bluuebunny/crossref_metadata_2025 huggingface dataset:
Agonist Ligands Expressed by Thymic Epithelium Enhance Positive Selection of Regulatory T Lymphocytes from Precursors with a Normally Diverse TCR Repertoire
Authors: Ribot Julie , Romagnoli Paola , van Meerwijk Joost P. M.
Published: April 2014

CD4+CD25+ regulatory T lymphocytes play a crucial role in inhibition of autoimmune pathology. In accordance with this physiological role, it is now well established that the repertoire of these lymphocytes is strongly enriched in autospecific cells. However, despite extensive investigation, the thymic mechanisms involved in development of regulatory T cells remain incompletely defined. To address the issue of selection of regulatory T cell precursors in mice with a naturally diverse TCR repertoire, we have analyzed development of superantigen-specific regulatory T cells in hemopoietic chimeras in which endogenous super-antigens are exclusively presented by thymic epithelial cells. Our results demonstrate that recognition of agonist ligands expressed by thymic epithelium does not lead to deletion but substantially enhances development of mature regulatory T cells. Interestingly, also development of a small subpopulation of CD25-expressing T cells lacking expression of the transcription factor Foxp3, thought to be autospecific, is enhanced by expression of the agonist ligand on thymic epithelium. Based on quantitative arguments, we propose that commitment to the regulatory T cell lineage is not dictated by the specificity of precursors, but that recognition of the agonist ligand expressed by thymic epithelium substantially enhances their positive selection.
HS1-Associated Protein X-1 Interacts with Membrane-Bound IgE: Impact on Receptor-Mediated Internalization
Authors: Oberndorfer Iris , Schmid Doris , Geisberger Roland , Achatz-Straussberger Gertrude , Crameri Reto , Lamers Marinus , Achatz Gernot
Published: April 2014

Engagement of the BCR triggers signals that control affinity maturation, memory induction, differentiation, and various other physiological processes in B cells. In previous work, we showed that truncation of the cytoplasmic tail of membrane-bound Ig (mIg)E in vivo resulted in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells, and the abrogation of specific secondary responses correlating with a defect in the selection of high-affinity Abs during the germinal center reaction. We concluded that the Ag receptor is necessary at all times during Ab responses not only for the maturation process, but also for the expansion of Ag-specific B cells. Based on these results, we asked whether the cytoplasmic tail of mIgE, or specific proteins binding the cytoplasmic tail in vivo commit a signal transduction accompanying the B cell along its differentiation process. In this study, we present the identification of HS1-associated protein X-1 as a novel protein interacting with the cytoplasmic tail of mIgE. ELISA, surface plasmon resonance analysis, and coimmunoprecipitation experiments confirmed the specific interaction in vitro. In functional assays, we clearly showed that HS1-associated protein X-1 expression levels influence the efficiency of BCR-mediated Ag internalization.
Mitogen-Activated Protein Kinase Pathway Activation by the CD6 Lymphocyte Surface Receptor
Authors: Ibáñez Anna , Sarrias Maria-Rosa , Farnós Montserrat , Gimferrer Idoia , Serra-Pagès Carles , Vives Jordi , Lozano Francisco
Published: April 2014

CD6 is a cell surface receptor primarily expressed on immature thymocytes and mature T and B1a lymphocytes. Through its binding to activated leukocyte cell adhesion molecule (ALCAM/CD166), CD6 is considered to play an important role in lymphocyte development and activation. Accordingly, CD6 associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse on T lymphocytes. Moreover, the CD6-ALCAM interaction has been shown to be critical for proper immunological synapse maturation and T cell proliferative responses. However, the precise biological effects of CD6 ligation and its signaling pathway are still not well understood. The present study shows that CD6 ligation with three different specific mAbs (161.8, SPV-L14.2, and MAE1-C10) induces time- and dose-dependent activation of ERK1/2 on normal and leukemic human T cells. This effect was also observed upon CD6 ligation with a chimerical ALCAM protein (ALCAM-Fc). The C-terminal cytoplasmic region of CD6, as well as Src tyrosine kinases, was critical for CD6-induced ERK1/2 activation. Synergistic effects were observed upon coligation of the TCR/CD3 complex with CD6. The ligation of CD6 induced the transcriptional activation of reporter genes under the control of the c-Fos serum responsive element and AP-1. Accordingly, CD6-mediated activation of p38 and JNK was also observed. These findings indicate that the CD6-ALCAM interaction results in activation of the three MAPK cascades, likely influencing the dynamic balance that determines whether resting or activated lymphocytes survive or undergo apoptosis.
Endogenous Tissue-Type Plasminogen Activator Is Protective duringEscherichia coli-Induced Abdominal Sepsis in Mice
Authors: Renckens Rosemarijn , Roelofs Joris J. T. H. , Florquin Sandrine , de Vos Alex F. , Pater Jennie M. , Lijnen H. Roger , Carmeliet Peter , van ’t Veer Cornelis , van der Poll Tom
Published: April 2014

