bluuebunny/crossref_metadata_2025 huggingface dataset:
Monocyte Activation by Circulating Fibronectin Fragments in HIV-1-Infected Patients
Authors: Trial JoAnn , Rubio Jose A. , Birdsall Holly H. , Rodriguez-Barradas Maria , Rossen Roger D.
Published: April 2014

To identify signals that can alter leukocyte function in patients receiving highly active antiretroviral therapy (HAART), we analyzed single blood samples from 74 HIV-1-infected patients and additional blood was collected at 90-day intervals from 51 HIV-1-infected patients over a 516 ± 172 (mean ± SD) day interval. Despite the absence of circulating immune complexes and normalization of phagocytic function, compared with controls, the fraction of patients’ monocytes expressing CD49e and CD62L was decreased and expression of CD11b and CD86 increased. Plasma from 63% of patients but none from normal controls contained 110–120 kDa fibronectin fragments (FNf). Presence of FNf did not reflect poor adherence to therapy. Addition of FNf to normal donor blood in vitro replicated changes in monocyte CD49e, CD62L, CD11b, and CD86 seen in vivo. FNf also induced monocytes to release a serine proteinase, nominally identified as proteinase-3, that hydrolyzed cell surface CD49e. α1-Antitrypsin blocked FNf-induced shedding of CD49e in a dose-dependent manner. Plasma with a normal frequency of CD49e+ monocytes contained antiproteases that partially blocked FNf-induced monocyte CD49e shedding, whereas plasma from patients with a low frequency of CD49e+ monocytes did not block this effect of FNf. Electrophoretic analyses of plasma from the latter group of patients suggested that a significant fraction of their α1-antitrypsin was tied up in high molecular mass complexes. These results suggest that monocyte behavior in HIV-1-infected patients may be influenced by FNf and the ratio of protease and antiproteases in the cells’ microenvironment.
Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa
Authors: Smith Donald W. , Nagler-Anderson Cathryn
Published: April 2014

The gut-associated lymphoid tissue (GALT) is constantly exposed to a variety of Ags and must therefore decipher a large number of distinct signals at all times. Responding correctly to each set of signals is crucial. When the GALT receives signals from the intestinal flora or food Ags, it must induce a state of nonresponsiveness (mucosal tolerance). In contrast, when pathogenic bacteria invade the intestinal mucosa, it is necessary to elicit strong T and B cell responses. The GALT is therefore in the position of constantly fighting intolerance to food and the commensal flora while effectively battling infectious microbes. Determining precisely which type of response to generate in each case is key to the prevention of immune dysregulation and tissue damage.
Cutting Edge: KIR2DL4 Transduces Signals into Human NK Cells through Association with the Fc Receptor γ Protein
Authors: Kikuchi-Maki Akiko , Catina Tracey L. , Campbell Kerry S.
Published: April 2014

KIR2DL4 (2DL4, CD158d), a member of the human killer cell Ig-like receptor (KIR) family, triggers potent IFN-γ responses but weak cytotoxicity in resting NK cells. 2DL4 mRNA has been detected in most NK cell clones from most humans examined, but surface protein expression is detectable only on CD56high NK cells from certain donors. The receptor possesses a transmembrane arginine residue, suggesting association with a signaling accessory protein that has remained elusive. We provide biochemical and functional evidence that FcεRI-γ (γ) associates with 2DL4 to promote surface expression and provide signal transducing function. Weak cytolytic responses triggered through 2DL4 may result from low stoichiometric association with γ. Selective association with γ distinguishes 2DL4 from all other activating forms of the KIR family, which alternatively associate with DNAX-activating protein (DAP)12.
Cutting Edge: IFN-γ Signaling to Macrophages Is Required for Optimal Vα14i NK T/NK Cell Cross-Talk
Authors: Wesley Johnna D. , Robbins Scott H. , Sidobre Stéphane , Kronenberg Mitchell , Terrizzi Stephanie , Brossay Laurent
Published: April 2014

Activated NK T cells are known to rapidly stimulate NK cells and, subsequently, CD8+ T cells and B cells. In this report, we first demonstrate that the downstream effects induced by α-galactosylceramide activated NK T cells on NK cells are mainly dependent on IFN-γ. We found that NK T cell activation of NK cells requires a functional IFN-γ signaling in macrophages and dendritic cells but not in B cells, NK cells, or NK T cells. NK T cell activation is dendritic cell-dependent whereas NK T cell activation of NK cells is indirect and in part mediated by macrophages. Interestingly, in this context, macrophage participation in the CD1d Ag presentation of α-galactosylceramide to NK T cells is not necessary. These data indicate that NK T cell-dependent activation of macrophages is required for optimal NK T cell-induced stimulation of NK cells.
Simultaneous LFA-1 and CD40 Ligand Antagonism Prevents Airway Remodeling in Orthotopic Airway Transplantation: Implications for the Role of Respiratory Epithelium as a Modulator of Fibrosis
Authors: Murakawa Tomohiro , Kerklo Michelle M. , Zamora Martin R. , Wei Yi , Gill Ronald G. , Henson Peter M. , Grover Frederick L. , Nicolls Mark R.
Published: April 2014

