factors in this protection was ruled out, as cell-to-cell contact was an essential component of this Th-mediated protection. Upon encounter with Ag-expressing tumor cells, CTL underwent significant apoptosis. However, in the presence of Th cells, the CTL not only were protected against death, but also had significantly greater lytic ability. In vivo tumor protection studies using peptide immunization showed that the activation of Ag-specific Th cells was crucial for optimal protection, but did not affect the magnitude of the CTL response in the lymphoid tissues. In this study, we examine the type of help that CD4 T cells may provide and propose a model of Th cell–CTL interaction that reduces CTL death. Our results show a novel role for Th cells in the maintenance of CTL responses.
Primitive Lymphoid Progenitors in Bone Marrow with T Lineage Reconstituting Potential
Authors: Perry S. Scott , Welner Robert S. , Kouro Taku , Kincade Paul W. , Sun Xiao-Hong
Published: April 2014

Multiple subsets of the bone marrow contain T cell precursors, but it remains unclear which is most likely to replenish the adult thymus. Therefore, RAG-1+ early lymphoid progenitors (RAG-1+ ELP), and CD62L/L-selectin+ progenitors (LSP), as well as common lymphoid progenitors from C57BL6-Thy1.1-RAG-1/GFP mouse bone marrow were directly compared in transplantation assays. The two c-Kithigh populations vigorously regenerated the thymus and were superior to common lymphoid progenitors in magnitude and frequency of thymic reconstitution. Regeneration was much faster than the 22 days described for transplanted stem cells, and RAG-1+ ELP produced small numbers of lymphocytes within 13 days. As previously reported, LSP were biased to a T cell fate, but this was not the case for RAG-1+ ELP. Although RAG-1+ ELP and LSP had reduced myeloid potential, they were both effective progenitors for T lymphocytes and NK cells. The LSP subset overlapped with and included most RAG-1+ ELP and many RAG-1−TdT+ ELP. LSP and RAG-1+ ELP were both present in the peripheral circulation, but RAG-1+ ELP had no exact counterpart among immature thymocytes. The most primitive of thymocytes were similar to Lin−c-KithighL-selectin+TdT+RAG-1− progenitors present in the marrow, suggesting that this population is normally important for sustaining the adult thymus.
Addition of a Prominent Epitope Affects Influenza A Virus-Specific CD8+ T Cell Immunodominance Hierarchies When Antigen Is Limiting
Authors: Jenkins Misty Rayna , Webby Richard , Doherty Peter C. , Turner Stephen J.
Published: April 2014

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA257–264) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HK×31 (H3N2) influenza A viruses. Initial characterization determined that KbOVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering Db nucleoprotein (NP)366 presentation. There were similar levels of KbOVA257- and DbNP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant KbOVA257-specific response resulted in diminished Db acidic polymerase224- and Kb basic polymerase subunit 1703-, but not DbNP366-specific responses and didn’t alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of KbOVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.
CD38 Is Required for the Peripheral Survival of Immunotolerogenic CD4+ Invariant NK T Cells in Nonobese Diabetic Mice
Authors: Chen Yi-Guang , Chen Jing , Osborne Melissa A. , Chapman Harold D. , Besra Gurdyal S. , Porcelli Steven A. , Leiter Edward H. , Wilson S. Brian , Serreze David V.
Published: April 2014

T cell-mediated autoimmune type-1 diabetes (T1D) in NOD mice partly results from this strain’s numerical and functional defects in invariant NK T (iNKT) cells. T1D is inhibited in NOD mice treated with the iNKT cell superagonist α-galactosylceramide through a process involving enhanced accumulation of immunotolerogenic dendritic cells in pancreatic lymph nodes. Conversely, T1D is accelerated in NOD mice lacking CD38 molecules that play a role in dendritic cell migration to inflamed tissues. Unlike in standard NOD mice, α-galactosylceramide pretreatment did not protect the CD38-deficient stock from T1D induced by an adoptively transferred pancreatic β cell-autoreactive CD8 T cell clone (AI4). We found that in the absence of CD38, ADP-ribosyltransferase 2 preferentially activates apoptotic deletion of peripheral iNKT cells, especially the CD4+ subset. Therefore, this study documents a previously unrecognized role for CD38 in maintaining survival of an iNKT cell subset that preferentially contributes to the maintenance of immunological tolerance.
CD8+ Cytotoxic T-APC Stimulate Central Memory CD8+ T Cell Responses via Acquired Peptide-MHC Class I Complexes and CD80 Costimulation, and IL-2 Secretion
Authors: Xia Dajing , Hao Siguo , Xiang Jim
Published: April 2014