Sepsis is associated with enhanced production of tissue-type plasminogen activator (tPA). We investigated the function of endogenous tPA in the immune responses to Escherichia coli-induced abdominal sepsis using tPA gene-deficient (tPA−/−) and normal wild-type (WT) mice. tPA−/− mice demonstrated an impaired defense against E. coli peritonitis as indicated by higher bacterial loads at the primary site of the infection, enhanced dissemination, and reduced survival. The protective function of tPA was independent of plasmin since plasminogen gene-deficient (Plg−/−) mice were indistinguishable from WT mice. Relative to WT mice, tPA−/− mice demonstrated similar neutrophil counts in the peritoneal cavity despite much higher bacterial loads and higher local concentrations of neutrophil attracting chemokines, suggesting a reduced migratory response. In line, tPA−/− mice demonstrated a reduced thioglycolate-induced neutrophil influx into the peritoneal cavity and i.p. injection of WT mice with a replication-defective adenoviral vector expressing tPA caused an enhanced cell migration to the peritoneal cavity during E. coli peritonitis. These findings identify a novel protective function of tPA in abdominal sepsis caused by E. coli that seems independent of its role in the generation of plasmin.
Lymph Node Resident Rather Than Skin-Derived Dendritic Cells Initiate Specific T Cell Responses after Leishmania major Infection
Authors: Iezzi Giandomenica , Fröhlich Anja , Ernst Bettina , Ampenberger Franziska , Saeland Sem , Glaichenhaus Nicolas , Kopf Manfred
Published: April 2014

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11chighCD11bhighGr-1−CD8−langerin− DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11chigh CD11bhigh DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.
Pathogenic Consequences in Semen Quality of an Autoimmune Response against the Prostate Gland: From Animal Models to Human Disease
Authors: Motrich Ruben D. , Maccioni Mariana , Ponce Andres A. , Gatti Gerardo A. , Oberti Juan P. Mackern , Rivero Virginia E.
Published: April 2014

We have recently proposed an autoimmune etiology in ∼35% of chronic nonbacterial prostatitis patients, the most frequent form of prostatitis observed, because they exhibit IFN-γ-secreting lymphocytes specific to prostate Ags. Interestingly, this particular group of patients, but not the rest of chronic nonbacterial prostatitis patients, also presented striking abnormalities in their semen quality.

In this work, we use an experimental animal model of autoimmune prostatitis on Wistar rats developed in our laboratory to investigate when, where, and how sperm cells from autoimmune prostatitis individuals are being damaged. As in patients, a marked reduction in sperm concentration, almost null sperm motility and viability, and an increased percentage of apoptotic spermatozoa were detected in samples from animals with the disease. Prostate-specific autoantibodies as well as elevated levels of NO, TNF-α, and IFN-γ were also detected in their seminal plasma. In contrast, epididymal spermatozoa remain intact, indicating that sperm damage occurs at the moment of joining of prostate secretion to sperm cells during ejaculation. These results were further supported by experiments in which mixture of normal sperm cells with autoimmune seminal plasma were performed.