Airway remodeling is a prominent feature of certain immune-mediated lung diseases such as asthma and chronic lung transplant rejection. Under conditions of airway inflammation, the respiratory epithelium may serve an important role in this remodeling process. Given the proposed role of respiratory epithelium in nonspecific injury models, we investigated the respiratory epithelium in an immune-specific orthotopic airway transplant model. MHC-mismatched tracheal transplants in mice were used to generate alloimmune-mediated airway lesions. Attenuation of this immune injury and alteration of antidonor reactivity were achieved by the administration of combined anti-LFA-1/anti-CD40L mAbs. By contrast, without immunotherapy, transplanted airways remodeled with a flattening of respiratory epithelium and significant subepithelial fibrosis. Unopposed alloimmune injury for 10 days was associated with subsequent epithelial transformation and subepithelial fibrosis that could not be reversed with immunotherapy. The relining of donor airways with recipient-derived epithelium was delayed with immunotherapy resulting in partially chimeric airways by 28 days. Partial epithelial cell chimerism was sufficient to prevent luminal fibrosis. However, epithelial chimerism was also associated with airway remodeling. Therefore, there appears to be an intimate relationship between the morphology and level of chimerism of the respiratory epithelium and the degree of airway remodeling following alloimmune injury.
Extracellular ATP Induces Cytokine Expression and Apoptosis through P2X7 Receptor in Murine Mast Cells
Authors: Bulanova Elena , Budagian Vadim , Orinska Zane , Hein Martina , Petersen Frank , Thon Lutz , Adam Dieter , Bulfone-Paus Silvia
Published: April 2014

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X7, and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca2+ influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-α. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4′-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X7 receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X7 receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X7-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.
IL-12 Induction of mRNA Encoding Substance P in Murine Macrophages from the Spleen and Sites of Inflammation
Authors: Arsenescu Razvan , Blum Arthur M. , Metwali Ahmed , Elliott David E. , Weinstock Joel V.
Published: April 2014

Substance P (SP), a neuropeptide, interacts with the neurokinin 1 receptor (NK-1R) on immune cells to help control IFN-γ production. In murine schistosomiasis mansoni, schistosome worms produce ova that incite focal Th2-type granulomatous inflammation within the liver and intestines. Normal gut is characterized by a controlled state of inflammation. IL-10 knockout mice develop chronic Th1-type colitis spontaneously. Both schistosome granulomas and gut mucosa display an SP immune regulatory circuit. However, the origin and regulation of SP production at these sites of inflammation are poorly understood. Macrophages are a potential source of SP. We therefore studied macrophages (F4/80+) from these models of inflammation. SP mRNA (preprotachykinin A (PPT A)) was detected within the schistosome granuloma, spleen, and lamina propria macrophages. Compared with those from wild-type mice, granuloma macrophages from STAT6−/− mice had 10-fold higher PPT A mRNA expression, whereas in STAT4−/− animals, PPT A mRNA expression was nearly abolished. IL-12 signals via STAT4 to induce Th1-type inflammation. It was demonstrated that IL-12, but not IL-18, induces SP mRNA expression in resting splenic macrophages from Schistosoma-infected mice and in wild-type lamina propria mononuclear cells. Thus, macrophages are a source for SP at these sites of chronic inflammation, and IL-12 and STAT4 are regulators of macrophage SP mRNA expression.
Anopheles Mosquito Bites Activate Cutaneous Mast Cells Leading to a Local Inflammatory Response and Lymph Node Hyperplasia
Authors: Demeure Christian E. , Brahimi Karima , Hacini Feriel , Marchand Françoise , Péronet Roger , Huerre Michel , St.-Mezard Pierre , Nicolas Jean-François , Brey Paul , Delespesse Guy , Mécheri Salaheddine
Published: April 2014

When Anopheles mosquitoes probe the skin for blood feeding, they inject saliva in dermal tissue. Mosquito saliva is known to exert various biological activities, but its perception by the immune system and its role in parasite transmission remain poorly understood. In the present study, we report on the cellular changes occurring in the mouse skin and draining lymph nodes after a Anopheles stephensi mosquito bite. We show that mosquito bites induce dermal mast cell degranulation, leading to fluid extravasation and neutrophil influx. This inflammatory response does not occur in mast cell-deficient W/Wv mice, unless these are reconstituted specifically with mast cells. Mast cell activation caused by A. stephensi mosquito bites is followed by hyperplasia of the draining lymph node due to the accumulation of CD3+, B220+, CD11b+, and CD11c+ leukocytes. The T cell enrichment of the draining lymph nodes results from their sequestration from the circulation rather than local proliferation. These data demonstrate that mosquito bites and very likely saliva rapidly trigger the immune system, emphasizing the critical contribution of peripheral mast cells in inducing T cell and dendritic cell recruitment within draining lymph nodes, a prerequisite for the elicitation of T and B lymphocyte priming.
Simultaneous Induction of CD4 T Cell Tolerance and CD8 T Cell Immunity by Semimature Dendritic Cells
Authors: Kleindienst Petra , Wiethe Carsten , Lutz Manfred B. , Brocker Thomas
Published: April 2014

