bluuebunny/crossref_metadata_2025 huggingface dataset:
Viral Activation of Macrophages through TLR-Dependent and -Independent Pathways
Authors: Malmgaard Lene , Melchjorsen Jesper , Bowie Andrew G. , Mogensen Søren C. , Paludan Søren R.
Published: April 2014

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-αβ expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-β is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-α was totally blocked in this cell line whereas IFN-αβ expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-α and RANTES expression dramatically whereas IFN-αβ induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-αβ induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-αβ on infection.
Interaction between Complement Regulators andStreptococcus pyogenes: Binding of C4b-Binding Protein and Factor H/Factor H-Like Protein 1 to M18 Strains Involves Two Different Cell Surface Molecules
Authors: Pérez-Caballero David , García-Laorden Isabel , Cortés Guadalupe , Wessels Michael R. , de Córdoba Santiago Rodríguez , Albertí Sebastián
Published: April 2014

Streptococcus pyogenes, or group A Streptococcus, is one of the most frequent causes of pharyngitis and skin infections in humans. Many virulence mechanisms have been suggested to be involved in the infectious process. Among them is the binding to the bacterial cell surface of the complement regulatory proteins factor H, factor H-like protein 1 (FHL-1), and C4b-binding protein. Previous studies indicate that binding of these three regulators to the streptococcal cell involves the M protein encoded by the emm gene. M-type 18 strains are prevalent among clinical isolates and have been shown to interact with all three complement regulators simultaneously. Using isogenic strains lacking expression of the Emm18 or the Enn18 proteins, we demonstrate in this study that, in contradistinction to previously described S. pyogenes strains, M18 strains bind the complement regulators factor H, FHL-1, and C4b-binding protein through two distinct cell surface proteins. Factor H and FHL-1 bind to the Emm18 protein, while C4BP binds to the Enn18 protein. We propose that expression of two distinct surface structures that bind complement regulatory proteins represents a unique adaptation of M18 strains that enhances their resistance to opsonization by human plasma and increases survival of this particular S. pyogenes strain in the human host. These new findings illustrate that S. pyogenes has evolved diverse mechanisms for recruitment of complement regulatory proteins to the bacterial surface to evade immune clearance in the human host.
NF-κB Activation in Airways Modulates Allergic Inflammation but Not Hyperresponsiveness
Authors: Poynter Matthew E. , Cloots Roy , van Woerkom Tiest , Butnor Kelly J. , Vacek Pamela , Taatjes Douglas J. , Irvin Charles G. , Janssen-Heininger Yvonne M. W.
Published: April 2014

Airways display robust NF-κB activation and represent targets for anti-inflammatory asthma therapies, but the functional importance of NF-κB activation in airway epithelium remains enigmatic. Therefore, transgenic mice were created in which NF-κB activation is repressed specifically in airways (CC10-IκBαSR mice). In response to inhaled Ag, transgenic mice demonstrated significantly ameliorated inflammation, reduced levels of chemokines, T cell cytokines, mucus cell metaplasia, and circulating IgE compared with littermate controls. Despite these findings, Ag-driven airways hyperresponsiveness was not attenuated in CC10-IκBαSR mice. This study clearly demonstrates that airway epithelial NF-κB activation orchestrates Ag-induced inflammation and subsequent adaptive immune responses, but does not contribute to airways hyperresponsiveness, the cardinal feature that underlies asthma.
Combinations of Anti-LFA-1, Everolimus, Anti-CD40 Ligand, and Allogeneic Bone Marrow Induce Central Transplantation Tolerance through Hemopoietic Chimerism, Including Protection from Chronic Heart Allograft Rejection
Authors: Metzler Barbara , Gfeller Patrick , Bigaud Marc , Li Jianping , Wieczorek Grazyna , Heusser Christoph , Lake Philip , Katopodis Andreas
Published: April 2014

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vβ11+ T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening (“chronic rejection”).
Tracking of Proinflammatory Collagen-Specific T Cells in Early and Late Collagen-Induced Arthritis in Humanized Mice
Authors: Svendsen Pia , Andersen Claus B. , Willcox Nick , Coyle Anthony J. , Holmdahl Rikard , Kamradt Thomas , Fugger Lars
Published: April 2014