We hypothesize that sperm damage in experimental autoimmune prostatitis can be the consequence of an inflammatory milieu, originally produced by an autoimmune response in the prostate; a diminished prostate functionality, evidenced by reduced levels of citric acid in semen or by both mechanisms simultaneously. Once more, we suggest that autoimmunity to prostate may have consequences on fertility.
Role of Immunoproteasomes in Cross-Presentation
Authors: Palmowski Michael J. , Gileadi Uzi , Salio Mariolina , Gallimore Awen , Millrain Maggie , James Edward , Addey Caroline , Scott Diane , Dyson Julian , Simpson Elizabeth , Cerundolo Vincenzo
Published: April 2014

The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty246–254 epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7−/−) skin grafts by female LMP7−/− mice. Second, we ruled out an altered Uty246–254-specific T cell repertoire in LMP7−/− female mice and demonstrated efficient Uty246–254 presentation by re-expressing LMP7 in male LMP7−/− cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty246–254-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7−/− female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.
Cutting Edge: Inhibition of TLR and FcR Responses in Macrophages by Triggering Receptor Expressed on Myeloid Cells (TREM)-2 and DAP12
Authors: Hamerman Jessica A. , Jarjoura Jessica R. , Humphrey Mary Beth , Nakamura Mary C. , Seaman William E. , Lanier Lewis L.
Published: April 2014

DAP12 is an ITAM-containing adapter that associates with receptors in myeloid and NK cells. DAP12-associated receptors can give activation signals leading to cytokine production; however, in some situations, DAP12 inhibits cytokine production stimulated through TLRs and FcRs. Here we show that Triggering Receptor Expressed on Myeloid cells (TREM)-2 is responsible for the DAP12-mediated inhibition in mouse macrophages. A chimeric receptor composed of the extracellular domain of TREM-2 and the cytoplasmic domain of DAP12 inhibited the TLR- and FcR-induced TNF production of DAP12-deficient macrophages, whereas a TREM-1 chimera did not. In wild-type macrophages, TREM-2 knockdown increased TLR-induced TNF production. A TREM-2 Fc fusion protein bound to macrophages, indicating that macrophages express a TREM-2 ligand. Thus, the interaction of TREM-2 and its ligand results in an inhibitory signal that can reduce the inflammatory response.
Modulation of Monocyte Function by Activated Protein C, a Natural Anticoagulant
Authors: Stephenson Daniel A. , Toltl Lisa J. , Beaudin Suzanne , Liaw Patricia C.
Published: April 2014

Activated protein C is the first effective biological therapy for the treatment of severe sepsis. Although activated protein C is well established as a physiological anticoagulant, emerging data suggest that it also exerts anti-inflammatory and antiapoptotic effects. In this study, we investigated the ability of activated protein C to modulate monocyte apoptosis, inflammation, phagocytosis, and adhesion. Using the immortalized human monocytic cell line THP-1, we demonstrated that activated protein C inhibited camptothecin-induced apoptosis in a dose-dependent manner. The antiapoptotic effect of activated protein C requires its serine protease domain and is dependent on the endothelial cell protein C receptor and protease-activated receptor-1. In primary blood monocytes from healthy individuals, activated protein C inhibited spontaneous apoptosis. With respect to inflammation, activated protein C inhibited the production of TNF, IL-1β, IL-6, and IL-8 by LPS-stimulated THP-1 cells. Activated protein C did not influence the phagocytic internalization of Gram-negative and Gram-positive bioparticles by THP-1 cells or by primary blood monocytes. Activated protein C also did not affect the expression of adhesion molecules by LPS-stimulated blood monocytes nor the ability of monocytes to adhere to LPS-stimulated endothelial cells. We hypothesize that the protective effect of activated protein C in sepsis reflects, in part, its ability to prolong monocyte survival in a manner that selectively inhibits inflammatory cytokine production while maintaining phagocytosis and adherence capabilities, thereby promoting antimicrobial properties while limiting tissue damage.
Regulatory and Effector T Cell Activation Levels Are Prime Determinants of In Vivo Immune Regulation
Authors: Billiard Fabienne , Litvinova Elena , Saadoun David , Djelti Fathia , Klatzmann David , Cohen José L. , Marodon Gilles , Salomon Benoı̂t L.
Published: April 2014