Previous studies suggested that depending on their maturation state, dendritic cells (DC) could either induce T cell tolerance (immature and semimature DC) or T cell activation (mature DC). Pretreatment of C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG)35–55 peptide-loaded semimature DC protected from MOG-induced autoimmune encephalomyelitis. This protection was mediated by IL-10-producing CD4 T cells specific for the self Ag. Here we show that semimature DC loaded with the MHC class II-restricted nonself peptide Ag (OVA) induce an identical regulatory T cell cytokine pattern. However, semimature DC loaded simultaneously with MHC class II- and MHC class I-restricted peptides, could efficiently initiate CD8 T cell responses leading to autoimmune diabetes in a TCR-transgenic adoptive transfer model. Double-peptide-loaded semimature DC also induced simultaneously in the same animal partially activated CD8 T cells with cytolytic function as well as protection from MOG-induced autoimmune encephalomyelitis. Our study suggests that the decision between tolerance and immunity not only depends on the DC, but also on the type and activation requirements of the responding T cell.
IFN Regulatory Factor-2 Cooperates with STAT1 to Regulate Transporter Associated with Antigen Processing-1 Promoter Activity
Authors: Rouyez Marie-Christine , Lestingi Marta , Charon Martine , Fichelson Serge , Buzyn Agnès , Dusanter-Fourt Isabelle
Published: April 2014

Class I MHC complexes (MHCI) are essential in mediating immune response. The transport of antigenic peptides (TAP) to MHCI and the stable expression of MHCI on the cell surface require the presence of a dedicated TAP. In this study we report that IFN-γ and thrombopoietin (TPO) strongly increase TAP1 protein expression in megakaryocytes, followed by an enhanced expression of MHCI on the cell surface. This expression parallels the enhanced TAP1 promoter activity and TAP1 mRNA expression, which are independent of protein synthesis. We also show that this cytokine-dependent expression of TAP1 transcripts depends on STAT1 and IFN regulatory factor-2 (IRF-2), but not on IRF-1, and provide evidence that IRF-2 constitutively binds to the TAP1 gene promoter and enhances TAP1 promoter activity. We show that IRF-2 forms a complex with STAT1 and the cytokine-responsive region of the TAP1 promoter in any TPO or IFN-γ target cells tested. Interaction of IRF-2 and STAT1 on the promoter depends on the DNA-binding domain of IRF-2. Overall, our data indicate that TPO and IFN-γ activate the expression of TAP1 via a new mechanism that involves functional cooperation between STAT1 and IRF-2 on the TAP1 promoter.
Inducing P-Selectin Ligand Formation in CD8 T Cells: IL-2 and IL-12 Are Active In Vitro but Not Required In Vivo
Authors: Carlow Douglas A. , Williams Michael J. , Ziltener Hermann J.
Published: April 2014

In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2null CD8 T cells responding in doubly deficient IL-2nullIL-12null or IL-2nullIL-15null male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.
Ex Vivo Characterization of the Autoimmune T Cell Response in the HLA-DR1 Mouse Model of Collagen-Induced Arthritis Reveals Long-Term Activation of Type II Collagen-Specific Cells and Their Presence in Arthritic Joints
Authors: Latham Kary A. , Whittington Karen B. , Zhou Ruohong , Qian Zhaohui , Rosloniec Edward F.
Published: April 2014

Although the pathogenesis of collagen-induced arthritis (CIA), a model of rheumatoid arthritis, is mediated by both collagen-specific CD4+ T cells and Ab specific for type II collagen (CII), the role of CII-specific T cells in the pathogenesis of CIA remains unclear. Using tetrameric HLA-DR1 with a covalently bound immunodominant CII peptide, CII259–273, we studied the development of the CII-specific T cell response in the periphery and arthritic joints of DR1 transgenic mice. Although the maximum number of DR1-CII-tetramer+ cells was detected in draining lymph nodes 10 days postimmunization, these T cells accounted for only 1% or less of the CD4+ population. After day 10, their numbers gradually decreased, but were still detectable on day 130. Examination of TCR expression and changes in CD62L, CD44high, and CD69 expression by these T cells indicated that they expressed a limited TCR-BV repertoire and had clearly undergone activation. RT-PCR analysis of cytokine expression by the tetramer+ T cells compared with tetramer− cells indicated the tetramer+ cells expressed high levels of Th1 and proinflammatory cytokines, including IL-2, IFN-γ, IL-6, TNF-α, and especially IL-17. Additionally, analysis of the synovium from arthritic paws indicated that the same CD4+/BV8+/BV14+/tetramer+ T cells were present in the arthritic joints. These data demonstrate that although only small numbers of CII-specific T cells are generated during the development of CIA, these cells express very high levels of cytokine mRNA and appear to preferentially migrate to the arthritic joint, indicating a potential direct role of CII-specific T cells in the pathogenesis of CIA.
Effector CD4+ T Cells Generate Intermediate Caspase Activity and Cleavage of Caspase-8 Substrates
Authors: Misra Ravi S. , Jelley-Gibbs Dawn M. , Russell Jennifer Q. , Huston Gail , Swain Susan L. , Budd Ralph C.
Published: April 2014