We previously showed that naive CD4+ Th cells acquire peptide-MHC class I (pMHC I) and costimulatory molecules from OVA-pulsed dendritic cells (DCOVA), and act as Th-APCs in stimulation of CD8+CTL responses. In this study, we further demonstrated that naive CD8+ cytotoxic T (Tc) cells also acquire pMHC I and costimulatory CD54 and CD80 molecules by DCOVA stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DCOVA and neighboring Tc-APC, and stimulating OVA-specific CD8+ central memory T responses and antitumor immunity. Interestingly, the stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).
G12/G13 Family G Proteins Regulate Marginal Zone B Cell Maturation, Migration, and Polarization
Authors: Rieken Stefan , Sassmann Antonia , Herroeder Susanne , Wallenwein Barbara , Moers Alexandra , Offermanns Stefan , Wettschureck Nina
Published: April 2014

G protein-coupled receptors play an important role in the regulation of lymphocyte functions such as migration, adhesion, proliferation, and differentiation. Although the role of Gi family G proteins has been intensively studied, no in vivo data exist with respect to G12/G13 family G proteins. We show in this study that mice that lack the G protein α-subunits Gα12 and Gα13 selectively in B cells show significantly reduced numbers of splenic marginal zone B (MZB) cells, resulting in a delay of Ab production in response to thymus-independent Ags. Basal and chemokine-induced adhesion to ICAM-1 and VCAM-1, two adhesion molecules critically involved in MZB localization, is normal in mutant B cells, and the same is true for chemokine-induced migration. However, migration in response to serum and sphingosine 1-phosphate is strongly increased in mutant MZB cells, but not in mutant follicular B cells. Live-cell imaging studies revealed that Gα12/Gα13-deficient MZB cells assumed more frequently an ameboid form than wild-type cells, and pseudopod formation was enhanced. In addition to their regulatory role in serum- and sphingosine 1-phosphate-induced migration, G12/G13 family G proteins seem to be involved in peripheral MZB cell maturation, because also splenic MZB cell precursors are reduced in mutant mice, although less prominently than mature MZB cells. These data suggest that G12/G13 family G proteins contribute to the formation of the mature MZB cell compartment both by controlling MZB cell migration and by regulating MZB cell precursor maturation.
Neonatal and Adult CD4+CD3− Cells Share Similar Gene Expression Profile, and Neonatal Cells Up-Regulate OX40 Ligand in Response to TL1A (TNFSF15)
Authors: Kim Mi-Yeon , Toellner Kai-Michael , White Andrea , McConnell Fiona M. , Gaspal Fabrina M. C. , Parnell Sonia M. , Jenkinson Eric , Anderson Graham , Lane Peter J. L.
Published: April 2014

We report here the quantitative expression of a set of immunity-related genes, including TNF family members, chemokine receptors, and transcription factors, in a CD4+CD3− accessory cell. By correlating gene expression between cell-sorted populations of defined phenotype, we show that the genetic fingerprint of these CD4+CD3− cells is distinct from dendritic cells, plasmacytoid dendritic cells, T cells, B cells, and NK cells. In contrast, it is highly similar to CD4+CD3− cells isolated from embryonic and neonatal tissues, with the exception that only adult populations express OX40L and CD30L. We have previously reported that IL-7 signals regulate CD30L expression. In the present study, we show that both neonatal and adult CD4+CD3− cells express the TNF family member, death receptor 3 (TNFRSF25), and that addition of TL1A (TNFSF15), the ligand for death receptor 3, up-regulates OX40L on neonatal CD4+CD3− cells. Finally, we demonstrate that this differentiation occurs in vivo: neonatal CD4+CD3− cells up-regulate both CD30L and OX40L after adoptive transfer into an adult recipient.
Expression and Release of HLA-E by Melanoma Cells and Melanocytes: Potential Impact on the Response of Cytotoxic Effector Cells
Authors: Derré Laurent , Corvaisier Murielle , Charreau Béatrice , Moreau Anne , Godefroy Emmanuelle , Moreau-Aubry Agnès , Jotereau Francine , Gervois Nadine
Published: April 2014