Little is known about the in vivo conditions in which CD4+CD25+ regulatory T cells (Treg) exert their suppressive effect in nonlymphopenic mice. To this end, we analyzed Treg-mediated suppression of expansion and cytokine production at different levels of Ag-specific CD4+CD25− T cell activation. Using Ab-mediated depletion of endogenous Treg, we show that basal immunosuppression is dependent on effector T cell activation. These polyclonal Treg, which were poorly activated in our immunization conditions, were effective in weak but not high T cell activation context. In contrast, the same immunization conditions led to proliferation of cotransferred Ag-specific Treg. Those efficiently inhibited T cell proliferation and cytokine production even in strong T cell activation context. Interestingly, Treg selectively suppressed expansion or cytokine production depending on the experimental approach. The importance of the immune context for efficient suppression is further supported by the observation that Treg depletion exacerbated diabetes of NOD mice only at the early stage of the disease. Overall, our study suggests that Treg-mediated suppression depends on the relative activation of Treg and effector T cells in vivo. This balance may be a critical factor in the regulation of immune responses.
Toxoplasma gondiiGenotype Determines MyD88-Dependent Signaling in Infected Macrophages
Authors: Kim Leesun , Butcher Barbara A. , Lee Chiang W. , Uematsu Satoshi , Akira Shizuo , Denkers Eric Y.
Published: April 2014

Infection of mouse macrophages with Toxoplasma gondii elicits MAPK activation and IL-12 production, but host cell signaling pathways have not been clearly delineated. Here, we compared macrophage signaling in response to high virulence type I (RH) vs low virulence type II (ME49) strain infection. Tachyzoites of both strains induced p38 MAPK-dependent macrophage IL-12 release, although ME49 elicited 2- to 3-fold more cytokine than RH. IL-12 production was largely restricted to infected cells in each case. RH-induced IL-12 release did not require MyD88, whereas ME49-triggered IL-12 production was substantially dependent on this TLR/IL-1R adaptor molecule. MyD88 was also not required for RH-stimulated p38 MAPK activation, which occurred in the absence of detectable upstream p38 MAPK kinase activity. In contrast, ME49-driven p38 MAPK activation displayed an MyD88-dependent component. This parasite strain also induced MyD88-dependent activation of MKK4, an upstream activator of p38 MAPK. The results suggest that RH triggers MAPK activation and IL-12 production using MyD88-independent signaling, whereas ME49 uses these pathways as well as MyD88-dependent signaling cascades. Differences in host signaling pathways triggered by RH vs ME49 may contribute to the high and low virulence characteristics displayed by these parasite strains.
Inflammation-Responsive Transcription Factor SAF-1 Activity Is Linked to the Development of Amyloid A Amyloidosis
Authors: Ray Alpana , Shakya Arvind , Kumar Deepak , Benson Merrill D. , Ray Bimal K.
Published: April 2014

Abundantly expressed serum amyloid A (SAA) protein under chronic inflammatory conditions gives rise to insoluble aggregates of SAA derivatives in multiple organs resulting in reactive amyloid A (AA) amyloidosis, a consequence of rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis, familial Mediterranean fever, and Castleman’s disease. An inflammation-responsive transcription factor, SAF (for SAA activating factor), has been implicated in the sustained expression of amyloidogenic SAA under chronic inflammatory conditions. However, its role in the pathogenesis of AA amyloidosis has thus far remained obscure. In this paper we have shown that SAF-1, a major member of the SAF family, is abundantly present in human AA amyloidosis patients. To assess whether SAF-1 is directly linked to the pathogenesis of AA amyloidosis, we have developed a SAF-1 transgenic mouse model. SAF-1-overexpressing mice spontaneously developed AA amyloidosis at the age of 14 mo or older. Immunohistochemical analysis confirmed the nature of the amyloid deposits as an AA type derived from amyloidogenic SAA1. Furthermore, SAF-1 transgenic mice rapidly developed severe AA amyloidosis in response to azocasein injection, indicating increased susceptibility to inflammation. Also, during inflammation SAF-1 transgenic mice exhibited a prolonged acute phase response, leading to an extended period of SAA synthesis. Together, these results provide direct evidence that SAF-1 plays a key role in the development of AA amyloidosis, a consequence of chronic inflammation.
Prostaglandin D2 Plays an Essential Role in Chronic Allergic Inflammation of the Skin via CRTH2 Receptor
Authors: Satoh Takahiro , Moroi Rie , Aritake Kosuke , Urade Yoshihiro , Kanai Yasumasa , Sumi Koji , Yokozeki Hiroo , Hirai Hiroyuki , Nagata Kinya , Hara Toshifumi , Utsuyama Masanori , Hirokawa Katsuiku , Sugamura Kazuo , Nishioka Kiyoshi , Nakamura Masataka
Published: April 2014