Caspase-8 activation promotes cell apoptosis but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4+ T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIPL, receptor interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4+ T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling.
T Cells in G1 Provide a Memory-Like Response to Secondary Stimulation
Authors: Munitic Ivana , Ryan Philip E. , Ashwell Jonathan D.
Published: April 2014

The commitment of naive T cells to proliferate is a function of the strength and duration of stimuli mediated by the TCR and coreceptors. Ranges of 2–20 h of stimulation have been reported as necessary in vitro. Whether T cells actually experience uninterrupted stimulation for such long periods under physiological conditions is controversial. Here we ask whether commitment to proliferate requires continuous stimulation, or can T cells integrate intermittent periods of stimulation. T cells were stimulated for two short-term (subthreshold) periods (5–7 h) either sequentially or separated by an interval of rest. Naive lymph node T cells were able to integrate interrupted stimulation, even when the duration of rest was as long as 2 days. Furthermore, when short-term-stimulated T cells were separated by density, three populations were observed: low density blasts, intermediate density G1 cells, and high density G0 cells. Low density cells progressed to division without further stimulation, whereas G0 and G1 cells remained undivided. However, after a period of rest, a second subthreshold stimulation caused the G1 but not the G0 fraction to quickly proceed through the cell cycle. We conclude that noncycling T cells in the G1 phase of the cell cycle remain in a state of readiness for prolonged periods of time, and may represent a population of memory-like effectors capable of responding rapidly to antigenic challenge.
IL-15 Activates Telomerase and Minimizes Telomere Loss and May Preserve the Replicative Life Span of Memory CD8+ T Cells In Vitro
Authors: Li Yu , Zhi Wang , Wareski Przemyslaw , Weng Nan-ping
Published: April 2014

The preservation of the replicative life span of memory CD8+ T cells is vital for long-term immune protection. Although IL-15 plays a key role in the homeostasis of memory CD8+ T cells, it is unknown whether IL-15 regulates the replicative life span of memory CD8+ T cells. In this study, we report an analysis of telomerase expression and telomere length in human memory phenotype CD8+ T cells maintained by IL-15 in vitro. We demonstrate that IL-15 is capable of activating telomerase in memory CD8+ T cells via Jak3 and PI3K signaling pathways. Furthermore, IL-15 induces a sustained level of telomerase activity over long periods of time, and in turn minimizes telomere loss in memory CD8+ T cells after substantial cell divisions. These findings suggest that IL-15 activates stable telomerase expression and compensates telomere loss in memory phenotype CD8+ T cells, and that telomerase may play an important role in memory CD8+ T cell homeostasis.
Increased Expression of CD27 on Activated Human Memory B Cells Correlates with Their Commitment to the Plasma Cell Lineage
Authors: Avery Danielle T. , Ellyard Julia I. , Mackay Fabienne , Corcoran Lynn M. , Hodgkin Philip D. , Tangye Stuart G.
Published: April 2014

Plasma cells (PC) or Ig-secreting cells (ISC) are terminally differentiated B cells responsible for the production of protective Ig. ISC can be generated in vitro by culturing human B cells with the T cell-derived stimuli CD40L, IL-2, and IL-10. ISC have traditionally been identified by the increased expression of CD38, analogous to primary human PC, and the acquired ability to secrete Ig. By tracking the proliferation history of activated B cells, we previously reported that the differentiation of memory B cells into CD38+ B cells is IL-10 dependent, and increases in frequency with cell division. However, <50% of CD38+ cells secreted Ig, and there was a population of CD38− ISC. Thus, the PC phenotype of CD38+ cells generated in vitro did not correlate with PC function. To address this, we have examined cultures of activated memory B cells to accurately identify the phenotype of ISC generated in vitro. We found that CD27 is also up-regulated on memory B cells in an IL-10-dependent and division-dependent manner, and that ISC segregated into the CD27high subset of activated memory B cells irrespective of the acquired expression of CD38. The ISC generated in these cultures expressed elevated levels of the transcription factors Blimp-1 and X box-binding protein-1 and reduced levels of Pax-5, and exhibited selective migration toward CXCL12, similar to primary PC. We propose that the differentiation of memory B cells into PC involves a transitional stage characterized by a CD27highCD38− phenotype with the acquired ability to secrete high levels of Ig.
An Alternate Core 2 β1,6-N-Acetylglucosaminyltransferase Selectively Contributes to P-Selectin Ligand Formation in Activated CD8 T Cells
Authors: Merzaban Jasmeen S. , Zuccolo Jonathan , Corbel Stéphane Y. , Williams Michael J. , Ziltener Hermann J.
Published: April 2014