Rheumatoid arthritis is a chronic inflammatory disease associated with certain HLA-DR4 subtypes. The target autoantigen(s) is unknown, but type II collagen (CII) is a candidate, with a single immunodominant DR4-restricted 261–273 T cell epitope (CII(261–273)). In the present study, we have prepared HLA-DR4:CII(261–273) tetramers and analyzed peripheral blood, lymph node, and synovial fluid cells from DR4-transgenic mice with early and late collagen-induced arthritis to draw a fuller picture of the role of CII-reactive Th cells in disease development. Their frequencies increased ∼20-fold in blood 1–2 wk postimmunization, and even more in acutely arthritic joints. Our data strongly suggest that CII-specific Th cells are necessary, but not sufficient for collagen-induced arthritis. The CII-specific Th cells displayed an activated proinflammatory Th1 phenotype, and their expansion correlated with onset and severity of arthritis and also with anti-CII Ab levels. Surprisingly, shortly after the first clinical signs of arthritis, activated HLA-DR4:CII tetramer+ cells became undetectable in the synovial fluid and rare in the blood, but persisted in lymph nodes. Consequently, future human studies should focus on patients with early arthritis, and on their synovial cells, to re-evaluate the occurrence and pathogenic importance of CII-specific or other Th cells in rheumatoid arthritis.
Cutting Edge: Distinct Roles for T Help and CD40/CD40 Ligand in Regulating Differentiation of Proliferation-Competent Memory CD8+ T Cells
Authors: Bachmann Martin F. , Schwarz Katrin , Wolint Petra , Meijerink Edwin , Martin Stephen , Manolova Vania , Oxenius Annette
Published: April 2014

Murine primary antiviral cytotoxic T cell (CTL) responses are often induced in the absence of Th cells. In this study, we show that virus-like particles, if combined with DNA rich in CpG motifs, efficiently trigger primary CTL responses and comparable frequencies of memory CTLs in the presence or absence of T help. However, memory CTLs primed in the absence of T help failed to proliferate upon viral challenge. Nevertheless, they were efficiently recruited to sites of inflammation, indicating that T help may regulate the balance between proliferation-competent and migration-competent memory CTLs. Surprisingly, generation of proliferation-competent memory CTLs was completely independent of CD40 or CD40L, molecules commonly assumed to be central for mediating the beneficial effects of Th cells on CTL development. Thus, Th cells but not CD40/CD40L are key for the differentiation of proliferation-competent central memory CD8+ T cells.
Relevance of CD6-Mediated Interactions in T Cell Activation and Proliferation
Authors: Gimferrer Idoia , Calvo Maria , Mittelbrunn María , Farnós Montse , Sarrias Maria Rosa , Enrich Carlos , Vives Jordi , Sánchez-Madrid Francisco , Lozano Francisco
Published: April 2014

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.
Immunoprevention of Mammary Carcinoma in HER-2/neu Transgenic Mice Is IFN-γ and B Cell Dependent
Authors: Nanni Patrizia , Landuzzi Lorena , Nicoletti Giordano , De Giovanni Carla , Rossi Ilaria , Croci Stefania , Astolfi Annalisa , Iezzi Manuela , Di Carlo Emma , Musiani Piero , Forni Guido , Lollini Pier-Luigi
Published: April 2014

A vaccine combining IL-12 and allogeneic mammary carcinoma cells expressing p185neu completely prevents tumor onset in HER-2/neu transgenic BALB/c mice (NeuT mice). The immune protection elicited was independent from CTL activity. We now formally prove that tumor prevention is mainly based on the production of anti-p185neu Abs. In the present studies, NeuT mice were crossed with knockout mice lacking IFN-γ production (IFN-γ−/−) or with B cell-deficient mice (μMT). Vaccination did not protect NeuT-IFN-γ−/− mice, thus confirming a central role of IFN-γ. The block of Ab production in NeuT-μMT mice was incomplete. About one third of NeuT-μMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-μMT mice that responded to the vaccine with a robust production of anti-p185neu Ab displayed a markedly delayed tumor onset. In these NeuT-μMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-μMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2q molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice.
Analyses of the In Vivo Trafficking of Stoichiometric Doses of an Anti-Complement Receptor 1/2 Monoclonal Antibody Infused Intravenously in Mice
Authors: Whipple Emily C. , Shanahan Ryan S. , Ditto Andrew H. , Taylor Ronald P. , Lindorfer Margaret A.
Published: April 2014

Complement plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 Ags with C3 breakdown product C3dg, a CR2-specific ligand. We used a C3dg-opsonized IC model, anti-CR1/2 mAb 7G6, to investigate how such substrates are processed. We used RIA, whole body imaging, flow cytometry, and fluorescence immunohistochemistry to examine the disposition of 0.1- to 2-μg quantities of mAb 7G6 infused i.v. into BALB/c mice. The mAb is rapidly taken up by the spleen and binds preferentially to marginal zone (MZ) B cells; within 24 h, the MZ B cells relocate and transfer mAb 7G6 to follicular dendritic cells (FDC). Transfer occurs coincident with loss of the extracellular portion of MZ B cell CR2, suggesting that the process may be mediated by proteolysis of CR2. Intravenous infusion of an FDC-specific mAb does not induce comparable splenic localization or cellular reorganization, emphasizing the importance of MZ B cells in intrasplenic trafficking of bound substrates. We propose the following mechanism: binding of C3dg-opsonized IC to noncognate MZ B cells promotes migration of these cells to the white pulp, followed by CR2 proteolysis, which allows transfer of the opsonized IC to FDC, thus facilitating presentation of intact Ags to cognate B cells.
Membrane-Associated Heparan Sulfate Proteoglycans Are Involved in the Recognition of Cellular Targets by NKp30 and NKp46
Authors: Bloushtain Noga , Qimron Udi , Bar-Ilan Ahuva , Hershkovitz Oren , Gazit Roi , Fima Eyal , Korc Murray , Vlodavsky Israel , Bovin Nicolai V. , Porgador Angel
Published: April 2014

Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.
CD8+ T Cells Specific for EBV, Cytomegalovirus, and Influenza Virus Are Activated during Primary HIV Infection
Authors: Doisne Jean-Marc , Urrutia Alejandra , Lacabaratz-Porret Christine , Goujard Cécile , Meyer Laurence , Chaix Marie-Laure , Sinet Martine , Venet Alain
Published: April 2014

Primary viral infections, including primary HIV infection, trigger intense activation of the immune system, with marked expansion of CD38+CD8+ T cells. Whether this expansion involves only viral-specific cells or includes a degree of bystander activation remains a matter of debate. We therefore examined the activation status of EBV-, CMV-, and influenza virus (FLU)-specific CD8+ T cells during primary HIV infection, in comparison to HIV-specific CD8+ T cells. The activation markers CD38 and HLA-DR were strongly expressed on HIV-specific CD8+ T cells. Surprisingly, CD38 expression was also up-regulated on CD8+ T cells specific for other viruses, albeit to a lesser extent. Activation marker expression returned to normal or near-normal values after 1 year of highly active antiretroviral therapy. HIV viral load correlated with CD38 expression on HIV-specific CD8+ T cells but also on EBV-, CMV-, and FLU-specific CD8+ T cells. In primary HIV infection, EBV-specific CD8+ T cells also showed increased Ki67 expression and decreased Bcl-2 expression, compared with values observed in HIV-seronegative control subjects. These results show that bystander activation occurs during primary HIV infection, even though HIV-specific CD8+ T cells express the highest level of activation. The role of this bystander activation in lymphocyte homeostasis and HIV pathogenesis remains to be determined.
Parasite-Induced Th2 Polarization Is Associated with Down-Regulated Dendritic Cell Responsiveness to Th1 Stimuli and a Transient Delay in T Lymphocyte Cycling
Authors: Jankovic Dragana , Kullberg Marika C. , Caspar Patricia , Sher Alan
Published: April 2014

The nature of the signals that bias Th effector choice is still not completely understood. Using parasite extracts from pathogens known to induce polarized Th1 or Th2 responses and an in vitro experimental model for priming murine CD4+ cells, we demonstrated that splenic dendritic cells (DC), but not B cells, promote Th1/Th2 differentiation of naive CD4+ lymphocytes. Th polarization in this system was found not to depend on DC secretion of the polarizing cytokines IL-12/IL-4, but instead correlated with distinct states of DC activation induced by the different parasite preparations. As expected, conditioning of DC for Th1 development was associated with up-regulation of costimulatory molecules and enhanced chemokine production and required intact MyD88 signaling. In contrast, conditioning of DC for Th2 differentiation correlated with down-regulation of many of the same functions and was MyD88 independent. This dampened DC activation was accompanied in the cocultures by a reduction in the frequency of CD4+ lymphocytes exiting the first division of the cell cycle. When the latter was mimicked by drug-induced arrest of peptide-primed CD4+ cells after the S phase of the first cycle, a marked Th2 polarization was also observed. Together, these findings suggest that the emergence of IL-4-producing CD4+ lymphocytes results from a suppression in DC function leading to a temporary delay in initial T cell cycling.
The 4-1BB Costimulation Augments the Proliferation of CD4+CD25+ Regulatory T Cells
Authors: Zheng Guoxing , Wang Bin , Chen Aoshuang
Published: April 2014

The thymus-derived CD4+CD25+ T cells belong to a subset of regulatory T cells potentially capable of suppressing the proliferation of pathogenic effector T cells. Intriguingly, these suppressor cells are themselves anergic, proliferating poorly to mitogenic stimulation in culture. In this study, we find that the 4-1BB costimulator receptor, best known for promoting the proliferation and survival of CD8+ T cells, also induces the proliferation of the CD4+CD25+ regulatory T cells both in culture and in vivo. The proliferating CD4+CD25+ T cells produce no detectable IL-2, suggesting that 4-1BB costimulation of these cells does not involve IL-2 production. The 4-1BB-expanded CD4+CD25+ T cells are functional, as they remain suppressive to other T cells in coculture. These results support the notion that the peripheral expansion of the CD4+CD25+ T cells is controlled in part by costimulation.
CD40 Ligand Rescues Inhibitor of Differentiation 3-Mediated G1 Arrest Induced by Anti-IgM in WEHI-231 B Lymphoma Cells
Authors: Hata Kikumi , Yoshimoto Takayuki , Mizuguchi Junichiro
Published: April 2014