PGD2 plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35–55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD2-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.
Increased Levels of Soluble CD40L in African Tick Bite Fever: Possible Involvement of TLRs in the Pathogenic Interaction betweenRickettsia africae, Endothelial Cells, and Platelets
Authors: Damås Jan K. , Jensenius Mogens , Ueland Thor , Otterdal Kari , Yndestad Arne , Frøland Stig S. , Rolain Jean-Marc , Myrvang Bjørn , Raoult Didier , Aukrust Pål
Published: April 2014

The pathophysiological hallmark of spotted fever group rickettsioses comprises infection of endothelial cells with subsequent infiltration of inflammatory cells. Based on its ability to promote inflammation and endothelial cell activation, we investigated the role of CD40L in African tick bite fever (ATBF), caused by Rickettsia africae, using different experimental approaches. Several significant findings were revealed. 1) Patients with ATBF (n = 15) had increased serum levels of soluble CD40 ligand (sCD40L), which decreased during follow-up. 2) These enhanced sCD40L levels seem to reflect both direct and indirect (through endothelial cell activation involving CX3CL1-related mechanisms) effects of R. africae on platelets. 3) In combination with sCD40L, R. africae promoted a procoagulant state in endothelial cells by up-regulating tissue factor and down-regulating thrombomodulin expression. 4) Although the R. africae-mediated activation of platelets involved TLR2, the combined procoagulant effects of R. africae and sCD40L on endothelial cells involved TLR4. 5) Doxycycline counteracted the combined procoagulant effects of R. africae and sCD40L on endothelial cells. Our findings suggest an inflammatory interaction between platelets and endothelial cells in ATBF, involving TLR-related mechanisms. This interaction, which includes additive effects between sCD40L and R. africae, may contribute to endothelial inflammation and hypercoagulation in this disorder.
Double-Negative T Regulatory Cells Can Develop Outside the Thymus and Do Not Mature from CD8+ T Cell Precursors
Authors: Ford Megan S. , Zhang Zhu-Xu , Chen Wenhao , Zhang Li
Published: April 2014

Recent studies have demonstrated that activated peripheral αβTCR+CD3+CD4−CD8−NK1.1− (double-negative, DN) regulatory T cells (Tregs) from both mice and humans are able to down-regulate immune responses in vitro and in vivo. However, the origin and developmental requirements of functional DN Tregs remain unclear. In this study, we investigated the requirement for CD8 expression as well as the presence of a thymus for the development of functional DN Tregs. We demonstrate that DN Tregs exist in CD8-deficient mice and that stimulation of CD8+ T cells in vivo with TCR-specific Ag does not convert CD8+ T cells into DN Tregs. In addition, we found that DN T cells are present in the spleens and lymph nodes of thymectomized mice that are irradiated and reconstituted with T cell-depleted bone marrow cells. Interestingly, DN Tregs that develop in thymectomized mice can suppress syngeneic CD8+ T cells more effectively than those that develop in sham-thymectomized mice. Taken together, our data suggest that DN Tregs are not derived from CD8+ T cell precursors and that functional DN Tregs may preferentially develop outside of the thymus. These data suggest that DN Tregs may represent a developmentally and functionally unique cell population.
Phosphatidylinositol Mannoside fromMycobacterium tuberculosisBinds α5β1 Integrin (VLA-5) on CD4+ T Cells and Induces Adhesion to Fibronectin
Authors: Rojas Roxana E. , Thomas Jeremy J. , Gehring Adam J. , Hill Preston J. , Belisle John T. , Harding Clifford V. , Boom W. Henry
Published: April 2014