Core 2 β1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) synthesizes essential core 2 O-glycans on selectin ligands, which mediate cell-cell adhesion required for lymphocyte trafficking. Although gene-deletion studies have implicated C2GlcNAcT-I in controlling selectin ligand-mediated cell trafficking, little is known about the role of the two other core 2 isoenzymes, C2GlcNAcT-II and C2GlcNAcT-III. We show that C2GlcNAcT-I-independent P-selectin ligand formation occurs in activated C2GlcNAcT-Inull CD8 T cells. These CD8 T cells were capable of rolling under shear flow on immobilized P-selectin in a P-selectin glycoprotein ligand 1-dependent manner. RT-PCR analysis identified significant levels of C2GlcNAcT-III RNA, identifying this enzyme as a possible source of core 2 enzyme activity. Up-regulation of P-selectin ligand correlated with altered cell surface binding of the core 2-sensitive mAb 1B11, indicating that CD43 and CD45 are also physiological targets for this alternate C2GlcNAcT enzyme. Furthermore, C2GlcNAcT-I-independent P-selectin ligand induction was observed in an in vivo model. HYtg CD8 T cells from C2GlcNAcT-Inull donors transferred into male recipients expressed P-selectin ligand in response to male Ag, although at reduced levels compared with wild-type HYtg CD8 T cells. Our data demonstrate that multiple C2GlcNAcT enzymes can contribute to P-selectin ligand formation and may cooperate with C2GlcNAcT-I in the control of CD8 T cell trafficking.
CD40 Engagement Prevents Peroxisome Proliferator-Activated Receptor γ Agonist-Induced Apoptosis of B Lymphocytes and B Lymphoma Cells by an NF-κB-Dependent Mechanism
Authors: Ray Denise M. , Akbiyik Filiz , Bernstein Steven H. , Phipps Richard P.
Published: April 2014

Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor important in fat metabolism and is emerging as an important regulator of immunity and inflammation. We previously demonstrated that normal and malignant B lineage cells express PPARγ and die by apoptosis after PPARγ agonist exposure. In this study, we used the WEHI-231 mouse B lymphoma and normal mouse spleen B lymphocytes to elucidate the mechanism of PPARγ agonist-induced apoptosis, and to determine whether an apoptosis rescue mechanism exists. In WEHI-231 cells, the natural PPARγ agonist 15-deoxy-Δ12,14-PGJ2 and the synthetic PPARγ agonist ciglitazone induced activation of caspase 3 and caspase 9, a decrease in mitochondrial membrane potential, and caused cleavage of the caspase substrate poly(ADP-ribose) polymerase. We next tested whether CD40, whose engagement delivers a potent prosurvival signal for B cells, could protect B cells from PPARγ agonist-induced apoptosis. CD40 engagement with CD40L significantly blunted the ability of PPARγ agonists to induce apoptosis of B lymphocytes and prevented the inhibition of NF-κB mobilization by 15-deoxy-Δ12,14-PGJ2 and ciglitazone. Interestingly, PPARγ agonists induced an increase in IκBα and IκBβ protein levels, which was prevented with CD40 engagement. The rescue mechanism induced by CD40 engagement was dependent on NF-κB, as an NF-κB inhibitor prevented rescue. Apoptosis induction by PPARγ ligands may be important for immune regulation by killing B lymphocytes as a rapid means to dampen inflammation. Moreover, the ability of PPARγ agonists to kill malignant B lineage cells has implications for their use as anti-B lymphoma agents.
Cross-Linking of CD45 on Suppressive/Regulatory T Cells Leads to the Abrogation of Their Suppressive Activity In Vitro
Authors: Shimizu Jun , Iida Ryuji , Sato Yuji , Moriizumi Eiko , Nishikawa Atsushi , Ishida Yasumasa
Published: April 2014

CD4+CD25+ T cells have immunoregulatory and suppressive functions and are responsible for suppressing self-reactive cells and maintaining self-tolerance. In addition to CD4+CD25+ T cells, there is some evidence that a fraction of CD4+CD25− T cells exhibit suppressive activity in vitro or in vivo. We have shown, using aged mice, that aging not only leads to a decline in the ability to mount CD4+CD25− T cell responses, but, at the same time, renders aged CD4+CD25− T cells suppressive. In this study we report two newly established mAbs that could abrogate the suppressive function of aged CD4+CD25− T cells. These mAbs recognized the same protein, the transmembrane phosphatase CD45. Cross-linking of CD45 on aged CD4+CD25− T cells was required for the disruption of their suppressive activity. Surprisingly, these mAbs also abrogated the suppressive action of CD4+CD25+ T cells in vitro. Our results demonstrate an unexpected function of CD45 as a negative regulator neutralizing the suppressive activity of aged CD4+CD25− and young CD4+CD25+ T cells.
Peripheral Deletion of Antigen-Specific T Cells Leads to Long-Term Tolerance Mediated by CD8+ Cytotoxic Cells
Authors: Herndon John M. , Stuart Patrick M. , Ferguson Thomas A.
Published: April 2014

Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8+ T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8+ CTLs.
Stat5a Inhibits IL-12-Induced Th1 Cell Differentiation through the Induction of Suppressor of Cytokine Signaling 3 Expression
Authors: Takatori Hiroaki , Nakajima Hiroshi , Kagami Shin-ichiro , Hirose Koichi , Suto Akira , Suzuki Kotaro , Kubo Masato , Yoshimura Akihiko , Saito Yasushi , Iwamoto Itsuo
Published: April 2014