HLA-E are nonclassical MHC molecules with poorly characterized tissue distribution and functions. Because of their capacity to bind the inhibitory receptor, CD94/NKG2A, expressed by NK cells and CTL, HLA-E molecules might play an important role in immunomodulation. In particular, expression of HLA-E might favor tumor cell escape from CTL and NK immunosurveillance. To address the potential role of HLA-E in melanoma immunobiology, we assessed the expression of these molecules ex vivo in human melanoma biopsies and in melanoma and melanocyte cell lines. Melanoma cell lines expressed no or low surface, but significant intracellular levels of HLA-E. We also report for the first time that some of them produced a soluble form of this molecule. IFN-γ significantly increased the surface expression of HLA-E and the shedding of soluble HLA-E by these cells, in a metalloproteinase-dependent fashion. In contrast, melanocyte cell lines constitutively expressed HLA-E molecules that were detectable both at the cell surface and in the soluble form, at levels that were poorly affected by IFN-γ treatment. On tumor sections, a majority of tumor cells of primary, but a low proportion of metastatic melanomas (30–70 and 10–20%, respectively), expressed HLA-E. Finally, HLA-E expression at the cell surface of melanoma cells decreased their susceptibility to CTL lysis. These data demonstrate that HLA-E expression and shedding are normal features of melanocytes, which are conserved in melanoma cells of primary tumors, but become dependent on IFN-γ induction after metastasis. The biological significance of these findings warrants further investigation.
Analysis of FOXP3 Reveals Multiple Domains Required for Its Function as a Transcriptional Repressor
Authors: Lopes Jared E. , Torgerson Troy R. , Schubert Lisa A. , Anover Stephanie D. , Ocheltree Elizabeth L. , Ochs Hans D. , Ziegler Steven F.
Published: April 2014

Foxp3 has been shown to be both necessary and sufficient for the development and function of naturally arising CD4+CD25+ regulatory T cells in mice. Mutation of Foxp3 in Scurfy mice and FOXP3 in humans with IPEX results in fatal, early onset autoimmune disease and demonstrates the critical role of FOXP3 in maintaining immune homeostasis. The FOXP3 protein encodes several functional domains, including a C2H2 zinc finger, a leucine zipper, and a winged-helix/forkhead (FKH) domain. We have shown previously that FOXP3 functions as a transcriptional repressor and inhibits activation-induced IL-2 gene transcription. To characterize the role of each predicted functional domain on the in vivo activity of FOXP3, we have evaluated the location of point mutations identified in a large cohort of patients with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) and found them to cluster primarily within the FKH domain and the leucine zipper, but also present within the poorly defined N-terminal portion of the protein. The molecular functions of each of the IPEX-targeted domains were investigated. We show that FOXP3 is constitutively localized to the nucleus and this localization requires sequences at both the amino and C-terminal ends of its FKH domain. Moreover, FOXP3 was found to homodimerize through its leucine zipper. We also identify a novel functional domain within the N-terminal half of FOXP3, which is required for FOXP3-mediated repression of transcription from both a constitutively active and a NF-AT-inducible promoter. Furthermore, we demonstrate that IPEX mutations in these domains correlate with deficiencies in FOXP3 repressor function, corroborating their in vivo relevance.
Leukotrienes Are Essential for the Control ofLeishmania amazonensisInfection and Contribute to Strain Variation in Susceptibility
Authors: Serezani Carlos H. , Perrela Joao H. , Russo Momtchilo , Peters-Golden Marc , Jancar Sonia
Published: April 2014

Leukotrienes (LTs) are known to be produced by macrophages when challenged with Leishmania, but it is not known whether these lipid mediators play a role in host defense against this important protozoan parasite. In this study, we investigated the involvement of LTs in the in vitro and in vivo response to Leishmania amazonensis infection in susceptible (BALB/c) and resistant (C3H/HePAS) mice. Pharmacologic or genetic deficiency of LTs resulted in impaired leishmanicidal activity of peritoneal macrophages in vitro. In contrast, addition of LTB4 increased leishmanicidal activity and this effect was dependent on the BLT1 receptor. LTB4 augmented NO production in response to L. amazonensis challenge, and studies with a NO synthesis inhibitor revealed that NO was critical for the enhancement of macrophage leishmanicidal activity. Interestingly, macrophages from resistant mice produced higher levels of LTB4 upon L. amazonensis challenge than did those from susceptible mice. In vivo infection severity, as assessed by footpad swelling following s.c. promastigote inoculation, was increased when endogenous LT synthesis was abrogated either pharmacologically or genetically. Taken together, these results for the first time reveal an important role for LTB4 in the protective response to L. amazonensis, identify relevant leishmanicidal mechanisms, and suggest that genetic variation in LTB4 synthesis might influence resistance and susceptibility patterns to infection.
CD40/CD154 Interactions Are Required for the Optimal Maturation of Skin-Derived APCs and the Induction of Helminth-Specific IFN-γ but Not IL-4
Authors: Hewitson James P. , Jenkins Gavin R. , Hamblin Paul A. , Mountford Adrian P.
Published: April 2014