The pathological hallmark of the host response to Mycobacterium tuberculosis is the granuloma where T cells and macrophages interact with the extracellular matrix (ECM) to control the infection. Recruitment and retention of T cells within inflamed tissues depend on adhesion to the ECM. T cells use integrins to adhere to the ECM, and fibronectin (FN) is one of its major components. We have found that the major M. tuberculosis cell wall glycolipid, phosphatidylinositol mannoside (PIM), induces homotypic adhesion of human CD4+ T cells and T cell adhesion to immobilized FN. Treatment with EDTA and cytochalasin D prevented PIM-induced T cell adhesion. PIM-induced T cell adhesion to FN was blocked with mAbs against α5 integrin chain and with RGD-containing peptides. α5β1 (VLA-5) is one of two major FN receptors on T cells. PIM was found to bind directly to purified human VLA-5. Thus, PIM interacts directly with VLA-5 on CD4+ T lymphocytes, inducing activation of the integrin, and promoting adhesion to the ECM glycoprotein, FN. This is the first report of direct binding of a M. tuberculosis molecule to a receptor on human T cells resulting in a change in CD4+ T cell function.
Factor I-Mediated Processing of Complement Fragments on HIV Immune Complexes Targets HIV to CR2-Expressing B Cells and Facilitates B Cell-Mediated Transmission of Opsonized HIV to T Cells
Authors: Bánki Zoltán , Wilflingseder Doris , Ammann Christoph G. , Pruenster Monika , Müllauer Brigitte , Holländer Karoline , Meyer Martina , Sprinzl Georg M. , van Lunzen Jan , Stellbrink Hans-Jürgen , Dierich Manfred P. , Stoiber Heribert
Published: April 2014

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.
Cutting Edge: TLR9 and TLR2 Signaling Together Account for MyD88-Dependent Control of Parasitemia in Trypanosoma cruzi Infection
Authors: Bafica Andre , Santiago Helton Costa , Goldszmid Romina , Ropert Catherine , Gazzinelli Ricardo T. , Sher Alan
Published: April 2014

Activation of innate immune cells by Trypanosoma cruzi-derived molecules such as GPI anchors and DNA induces proinflammatory cytokine production and host defense mechanisms. In this study, we demonstrate that DNA from T. cruzi stimulates cytokine production by APCs in a TLR9-dependent manner and synergizes with parasite-derived GPI anchor, a TLR2 agonist, in the induction of cytokines by macrophages. Compared with wild-type animals, T. cruzi-infected Tlr9−/− mice displayed elevated parasitemia and decreased survival. Strikingly, infected Tlr2−/−Tlr9−/− mice developed a parasitemia equivalent to animals lacking MyD88, an essential signaling molecule for most TLR, but did not show the acute mortality displayed by MyD88−/− animals. The enhanced susceptibility of Tlr9−/− and Tlr2−/−Tlr9−/− mice was associated with decreased in vivo IL-12/IFN-γ responses. Our results reveal that TLR2 and TLR9 cooperate in the control of parasite replication and that TLR9 has a primary role in the MyD88-dependent induction of IL-12/IFN-γ synthesis during infection with T. cruzi.
Cutting Edge: TREM-2 Attenuates Macrophage Activation
Authors: Turnbull Isaiah R. , Gilfillan Susan , Cella Marina , Aoshi Taiki , Miller Mark , Piccio Laura , Hernandez Maristela , Colonna Marco
Published: April 2014

The triggering receptor expressed on myeloid cells 2 (TREM-2) delivers intracellular signals through the adaptor DAP12 to regulate myeloid cell function both within and outside the immune system. The role of TREM-2 in immunity has been obscured by the failure to detect expression of the TREM-2 protein in vivo. In this study, we show that TREM-2 is expressed on macrophages infiltrating the tissues from the circulation and that alternative activation with IL-4 can induce TREM-2. TREM-2 expression is abrogated by macrophage maturation with LPS of IFN-γ. Using TREM-2−/− mice, we find that TREM-2 functions to inhibit cytokine production by macrophages in response to the TLR ligands LPS, zymosan, and CpG. Furthermore, we find that TREM-2 completely accounts for the increased cytokine production previously reported by DAP12−/− macrophages. Taken together, these data show that TREM-2 is expressed on newly differentiated and alternatively activated macrophages and functions to restrain macrophage activation.
IFN-β Inhibits T Cell Activation Capacity of Central Nervous System APCs
Authors: Teige Ingrid , Liu Yawei , Issazadeh-Navikas Shohreh
Published: April 2014