The engagement of membrane-bound Igs (mIgs) results in growth arrest, accompanied by apoptosis, in the WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells. Inhibitor of differentiation (Id) proteins, members of the helix-loop-helix protein family, functions in proliferation, differentiation, and apoptosis in a variety of cell types. In this study, we analyzed the involvement of Id protein in mIg-induced growth arrest and apoptosis in WEHI-231 cells. Following stimulation with anti-IgM, expression of Id3 was up-regulated at both the mRNA and protein levels; this up-regulation could be reversed by CD40L treatment. Retrovirus-mediated transduction of the Id3 gene into WEHI-231 cells resulted in an accumulation of the cells in G1 phase, but did not induce apoptosis. E box-binding activity decreased in response to anti-IgM administration, but increased after stimulation with either CD40L alone or anti-IgM plus CD40L, suggesting that E box-binding activity correlates with cell cycle progression. WEHI-231 cells overexpressing Id3 accumulated in G1 phase, which was accompanied by reduced levels of cyclin D2, cyclin E, and cyclin A, and a reciprocal up-regulation of p27Kip1. Both the helix-loop-helix and the C-terminal regions of Id3 were required for growth-suppressive activity. These data suggest that Id3 mimics mIg-mediated G1 arrest in WEHI-231 cells.
Ionizing Radiation Affects Human MART-1 Melanoma Antigen Processing and Presentation by Dendritic Cells
Authors: Liao Yu-Pei , Wang Chun-Chieh , Butterfield Lisa H. , Economou James S. , Ribas Antoni , Meng Wilson S. , Iwamoto Keisuke S. , McBride William H.
Published: April 2014

Radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes. In this study, we demonstrate the noncytotoxic effects of ionizing radiation on MHC class I Ag presentation by bone marrow-derived dendritic cells (DCs) that have divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-127–35 that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-127–35 also protected mice against growth of B16-A2/Kb tumors in HLA-A2.1/Kb transgenic mice. Taken together, these results suggest that radiation modulates MHC class I-mediated antitumor immunity by functionally affecting DC Ag presentation pathways.
The Positive Regulatory Effect of TGF-β2 on Primitive Murine Hemopoietic Stem and Progenitor Cells Is Dependent on Age, Genetic Background, and Serum Factors
Authors: Henckaerts Els , Langer Jessica C. , Orenstein Jonathan , Snoeck Hans-Willem
Published: April 2014

TGF-β is considered a negative regulator of hemopoietic stem and progenitor cells. We have previously shown that one TGF-β isoform, TGF-β2, is, in fact, a positive regulator of murine hemopoietic stem cell function in vivo. In vitro, TGF-β2, but not TGF-β1 and TGF-β3, had a biphasic dose response on the proliferation of purified lin-Sca1++kit+ (LSK) cells, with a stimulatory effect at low concentrations, which was subject to mouse strain-dependent variation. In this study we report that the stimulatory effect of TGF-β2 on the proliferation of LSK cells increases with age and after replicative stress in C57BL/6, but not in DBA/2, mice. The age-related changes in the TGF-β2 effect correlated with life span in BXD recombinant strains. The stimulatory effect of TGF-β2 on the proliferation of LSK cells requires one or more nonprotein, low m.w. factors present in fetal calf and mouse sera. The activity of this factor(s) in mouse serum increases with age. Taken together, our data suggest a role for TGF-β2 and as yet unknown serum factors in the aging of the hemopoietic stem cell compartment and possibly in organismal aging.
Murine B7-H3 Is a Negative Regulator of T Cells
Authors: Prasad Durbaka V. R. , Nguyen Thang , Li Zhaoxia , Yang Yang , Duong Julie , Wang Ying , Dong Chen
Published: April 2014

T cell activation is regulated by the innate immune system through positive and negative costimulatory molecules. B7-H3 is a novel B7-like molecule with a putative receptor on activated T cells. Human B7-H3 was first described as a positive costimulator, most potently inducing IFN-γ production and cellular immunity. In this study we examined the expression and function of mouse B7-H3. B7-H3 is mostly expressed on professional APCs; its expression on dendritic cells appears to be up-regulated by LPS. In contrast to human B7-H3, we found that mouse B7-H3 protein inhibited T cell activation and effector cytokine production. An antagonistic mAb to B7-H3 enhanced T cell proliferation in vitro and led to exacerbated experimental autoimmune encephalomyelitis in vivo. Therefore, mouse B7-H3 serves as a negative regulator of T cell activation and function.
Up-Regulation of Vascular Endothelial Growth Factor (VEGF) in Small-for-Size Liver Grafts Enhances Macrophage Activities through VEGF Receptor 2-Dependent Pathway
Authors: Yang Zhen Fan , Poon Ronnie T. , Luo Ying , Cheung Cindy K. , Ho David W. , Lo Chung Mau , Fan Sheung Tat
Published: April 2014