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a−/−) CD4+ T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-γ production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a−/− CD4+ T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a−/− CD4+ T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a−/− CD4+ T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4+ T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a−/− CD4+ T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3.
Receptors and Counterreceptors Involved in NK-B Cell Interactions
Authors: Gao Ning , Dang Tam , Dunnick Wesley A. , Collins John T. , Blazar Bruce R. , Yuan Dorothy
Published: April 2014

In addition to the well-documented effect of NK cells on B cell differentiation via their ability to secrete IFN-γ, NK cells can also induce, via direct cell-cell interactions, germline transcripts (Iγ2a) necessary for switch recombination to IgG2a. Analysis of the ligand-receptor pairs that could be involved in this induction revealed that the expression of CD48 on B cells is crucial for the induction. NK cells from mice with targeted deletions of either the CD2 or the CD244 gene, both of which encode ligands for CD48, are compromised in their ability to induce B cell Iγ2a expression. Interestingly, although CD244 can bind to CD48 with a higher affinity, the ability of NK cells from CD244−/− mice to stimulate Iγ2a is not as compromised as NK cells from CD2−/− mice. Despite the difference between cell surface receptors that are stimulated by NK cells vs those stimulated by the combination of LPS and IFN-γ, we show in this study that the initiation of γ2a germline transcription is regulated by similar cis-acting elements located at the 3′ end of the IgH locus. However, NK cells cannot induce the final steps of switch recombination resulting in the production of mature mRNA from recombined DNA. Our findings suggest that these different signaling pathways converge on regulatory elements that are common to germline transcription; however, because NK induction does not result in the final steps of switch recombination, some signals initiated by LPS plus IFN-γ are not induced by NK cells.
Stable Activation of Phosphatidylinositol 3-Kinase in the T Cell Immunological Synapse Stimulates Akt Signaling to FoxO1 Nuclear Exclusion and Cell Growth Control
Authors: Fabre Stéphanie , Lang Valérie , Harriague Julie , Jobart Aude , Unterman Terry G. , Trautmann Alain , Bismuth Georges
Published: April 2014

We have previously reported at the single cell level that PI3K is activated after conjugate formation between T lymphocytes and APCs. However, in contrast to cells exposed to an asymmetrical signal that usually increase 3′-phosphoinositides (3′-PI) transiently in the region of the activated receptors, T cells contacting APC accumulate 3′-PI across their whole plasma membrane far beyond the region of the immunological synapse (IS). Importantly, this effect is maintained over time, for hours, and although PI3K-dependent pathways translate in various cell types extracellular stimuli into a wide range of biological events, in primary T cells this stability is mostly required for cell division induced by Ag. Using imaging methodologies, the present article elucidates the molecular mechanisms responsible for this particular functioning of the PI3K pathway in primary human T lymphocytes interacting with APCs, especially with dendritic cells. The results reveal that the IS unremittingly recruits PI3K to maintain high 3′-PI levels in T cells through phosphotyrosine-dependent mechanisms, suggesting a major participation of class Ia PI3K. This persistent activation of PI3K results in the Akt-dependent sequestration of the FoxO transcription factor, FoxO1, outside the nucleus of T cells interacting with APCs. Using an active form of FoxO1, we demonstrate that this compartmentalization process can affect T cell growth after Ag recognition. We conclude that the need for sustained PI3K signaling within the consolidated IS is probably an undemanding tactic used by primary T cells critical for initiating cell cycle progression through the prolonged inactivation of FoxO1, one important factor that can control cell quiescence.
Linking C5 Deficiency to an Exonic Splicing Enhancer Mutation
Authors: Pfarr Nicole , Prawitt Dirk , Kirschfink Michael , Schroff Claudia , Knuf Markus , Habermehl Pirmin , Mannhardt Wilma , Zepp Fred , Fairbrother William , Loos Michael , Burge Christopher B. , Pohlenz Joachim
Published: April 2014

As an important component of the innate immune system, complement provides the initial response to prevent infections by pathogenic microorganisms. Patients with dysfunction of C5 display a propensity for severe recurrent infections. In this study, we present a patient with C5 deficiency demonstrated by immunochemical and functional analyses. Direct sequencing of all C5 exons displayed no mutation of obvious functional significance, except for an A to G transition in exon 10 predicting an exchange from lysine to arginine. This sequence alteration was present in only one allele of family members with a reduced serum C5 concentration and in both alleles of the patient with almost complete C5 deficiency, suggesting that this alteration may be producing the phenotype. Recent findings indicate that distinct nucleotide sequences, termed exonic splicing enhancers (ESEs), influence the splicing process. cDNA from all family members harboring the mutated allele showed skipping of exon 10, which resulted in a premature STOP codon, explaining the lack of C5 in the propositus. Sequence analysis of the mutated region revealed the substitution to be located within an ESE, as predicted by the RESCUE-ESE program. The altered ESE sequence is located close to the 5′ splicing site and also lowers the predicted strength of the splice site itself. This apparently inconsequential sequence alteration represents a noncanonical splicing mutation altering an ESE. Our finding sheds a new light on the role of putative silent/conservative mutations in disease-associated genes.
p38 MAPK Autophosphorylation Drives Macrophage IL-12 Production during Intracellular Infection
Authors: Kim Leesun , Rio Laura Del , Butcher Barbara A. , Mogensen Trine H. , Paludan Søren R. , Flavell Richard A. , Denkers Eric Y.
Published: April 2014