The mechanisms through which Schistosoma mansoni larvae induce Th1 rather than Th2 immune responses are not well understood. In this study, using CD154−/− mice exposed to radiation-attenuated S. mansoni larvae, we demonstrate roles for CD154/CD40 in the activation of skin-derived APCs and the development of Th1 cells in the skin-draining lymph nodes (sdLN). The presence of CD154 was important for optimal IL-12p40 and essential for Ag-specific IFN-γ, but CD154 expression by wild-type CD4− cells was insufficient to rescue recall responses of CD4+ cells from CD154−/− mice. This defect is probably due to impaired CD40-dependent IL-12 production in vivo, because administration of anti-CD40 Ab, or rIL-12, restored IFN-γ production by sdLN cells from CD154−/− mice. CD154 ligation of CD40 was not required for the migration of skin-derived APCs, but did have a limited role in their maturation (increased MHC II and CD86). Unexpectedly, although CD4 cells from CD154−/− mice were deficient in their ability to produce IFN-γ, they produced significant amounts of IL-4 and IL-5 in the presence of skin-derived APCs from wild-type and CD154−/− mice. Thus, in contrast to IFN-γ, the production of Th2-associated cytokines is (in this model) independent of CD154. We conclude that whereas the priming of Th1 responses soon after exposure to schistosome larvae is completely CD40/CD154 dependent, IL-4, IL-5, and IL-13 are independent of CD154, suggesting a dichotomy in the specific mechanisms that induce these cytokines by CD4+ cells in the sdLN.
IL-12, but Not IL-18, Is Critical to Neutrophil Activation and Resistance to Polymicrobial Sepsis Induced by Cecal Ligation and Puncture
Authors: Moreno Susana E. , Alves-Filho José C. , Alfaya Thais M. , da Silva João S. , Ferreira Sergio H. , Liew Foo Y.
Published: April 2014

Sepsis is a systemic inflammatory response resulting from local infection due, at least in part, to impaired neutrophil migration. IL-12 and IL-18 play an important role in neutrophil migration. We have investigated the mechanism and relative role of IL-12 and IL-18 in polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. Wild-type (WT) and IL-18−/− mice were resistant to sublethal CLP (SL-CLP) sepsis. In contrast, IL-12−/− mice were susceptible to SL-CLP sepsis with high bacteria load in peritoneal cavity and systemic inflammation (serum TNF-α and lung neutrophil infiltration). The magnitude of these events was similar to those observed in WT mice with lethal CLP sepsis. The inability of IL-12−/− mice to restrict the infection was not due to impairment of neutrophil migration, but correlated with decrease of phagocytosis, NO production, and microbicidal activities of their neutrophils, and with reduction of systemic IFN-γ synthesis. Consistent with this observation, IFN-γ−/− mice were as susceptible to SL-CLP as IL-12−/− mice. Moreover, addition of IFN-γ to cultures of neutrophils from IL-12−/− mice restored their phagocytic, microbicidal activities and NO production. Mortality of IL-12−/− mice to SL-CLP was prevented by treatment with IFN-γ. Thus we show that IL-12, but not IL-18, is critical to an efficient host defense in polymicrobial sepsis. IL-12 acts through induction of IFN-γ and stimulation of phagocytic and microbicidal activities of neutrophils, rather than neutrophil migration per se. Our data therefore provide further insight into the defense mechanism against this critical area of infectious disease.
Coordinated Function of Murine Cytomegalovirus Genes Completely Inhibits CTL Lysis
Authors: Pinto Amelia K. , Munks Michael W. , Koszinowski Ulrich H. , Hill Ann B.
Published: April 2014