This study aims to investigate the potential role of vascular endothelial growth factor (VEGF) and VEGF-R2 (fetal liver kinase (Flk)-1) in mediating macrophage activities in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed using either whole, 50, or 30% liver grafts (both 50 and 30% were regarded as small-for-size) in syngeneic or allogeneic combinations, respectively. Firstly, the mRNA and protein levels of VEGF and Flk-1 in liver grafts were detected by RT-PCR and Western blot, and the number of Flk-1+ macrophages (labeled by ED1) was determined by flow cytometry. It was found that the small-for-size isografts and allografts presented higher levels of VEGF and Flk-1 expression than the whole isograft and allograft. In addition, a higher number of Flk-1+ED1+ cells were detected in the small-for-size isografts and allografts than the whole isograft and allograft. Secondly, our study revealed that macrophage cell lines did not initially express detectable Flk-1, but could be induced by VEGF, and the inducible expression of Flk-1 in macrophages was related to their migration and proliferation activities. Finally, our study demonstrated that the induction of Flk-1 expression on macrophages by VEGF was associated with the expression of NF-κB and heat shock protein 90. In conclusion, the present study showed that the up-regulated expression of VEGF and its interaction with Flk-1 in small-for-size liver grafts might facilitate the activities of macrophages.
CD45-Deficient Mice Accumulate Pro-B Cells Both In Vivo and In Vitro
Authors: Fleming Heather E. , Milne Craig D. , Paige Christopher J.
Published: April 2014

Efficient generation of mature B lineage cells requires the participation of the BCR, the pre-BCR, accessory coreceptors, and growth factor receptors. Together these receptors integrate cell intrinsic signals with regulatory pathways initiated by surrounding cells and structures. CD45 is a receptor tyrosine phosphatase expressed at high levels on all hemopoietic cells, and has been shown to modulate many signaling cascades in both positive and negative manners. In the absence of B220, the B lineage isoform of CD45, differentiation to the mature B cell stage is incomplete. We demonstrate that CD45-deficient mice also accumulate pro-B cells in the bone marrow. In vitro differentiation is altered in that B lineage populations exhibit prolonged survival in the presence of high concentrations of IL-7. Cell lines derived from CD45-deficient animals experience prolonged JAK/STAT activation in response to IL-7 stimulation, and constitutively elevated levels of phosphorylated src kinases. Aberrant IL-7Rα expression is observed in vivo, and may be responsible for the skewed development present in CD45−/− animals. Demonstrating that CD45-deficient pro-B cells are affected by the absence of B220 highlights a previously unrecognized parallel in B and T lineage precursors, and emphasizes that the presence of normal numbers of peripheral B cells does not assure that the bone marrow compartment is intact.
Mechanisms Regulating the Positioning of Mouse p47 Resistance GTPases LRG-47 and IIGP1 on Cellular Membranes: Retargeting to Plasma Membrane Induced by Phagocytosis
Authors: Martens Sascha , Sabel Katja , Lange Rita , Uthaiah Revathy , Wolf Eva , Howard Jonathan C.
Published: April 2014

The recently identified p47 GTPases are one of the most effective cell-autonomous resistance systems known against intracellular pathogens in the mouse. One member of the family, LRG-47, has been shown to be essential for immune control in vivo of Listeria monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, and Mycobacterium avium, possibly by promoting acidification of the phagosome. However, the intracellular localization of LRG-47, and the nature of its association with the phagosomal or any other membrane system is unknown. In this study, we show that LRG-47 is a Golgi-associated protein in the IFN-stimulated cell, which is rapidly recruited to active plasma membrane upon phagocytosis and remains associated with phagosomes as they mature. We show that the Golgi localization of LRG-47 is dependent on the integrity of an amphipathic helix near the C terminus, whereas the plasma membrane localization depends on an unidentified signal associated with the G domain. Unlike LRG-47, but like the published p47 resistance GTPase, IGTP, a further p47 GTPase, IIGP1, is associated with the endoplasmic reticulum. However, unlike IGTP, IIGP1 is associated with the endoplasmic reticulum by an N-terminal myristoylation modification. Thus, the p47 GTPases are a diverse battery of intracellular defense factors dynamically associated with different membrane systems.
Cytoplasmic Entry of Listeria monocytogenes Enhances Dendritic Cell Maturation and T Cell Differentiation and Function
Authors: Brzoza Kristina L. , Rockel Andrea B. , Hiltbold Elizabeth M.
Published: April 2014