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mφ). We used synthetic inhibitors and dominant-negative Mφ mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3−/− Mφ, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38α MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38α MAPK association with adaptor protein, TGF-β-activated protein kinase 1-binding protein-1. We observed TGF-β-activated protein kinase 1-binding protein-1-p38α MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mφ. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38α MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.
Maintenance of Pulmonary Th1 Effector Function in Chronic Tuberculosis Requires Persistent IL-12 Production
Authors: Feng Carl G. , Jankovic Dragana , Kullberg Marika , Cheever Allen , Scanga Charles A. , Hieny Sara , Caspar Patricia , Yap George S. , Sher Alan
Published: April 2014

The mechanisms that prevent reactivation of latent Mycobacterium tuberculosis infection in asymptomatic individuals are poorly understood. Although IL-12 is critical for the induction of IFN-γ-dependent host control of M. tuberculosis, the requirement for the cytokine in the maintenance of host resistance and pulmonary Th1 effector function has not yet been formally examined. In this study, we reconstituted IL-12p40-deficient mice with IL-12 during the first 4 wk of infection and then assessed the effects of cytokine withdrawal. Although IL-12 administration initially resulted in restricted mycobacterial growth and prolonged survival, the reconstituted animals eventually succumbed to infection. This breakdown in bacterial control was accompanied by a marked reduction in the numbers of IFN-γ-producing CD4+ T cells in lungs. Moreover, whereas CD4+ T cells isolated from chronically infected wild-type mice expanded and transferred long-term protection to M. tuberculosis-challenged RAG−/− mice, they failed to do so in IL-12p40-deficient RAG−/− recipients and were clearly reduced in frequency within pulmonary granulomas in the latter animals. These studies establish that continuous IL-12 production is necessary for maintenance of the pulmonary Th1 cells required for host control of persistent M. tuberculosis infection and suggest that breakdown of this mechanism could be a contributing factor in reactivated disease.
Smad3 Deficiency in Mast Cells Provides Efficient Host Protection against Acute Septic Peritonitis
Authors: Kanamaru Yutaka , Sumiyoshi Koji , Ushio Hiroko , Ogawa Hideoki , Okumura Ko , Nakao Atsuhito
Published: April 2014

Mast cells play an important role in innate immunity as well as in allergic reaction. However, regulatory mechanisms underlying mast cell-mediated innate immune responses remain largely unknown. Here we determined whether Smad3, a major signal transducer of TGF-β, regulates innate immune response by mast cells against Gram-negative bacteria. Bone marrow-derived mast cells (BMMC) obtained from Smad3 null mutant mice showed augmented capacity to produce proinflammatory cytokines upon stimulation with a Gram-negative bacteria-associated product, LPS. In acute septic peritonitis model induced by cecal ligation and puncture, mast cell-deficient W/Wv mice reconstituted with Smad3 null BMMC had significantly higher survival rate than W/Wv mice reconstituted with wild-type BMMC, which was associated with higher production of proinflammatory cytokines in the peritoneal cavity. These in vitro and in vivo results suggest that Smad3 in mast cells functions as inhibitory for mast cell-mediated innate immune response against Gram-negative bacteria. Suppression of Smad3 expression in mast cells may thus have therapeutic potential for Gram-negative bacterial infection such as acute septic peritonitis by augmenting innate immune responses of mast cells.
Inverse Correlation of Maturity and Antibacterial Activity in Human Dendritic Cells
Authors: Buettner Maike , Meinken Christoph , Bastian Max , Bhat Rauf , Stössel Elmar , Faller Gerhard , Cianciolo George , Ficker Joachim , Wagner Manfred , Röllinghoff Martin , Stenger Steffen
Published: April 2014

Dendritic cells (DCs) are a key part of host defense against microbial pathogens, being part of the innate immune system, but also instructing the adaptive T cell response. This study was designed to evaluate whether human DCs directly contribute to innate immunity by killing intracellular bacteria, using tuberculosis as a model. DCs were detected in bronchoalveolar lavage samples indicating that DCs are available for immediate interaction with Mycobacterium tuberculosis (M. Tb) after inhalation of the pathogen. The phenotype of DC in bronchoalveolar lavage closely resembles monocyte-derived immature DC (iDC) according to the expression of CD1a, CD83, and CCR7. The antimicrobial activity of iDC against intracellular M. Tb inversely correlated with TNF-α-release and was enhanced by treatment with anti-TNF-α Abs. Differentiation of iDC into mature DC by addition of TNF-α or activation via Toll-like receptors further reduced killing of M. Tb. The antibacterial activity against intracellular M. Tb of all DCs was significantly lower than alveolar macrophages. Therefore, the maintenance of a pool of DCs at the site of disease activity in tuberculosis, and the maturation of these DC by TNF-α provides a mechanism by which M. Tb escapes the innate immune system.
Cross-Talk between CD14 and Complement Receptor 3 Promotes Phagocytosis of Mycobacteria: Regulation by Phosphatidylinositol 3-Kinase and Cytohesin-1
Authors: Sendide Khalid , Reiner Neil E. , Lee Jimmy S. I. , Bourgoin Sylvain , Talal Amina , Hmama Zakaria
Published: April 2014