Murine CMV (MCMV) encodes three viral genes that interfere with Ag presentation (VIPRs) to CD8 T cells, m04, m06, and m152. Because the functional impact of these genes during normal infection of C57BL/6 mice is surprisingly modest, we wanted to determine whether the VIPRs are equally effective against the entire spectrum of H-2b-restricted CD8 T cell epitopes. We also wanted to understand how the VIPRs interact at a functional level. To address these questions, we used a panel of MCMV mutants lacking each VIPR in all possible combinations, and CTL specific for 15 H-2b-restricted MCMV epitopes. Only expression of all three MCMV VIPRs completely inhibited killing by CTL specific for all 15 epitopes, but removal of any one VIPR enabled lysis by at least some CTL. The dominant interaction between the VIPRs was cooperation: m06 increased the inhibition of lysis achieved by either m152 or m04. However, for 1 of 15 epitopes m04 functionally antagonized m152. There was little differential impact of any of the VIPRs on Kb vs Db, but a surprising degree of differential impact of the three VIPRs for different epitopes. These epitope-specific differences did not correlate with functional avidity, or with timing of VIPR expression in relation to Ag expression in the virus replication cycle. Although questions remain about the molecular mechanism and in vivo role of these genes, we conclude that the coordinated function of MCMV’s three VIPRs results in a powerful inhibition of lysis of infected cells by CD8 T cells.
Activation and Regulation of Platelet-Activating Factor Receptor: Role of Gi and Gq in Receptor-Mediated Chemotactic, Cytotoxic, and Cross-Regulatory Signals
Authors: Brown Stephan L. , Jala Venkatakrishna R. , Raghuwanshi Sandeep K. , Nasser Mohd W. , Haribabu Bodduluri , Richardson Ricardo M.
Published: April 2014

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of Gi and Gq in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing Gαi3 (PAFR-Gαi3) or Gαq (PAFR-Gαq) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-Gαi3, or PAFR-Gαq was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of β-hexosaminidase. PAFR-Gαi3 and PAFR-Gαq mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-Gαi3, but not PAFR-Gαq, mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated βarrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-Gαi3 activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that Gi exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both Gi and Gq activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by Gi, the data suggest that differential activation of both Gi and Gq by PAFR likely mediate specific as well as redundant signaling pathways.
Protective Roles of Mast Cells and Mast Cell-Derived TNF in Murine Malaria
Authors: Furuta Takahisa , Kikuchi Takane , Iwakura Yoichiro , Watanabe Naohiro
Published: April 2014

TNF plays important roles in the protection and onset of malaria. Although mast cells are known as a source of TNF, little is known about the relationship between mast cells and pathogenesis of malaria. In this study, mast cell-deficient WBB6F1-W/Wv (W/Wv) and the control littermate WBB6F1+/+ (+/+) mice were infected with 1 × 105 of Plasmodium berghei ANKA. +/+ mice had lower parasitemia with higher TNF levels, as compared with W/Wv mice. Diminished resistance in W/Wv mice was considered to be due to mast cells and TNF. This fact was confirmed by experiments in W/Wv mice reconstituted with bone marrow-derived mast cells (BMMCs) of +/+ mice or of TNF−/− mice. W/Wv mice with BMMCs of +/+ mice exhibit lower parasitemia and mortality accompanying significantly higher TNF levels than those of W/Wv mice. Parasitemia in W/Wv mice with BMMCs of TNF−/− mice was higher than that in +/+ mice. Activation of mast cells by anti-IgE or compound 48/80 resulted in release of TNF and decrease of parasitemia. In addition, splenic hypertrophy and increased number of mast cells in the spleen were observed after infection in +/+ mice and W/Wv mice reconstituted with BMMCs of +/+ mice as compared with W/Wv mice. These findings propose a novel mechanism that mast cells and mast cell-derived TNF play protective roles in malaria.
Targeted Deletion of CCR2 Impairs Deep Vein Thombosis Resolution in a Mouse Model
Authors: Henke Peter K. , Pearce Charles G. , Moaveni Daria M. , Moore Andrea J. , Lynch Erin M. , Longo Christopher , Varma Manu , Dewyer Nicholas A. , Deatrick K. Barry , Upchurch Gilbert R. , Wakefield Thomas W. , Hogaboam Cory , Kunkel Steven L.
Published: April 2014

CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring Th1 lymphokine responses. Deep vein thrombosis (DVT) resolution represents a specific inflammatory response whereby the thrombus must be dissolved for restoration of blood flow. Using a stasis model of DVT in the mouse, we investigated the role of CCR2 on DVT resolution. Genetic deletion of CCR2 (CCR2−/−) was associated with larger thrombi at early and later time points, increased thrombus collagen, fewer thrombus monocytes (F4/80), and significantly impaired neovascularization. IL-2 and IFN-γ were significantly reduced in early CCR2−/− thrombi, whereas MCP-1 was significantly increased, and Th2 lymphokines were unaffected. Supplementation of CCR2−/− mice with IFN-γ normalized early thrombus resolution without increasing monocyte influx. Neither Ab depletion of IFN-γ nor genetic deletion of IFN-γ impaired early DVT resolution. Early fibrinolysis was not impaired in CCR2−/− mice, but a significant reduction in both matrix metalloproteinase (MMP)-2 and MMP-9 activity was observed. However, only MMP-9 activity was restored with administration of IFN-γ. We conclude that an early CCR2-dependent Th1 lymphokine response predominates in normal DVT resolution, mediates this in part by MMP-9 activation, but is not solely dependent on IFN-γ.
Induction of Complement-Fixing Autoantibodies against Type VII Collagen Results in Subepidermal Blistering in Mice
Authors: Sitaru Cassian , Chiriac Mircea T. , Mihai Sidonia , Büning Jürgen , Gebert Andreas , Ishiko Akira , Zillikens Detlef
Published: April 2014

Experimental models reproducing an autoimmune response resulting in skin blistering in immunocompetent animals are lacking. Epidermolysis bullosa acquisita (EBA) is a bullous skin disease caused by autoantibodies to type VII collagen. In this study, we describe an active disease model of EBA by immunizing mice of different strains with murine type VII collagen. All mice developed circulating IgG autoantibodies that recognized type VII collagen and bound to the lamina densa of the dermal-epidermal junction. Importantly, subepidermal blisters developed in 82% of SJL-1, 56% of BALB/c mice, and 45% of FcγRIIb-deficient mice, but not in SKH-1 mice. In susceptible animals, deposits of IgG1, IgG2, and complement C3 were detected at the dermal-epidermal junction. In contrast, in the nondiseased mice, tissue-bound autoantibodies were predominantly of the IgG1 subclass and complement activation was weak or absent. This active disease model reproduces in mice the clinical, histopathological, and immunopathological findings in EBA patients. This robust experimental system should greatly facilitate further studies on the pathogenesis of EBA and the development of novel immunomodulatory therapies for this and other autoimmune diseases.
Phase II Enzymes Induction Blocks the Enhanced IgE Production in B Cells by Diesel Exhaust Particles
Authors: Wan Junxiang , Diaz-Sanchez David
Published: April 2014

Oxidant pollutants such as diesel exhaust particles (DEPs) can initiate and exacerbate airway allergic responses through enhanced IgE production. These effects are especially pronounced in individuals in whom phase II antioxidant enzyme responses are impaired. We confirmed that DEPs and DEP extracts (DEPX) can act directly on B lymphocytes and showed that DEPX could enhance IgH ε germline transcription in a B cell line and in PBMCs. We therefore studied the regulation in B cells of NAD(P)H: quinone oxidoreductase (NQO1) as a typical model phase II enzyme and its role in modulating DEPX-enhanced IgE responses. DEPX increased NQO1 mRNA expression in a dose-dependent manner. NQO1 protein induction by DEPX was confirmed by Western blot. DEPs induced activity of the antioxidant response element located in the NQO1 gene promoter. Induction of both NQO1 mRNA and protein expression could be blocked by coculture with an antioxidant and partly repressed by inhibitors of PI3K and p38 MAPK, but not by inhibitors of MAPK/ERK kinase (MEK/ERK) or protein kinase C. The ability of DEPX to enhance IgE production was blocked by the induction of phase II enzymes, including NQO1 in B cells by the chemical sulforaphane. These findings suggest that a natural protective mechanism in B cells from oxidant pollutants such as diesel particles is the expression of phase II enzymes through induction of antioxidant response elements and support the approach of overexpression of these enzymes as a potential future chemopreventative strategy.
Induction of Efficient Antitumor Immunity Using Dendritic Cells Activated by Recombinant Sendai Virus and Its Modulation by Exogenous IFN-β Gene
Authors: Shibata Satoko , Okano Shinji , Yonemitsu Yoshikazu , Onimaru Mitsuho , Sata Shihoko , Nagata-Takeshita Hiroko , Inoue Makoto , Zhu Tsugumine , Hasegawa Mamoru , Moroi Yoichi , Furue Masutaka , Sueishi Katsuo
Published: April 2014