Protective immunity to the intracellular bacterial pathogen, Listeria monocytogenes, is mediated by a vigorous T cell response. In particular, CD8+ cytolytic T cells provide essential effector function in the clearance of bacterial infection. The cytoplasmic entry of Listeria facilitated by listeriolysin O is an essential feature not only of the bacteria’s virulence, but of the ability of the bacteria to elicit protective immunity in the host. To determine how cytoplasmic entry of Listeria regulates the development of protective immunity, we examined the effects of this process on the maturation of murine dendritic cells (DC) and on their ability to prime naive CD8+ T cell responses. Costimulatory molecules (CD40, CD80, and CD86) were induced by listerial infection only when the bacteria invaded the cytoplasm. In addition, the production of IL-12, IL-10, IL-6, and TNF-α was most efficiently triggered by cytosolic Listeria. Naive T cells primed by peptide-loaded DC infected with either wild-type or nonhemolytic mutant Listeria proliferated equivalently, but a much larger proportion of those primed by wild-type Listeria monocytogenes produced IFN-γ. Costimulatory molecules induced by cytosolic entry regulated T cell proliferation and, as a result, the number of functional T cells generated. DC-produced cytokines (specifically IL-12 and IL-10) were the major factors determining the proportion of T cells producing IFN-γ. These data highlight the requirement for listerial cytoplasmic invasion for the optimal priming of T cell cytokine production and attest to the importance of this event to the development of protective CTL responses to this pathogen.
Temporin A and Related Frog Antimicrobial Peptides Use Formyl Peptide Receptor-Like 1 as a Receptor to Chemoattract Phagocytes
Authors: Chen Qian , Wade David , Kurosaka Kahori , Wang Zhao Yuan , Oppenheim Joost J. , Yang De
Published: April 2014

Many mammalian antimicrobial peptides (AMPs) have multiple effects on antimicrobial immunity. We found that temporin A (TA), a representative frog-derived AMP, induced the migration of human monocytes, neutrophils, and macrophages with a bell-shaped response curve in a pertussis toxin-sensitive manner, activated p44/42 MAPK, and stimulated Ca2+ flux in monocytes, suggesting that TA is capable of chemoattracting phagocytic leukocytes by the use of a Giα protein-coupled receptor. TA-induced Ca2+ flux in monocytes was cross-desensitized by an agonistic ligand MMK-1 specific for formyl peptide receptor-like 1 (FPRL1) and vice versa, suggesting that TA uses FPRL1 as a receptor. This conclusion was confirmed by data showing that TA selectively stimulated chemotaxis of HEK 293 cells transfected with human FPRL1 or its mouse ortholog, murine formyl peptide receptor 2. In addition, TA elicited the infiltration of neutrophils and monocytes into the injection site of mice, indicating that TA is also functionally chemotactic in vivo. Examination of two additional temporins revealed that Rana-6 was also able to attract human phagocytes using FPRL1, but temporin 1P selectively induced the migration of neutrophils using a distinct receptor. Comparison of the chemotactic and antimicrobial activities of several synthetic analogues suggested that these activities are likely to rely on different structural characteristics. Overall, the results demonstrate that certain frog-derived temporins have the capacity to chemoattract phagocytes by the use of human FPRL1 (or its orthologs in other species), providing the first evidence suggesting the potential participation of certain amphibian antimicrobial peptides in host antimicrobial immunity.
Partial Activation of Neonatal CD11c+ Dendritic Cells and Induction of Adult-Like CD8+ Cytotoxic T Cell Responses by Synthetic Microspheres
Authors: Regner Matthias , Martinez Xavier , Belnoue Elodie , Sun Cheng-Ming , Boisgerault Florence , Lambert Paul-Henri , Leclerc Claude , Siegrist Claire-Anne
Published: April 2014

Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8+ cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8+ T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-γ-secreting CD8+ splenocytes. Neonatal CD8+ T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c+ dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c+ DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4+ T cell help. The effective induction of neonatal CD8+ T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c+ DC may mature sufficiently to stimulate naive CD8+ neonatal T cells, even in the absence of strong maturation signals.
Lipopolysaccharide Binding Protein Binds to Triacylated and Diacylated Lipopeptides and Mediates Innate Immune Responses
Authors: Schröder Nicolas W. J. , Heine Holger , Alexander Christian , Manukyan Maria , Eckert Jana , Hamann Lutz , Göbel Ulf B. , Schumann Ralf R.
Published: April 2014