The glycosylphosphatidyl anchored molecule CD14 to the monocyte membrane plays a prominent role in innate immunity, and the paradigms for CD14 selective signaling are beginning to be elucidated. In this study, transfected human monocytic cell line THP-1 and Chinese hamster ovary (CHO) fibroblastic cells were used to examine phagocytosis of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Flow cytometry was combined with molecular and biochemical approaches to demonstrate a dual mechanism for BCG internalization involving either CD14 alone or a CD14-regulated complement receptor (CR)3-dependent pathway. Phagocytosis by CD14-positive THP-1 cells was attenuated by phosphatidylinositol-3 inhibitors LY294002 and wortmannin and experiments using transfected CHO cells showed substantial accumulation of phosphatidylinositol-3,4,5-trisphosphate at the BCG attachment site in CHO cells expressing CD14 and TLR2 suggesting that bacteria bind to CD14 and use TLR2 to initiate a PI3K signaling pathway. Additional experiments using blocking Abs showed that anti-TLR2 Abs inhibit phagocytosis of BCG by THP-1 cells. Furthermore, knockdown of cytohesin-1, a PI3K-regulated adaptor molecule for β2 integrin activation, specifically abrogated CD14-regulated CR3 ingestion of BCG consistent with the observation of physical association between CR3 and cytohesin-1 in cells stimulated with mycobacterial surface components. These findings reveal that mycobacteria promote their uptake through a process of “inside-out” signaling involving CD14, TLR2, PI3K, and cytohesin-1. This converts low avidity CR3 into an active receptor leading to increased bacterial internalization.
Uptake of Granulysin via Lipid Rafts Leads to Lysis of Intracellular Listeria innocua
Authors: Walch Michael , Eppler Elisabeth , Dumrese Claudia , Barman Hanna , Groscurth Peter , Ziegler Urs
Published: April 2014

The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis in dendritic cells (DC) with high efficiency. Despite that crucial effector function, the killing mechanism and uptake of granulysin into target cells have not been well investigated. To this end we analyzed granulysin binding, uptake, and the subsequent lysis of intracellular Listeria innocua in human DC. Recombinant granulysin was found to be actively taken up by DC into early endosomal Ag 1-labeled endosomes, as detected by immunofluorescence. Further transfer to L. innocua-containing phagosomes was indicated by colocalization of bacterial DNA with granulysin. After uptake of granulysin by DC, lysis of L. innocua was found in a dose-dependent manner. Uptake as well as lysis of Listeria were inhibited after blocking endocytosis by lowering the temperature and by cholesterol depletion of DC. Colocalization of granulysin with cholera toxin during uptake showed binding to and internalization via lipid rafts. In contrast to cholera toxin, which was targeted to the perinuclear compartment, granulysin was found exclusively in endosomal-phagosomal vesicles. Lipid raft microdomains, enriched in the immunological synapse, may thus enhance uptake and transfer of granulysin into bacterial infected host cells.
Early Role of CD4+ Th1 Cells and Antibodies in HER-2 Adenovirus Vaccine Protection against Autochthonous Mammary Carcinomas
Authors: Park Jong Myun , Terabe Masaki , Sakai Yoshio , Munasinghe Jeeva , Forni Guido , Morris John C. , Berzofsky Jay A.
Published: April 2014

HER-2 is an oncogenic tumor-associated Ag that is overexpressed in several human tumors including breast and ovarian cancer. The efficacy and mechanism of a HER-2-expressing recombinant adenoviral vaccine to protect against tumorigenesis was examined using HER-2 transgenic (BALB-neuT) mice, which develop spontaneous breast tumors in all 10 mammary glands, and also using a transplantable mouse tumor model. Vaccination beginning at 6–8 wk of age (through 19 wk of age) prevented development of spontaneous mammary tumors even after 50 wk, whereas the animals in the control groups had tumors in all mammary glands by 25 wk. Such long-term protection after the last boost has not been achieved previously in this transgenic mouse in which the oncogene is continuously spawning tumorigenesis. Using β2-microglobulin-knockout, IFN-γ-knockout, and B cell-deficient mice, CD4+ and CD8+ cell depletion, and Ab transfer studies, we show that induction of anti-HER-2/neu Abs are both necessary and sufficient for protection, and the IgG2a isotype is most effective. In contrast, CD8+ T cells are not necessary at all, and CD4+ T cells are necessary for only 36–48 h after immunization to provide help for B cells but not as effector cells. Equal protection in immunized mice deficient in FcγRI/III excluded an FcR-mediated mechanism. Anti-HER-2 serum not only inhibited growth of mammary tumor cell lines expressing HER-2 in vitro but also protected mice from tumors in vivo, suggesting a direct action of Ab on the tumor cells. Such a vaccine may provide Ab-mediated protection against HER-2-expressing breast cancers in humans.