Dendritic cell (DC)-based cancer immunotherapy has been paid much attention as a new and cancer cell-specific therapeutic in the last decade; however, little clinical outcome has been reported. Current limitations of DC-based cancer immunotherapy include sparse information about which DC phenotype should be administered. We here report a unique, representative, and powerful method to activate DCs, namely recombinant Sendai virus-modified DCs (SeV/DC), for cancer immunotherapy. In vitro treatment of SeV without any bioactive gene solely led DCs to a mature phenotype. Even though the expression of surface markers for DC activation ex vivo did not always reach the level attained by an optimized amount of LPS, superior antitumor effects to B16F1 melanoma, namely tumor elimination and survival, were obtained with use of SeV-GFP/DC as compared with those seen with LPS/DC in vivo, and the effect was enhanced by SeV/DC-expressing IFN-β (SeV-murine IFN-β (mIFN-β)/DC). In case of the treatment of an established tumor of B16F10 (7–9 mm in diameter), a highly malignant subline of B16 melanoma, SeV-modified DCs (both SeV-GFP/DC and SeV-mIFN-β/DC), but not immature DC and LPS/DC, dramatically improved the survival of animals. Furthermore, SeV-mIFN-β/DC but not other DCs could lead B16F10 tumor to the dormancy, associated with strongly enhanced CD8+ CTL responses. These results indicate that rSeV is a new and powerful tool as an immune booster for DC-based cancer immunotherapy that can be significantly modified by IFN-β, and SeV/DC, therefore, warrants further investigation as a promising alternative for cancer immunotherapy.
Dendritic Cell Modulation by Mast Cells Controls the Th1/Th2 Balance in Responding T Cells
Authors: Mazzoni Alessandra , Siraganian Reuben P. , Leifer Cynthia A. , Segal David M.
Published: April 2014

The cytokines secreted by pathogen-activated human dendritic cells (DC) are strongly regulated in vitro by histamine, a major component of mast cell granules, ultimately modulating the capacity of the DC to polarize naive T cells. Because DC and mast cells are located in close proximity in peripheral compartments, we hypothesized that mast cell products would influence the maturation of DC and hence the Th balance of an immune response in vivo. In this study, we show that specific mast cell degranulation stimuli, given s.c. in mice with Ag and adjuvant, produce effector T cells that proliferate to Ag but secrete dramatically reduced levels of IFN-γ and increased amounts of IL-4 compared with control T cells primed in the absence of a mast cell stimulus. Immunization with Ag and adjuvant in the presence of a degranulation stimulus also resulted in the accumulation of DC in the draining lymph nodes that had reduced capacity to induce Ag-specific Th1 cells, in comparison with DC from mice lacking a degranulation stimulus. Therefore, by acting upon DC at sites of inflammation, mast cells play a critical role in determining the polarity of Ag-specific T cell responses in vivo.
T Cell-Dependent Activation of Dendritic Cells Requires IL-12 and IFN-γ Signaling in T Cells
Authors: Miro Francesc , Nobile Cinzia , Blanchard Nicolas , Lind Marianne , Filipe-Santos Orchidée , Fieschi Claire , Chapgier Ariane , Vogt Guillaume , de Beaucoudrey Ludovic , Kumararatne Dinakantha S. , Le Deist Françoise , Casanova Jean-Laurent , Amigorena Sebastian , Hivroz Claire
Published: April 2014

Patients presenting with genetic deficiencies in IFNGR1, IFNGR2, IL-12B, and IL-12RB1 display increased susceptibility to mycobacterial infections. We analyzed in this group of patients the cross-talk between human CD4+ T lymphocytes and dendritic cells (DCs) that leads to maturation of DC into producers of bioactive IL-12 and to activation of T cells into IFN-γ producers. We found that this cross-talk is defective in all patients from this group. Unraveling the mechanisms underlying this deficiency, we showed that IL-12 signaling in T cells is required to induce expression of costimulatory molecules and secretion of IL-12 by DCs and that IFNGR expression is required on both DCs and CD4+ T cells to induce IL-12 secretion by DCs. These data suggest that CD4+ T cell-mediated activation of DCs plays a critical role in the defense against mycobacterial infections in humans.