LPS binding protein (LBP) is an acute-phase protein synthesized predominantly in the liver of the mammalian host. It was first described to bind LPS of Gram-negative bacteria and transfer it via a CD14-enhanced mechanism to a receptor complex including TLR-4 and MD-2, initiating a signal transduction cascade leading to the release of proinflammatory cytokines. In recent studies, we found that LBP also mediates cytokine induction caused by compounds derived from Gram-positive bacteria, including lipoteichoic acid and peptidoglycan fragments. Lipoproteins and lipopeptides have repeatedly been shown to act as potent cytokine inducers, interacting with TLR-2, in synergy with TLR-1 or -6. In this study, we show that these compounds also interact with LBP and CD14. We used triacylated lipopeptides, corresponding to lipoproteins of Borrelia burgdorferi, mycobacteria, and Escherichia coli, as well as diacylated lipopeptides, corresponding to, e.g., 2-kDa macrophage activating lipopeptide of Mycoplasma spp. Activation of Chinese hamster ovary cells transfected with TLR-2 by both lipopeptides was enhanced by cotransfection of CD14. Responsiveness of human mononuclear cells to these compounds was greatly enhanced in the presence of human LBP. Binding of lipopeptides to LBP as well as competitive inhibition of this interaction by LPS was demonstrated in a microplate assay. Furthermore, we were able to show that LBP transfers lipopeptides to CD14 on human monocytes using FACS analysis. These results support that LBP is a pattern recognition receptor transferring a variety of bacterial ligands including the two major types of lipopeptides to CD14 present in different receptor complexes.
Oral Administration with Papillomavirus Pseudovirus Encoding IL-2 Fully Restores Mucosal and Systemic Immune Responses to Vaccinations in Aged Mice
Authors: Fayad Raja , Zhang Hongtao , Quinn Daniel , Huang Yujun , Qiao Liang
Published: April 2014

Infectious diseases are one of the major threats for the elderly because their immune system is often compromised, and vaccinations to prevent these infections are not effective. A major defect in their immune system seems to be the inability of T cells to produce IL-2. We used papillomavirus (PV) pseudoviruses (PSVs) as a model vaccine and a gene delivery vector to address how to enhance immune responses to vaccinations. We found that oral immunization with PV PSV induced minimal mucosal and systemic Abs and CTLs specific for the PSVs in aged mice compared with young adult mice. In addition, fewer specific Th cells were generated in the aged mice. When aged mice were immunized with PV PSVs encoding human IL-2, specific Th cells were generated, producing murine IL-2, IL-4, and IFN-γ. Further, specific Abs and CTLs were induced, resulting in protection against mucosal viral challenge. Thus, this study provided a basis for clinical trials using PV PSVs encoding IL-2 for vaccination of the elderly.
Mature Human Langerhans Cells Derived from CD34+ Hematopoietic Progenitors Stimulate Greater Cytolytic T Lymphocyte Activity in the Absence of Bioactive IL-12p70, by Either Single Peptide Presentation or Cross-Priming, Than Do Dermal-Interstitial or Monocyte-Derived Dendritic Cells
Authors: Ratzinger Gudrun , Baggers Jan , de Cos Maria A. , Yuan Jianda , Dao Tao , Reagan John L. , Münz Christian , Heller Glenn , Young James W.
Published: April 2014

The emerging heterogeneity of dendritic cells (DCs) mirrors their increasingly recognized division of labor at myriad control points in innate and acquired cellular immunity. We separately generated blood monocyte-derived DCs (moDCs), as well as Langerhans cells (LCs) and dermal-interstitial DCs (DDC-IDCs) from CD34+ hematopoietic progenitor cells. Differential expression of CD11b, CD52, CD91, and the CD1 isoforms proved useful in distinguishing these three DC types. All mature DCs uniformly expressed comparable levels of HLA-DR, CD83, CD80, and CD86, and were potent stimulators of allogeneic T cells after exposure either to recombinant human CD40L trimer or a combination of inflammatory cytokines with PGE2. moDCs, however, required 0.5–1 log greater numbers than LCs or DDC-IDCs to stimulate comparable T cell proliferation. Only moDCs secreted the bioactive heterodimer IL-12p70, and moDCs phagocytosed significantly more dying tumor cells than did either LCs or DDC-IDCs. LCs nevertheless proved superior to moDCs and DDC-IDCs in stimulating CTL against a recall viral Ag by presenting passively loaded peptide or against tumor Ag by cross-priming autologous CD8+ T cells. LCs also secreted significantly more IL-15 than did either moDCs or DDC-IDCs, which is especially important to the generation of CTL. These findings merit further comparisons in clinical trials designed to determine the physiologic relevance of these distinctions in activity between LCs and other DCs.
Characterization and Recruitment of Plasmacytoid Dendritic Cells in Synovial Fluid and Tissue of Patients with Chronic Inflammatory Arthritis
Authors: Lande Roberto , Giacomini Elena , Serafini Barbara , Rosicarelli Barbara , Sebastiani Gian Domenico , Minisola Giovanni , Tarantino Umberto , Riccieri Valeria , Valesini Guido , Coccia Eliana M.
Published: April 2014

Dendritic cells (DCs) are thought to play a key role in driving the immunopathogenic response underlying chronic inflammatory arthritis. In this study, we have examined the presence and phenotype of plasmacytoid DCs (pDCs) in the synovial fluids (SF) of patients with rheumatoid arthritis (RA), psoriatic arthritis (PA), and osteoarthritis (OA) and determined the chemotactic properties of SF from these patients toward pDCs. Flow cytometry analysis showed that the percentage of pDCs, identified as a population of Lin−CD123++ cells, is 4- to 5-fold higher in RA SF and PA SF than in OA SF. The morphological and immunophenotypic characterization of pDCs isolated from PA and RA SF indicates that they are in an immature state, most likely due to inhibitory factors present in RA SF, but are still able to undergo maturation when exposed ex vivo to viral agent or unmethylated DNA. CD123+ and BDCA2+ pDCs were detected by immunohistochemistry in RA synovial tissue in which expression of the IFN-α-inducible protein MxA was also found, suggesting production of type I IFN by maturing pDCs. We also show that CXCR3 and CXCR4 are expressed by both blood-derived pDCs and pDCs isolated from RA and PA SF and that CXCL-10, CXCL-11, and CXCL-12 present in RA and PA SF stimulate chemotaxis of blood-derived pDCs. Altogether, these findings suggest that chemokine-driven recruitment of pDCs from the blood to the inflamed synovium could be important in the regulation of the immune response in chronic inflammatory arthritis.
Epitope-Dependent Inhibition of T Cell Activation by theEaTransgene: An Explanation for Transgene-Mediated Protection from Murine Lupus
Authors: Martinez-Soría Eduardo , Ibnou-Zekri Nabila , Iwamoto Masahiro , Santiago-Raber Marie-Laure , Kikuchi Shuichi , Kosco-Vilbois Marie , Izui Shozo
Published: April 2014

A high level expression of the Ead transgene encoding the I-E α-chain is highly effective in the suppression of lupus autoantibody production in mice. To explore the possible modulation of the Ag-presenting capacity of B cells as a result of the transgene expression, we assessed the ability of the transgenic B cells to activate Ag-specific T cells in vitro. By using four different model Ag-MHC class II combinations, this analysis revealed that a high transgene expression in B cells markedly inhibits the activation of T cells in an epitope-dependent manner, without modulation of the I-E expression. The transgene-mediated suppression of T cell responses is likely to be related to the relative affinity of peptides derived from transgenic I-E α-chains (Eα peptides) vs antigenic peptides to individual class II molecules. Our results support a model of autoimmunity prevention based on competition for Ag presentation, in which the generation of large amounts of Eα peptides with high affinity to I-A molecules decreases the use of I-A for presentation of pathogenic self-peptides by B cells, thereby preventing excessive activation of autoreactive T and B cells.
License to Heal: Bidirectional Interaction of Antigen-Specific Regulatory T Cells and Tolerogenic APC
Authors: Vlad George , Cortesini Raffaello , Suciu-Foca Nicole
Published: April 2014

Naturally occurring CD4+CD25+ regulatory T (TR) cells, a component of the innate immune response, which play a key role in the maintenance of self-tolerance, have become the focus of numerous studies over the last decade. These cells inhibit the immune response in an Ag-nonspecific manner, interacting with other T cells. Much less is known about adaptive TR cells, which develop in response to chronic antigenic stimulation, and act directly on professional and nonprofessional APC, rendering them tolerogenic and able to elicit the differentiation of CD8+ and CD4+ T cells with suppressive activity. In this review, we will discuss data pertaining to the bidirectional interaction between Ag-specific TR with APC and their clinical relevance.
Cutting Edge: The Acquisition of TLR Tolerance during Malaria Infection Impacts T Cell Activation
Authors: Perry James A. , Olver Christine S. , Burnett Robert C. , Avery Anne C.
Published: April 2014

An effective immune response to infection requires control of pathogen growth while minimizing inflammation-associated pathology. During malaria infection, this balance is particularly important. Murine malaria is characterized by early production of proinflammatory cytokines, which declines in the face of continuing parasitemia. The mechanism by which this occurs remains poorly understood. In this study, we investigated the role of dendritic cells (DCs) in regulating pro- and anti-inflammatory cytokine responses. As malaria infection progresses, DCs become refractory to TLR-mediated IL-12 and TNF-α production, while increasing their ability to produce IL-10 and retaining the capacity for activation of naive T cells. IL-12-secreting DCs from early infection stimulate an IFN-γ-dominated T cell response, whereas IL-10-secreting DCs from later stages induce an IL-10-dominated T cell response. We suggest that phenotypic changes in DCs during Plasmodium yoelii infection represent a mechanism of controlling host inflammation while maintaining effective adaptive immunity.