We have previously investigated the physiological effects of IFN-β on chronic CNS inflammation and shown that IFN-β−/− mice develop a more severe experimental autoimmune encephalomyelitis than their IFN-β+/− littermates. This result was shown to be associated with a higher activation state of the glial cells and a higher T cell cytokine production in the CNS. Because this state suggested a down-regulatory effect of IFN-β on CNS-specific APCs, these results were investigated further. We report that IFN-β pretreatment of astrocytes and microglia (glial cells) indeed down-modulate their capacity to activate autoreactive Th1 cells. First, we investigated the intrinsic ability of glial cells as APCs and report that glial cells prevent autoreactive Th1 cells expansion while maintaining Ag-specific T cell effector functions. However, when the glial cells are treated with IFN-β before coculture with T cells, the effector functions of T cells are impaired as IFN-γ, TNF-α, and NO productions are decreased. Induction of the T cell activation marker, CD25 is also reduced. This suppression of T cell response is cell-cell dependent, but it is not dependent on a decrease in glial expression of MHC class II or costimulatory molecules. We propose that IFN-β might exert its beneficial effects mainly by reducing the Ag-presenting capacity of CNS-specific APCs, which in turn inhibits the effector functions of encephalitogenic T cells. This affect is of importance because activation of encephalitogenic T cells within the CNS is a prerequisite for the development of a chronic progressive CNS inflammation.
The Extracellular Membrane-Proximal Domain of Human Membrane IgE Controls Apoptotic Signaling of the B Cell Receptor in the Mature B Cell Line A20
Authors: Poggianella Monica , Bestagno Marco , Burrone Oscar R.
Published: April 2014

Ag engagement of BCR in mature B cells can deliver specific signals, which decide cell survival or cell death. Circulating membrane IgE+ (mIgE+) cells are found in extremely low numbers. We hypothesized that engagement of an εBCR in a mature isotype-switched B cell could induce apoptosis. We studied the role of the extracellular membrane-proximal domain (EMPD) of human mIgE upon BCR engagement with anti-Id Abs. Using mutants lacking the EMPD, we show that this domain is involved in controlling Ca2+ mobilization in immunoreceptors of both γ and ε isotypes, as well as apoptosis in signaling originated only from the εBCR. We mapped to the εCH4 ectodomain the region responsible for apoptosis in EMPD-deleted receptors. Ca2+ mobilization was not related to apoptotic signaling. This apoptotic pathway was caspase independent, involved ERK1/2 phosphorylation and was partially rescued by CD40 costimulation. We therefore conclude that the EMPD of human mIgE is a key control element of apoptotic signaling delivered through engagement of εBCR within the context of a mature B cell.
Protection from Type 1 Diabetes by Invariant NK T Cells Requires the Activity of CD4+CD25+ Regulatory T Cells
Authors: Ly Dalam , Mi Qing-Sheng , Hussain Shabbir , Delovitch Terry L.
Published: April 2014

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by α-galactosylceramide (α-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+CD25+ Treg, for this protection. We found that CD4+CD25+ T cells from NOD.CD1d−/− mice deficient in iNKT cell function similarly in vitro to CD4+CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon α-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+CD25+ Tregs from NOD mice pretreated with α-GalCer demonstrated that activated iNKT cells do not influence the ability of Tregs to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+CD25+ T cells, because splenocytes pretreated with α-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+CD25+ T cells before α-GalCer treatment were also not protected from T1D. Our data suggest that CD4+CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+CD25+ Tregs is required for iNKT cells to transfer protection from T1D.
Reduced Susceptibility of Recombinant Polyclonal Antibodies to Inhibitory Anti-Variable Domain Antibody Responses
Authors: Klitgaard Josephine L. , Coljee Vincent W. , Andersen Peter S. , Rasmussen Lone K. , Nielsen Lars S. , Haurum John S. , Bregenholt Søren
Published: April 2014

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.
Evidence That Marginal Zone B Cells Possess an Enhanced Secretory Apparatus and Exhibit Superior Secretory Activity
Authors: Gunn Kathryn E. , Brewer Joseph W.
Published: April 2014

Marginal zone B (MZB) cells are the first splenic B cells to initiate Ab secretion against polysaccharide-encapsulated Ags in vivo. This swift MZB cell response can be reproduced in vitro as LPS treatment induces Ab secretion in as little as 12 h. Conversely, in vitro LPS treatment of splenic follicular B (FOB) cells results in Ab secretion after 2–3 days. The basis for these distinct response kinetics is not understood. We performed ex vivo analysis of resting and LPS-stimulated murine MZB and FOB cells and found that MZB cells express higher levels of the LPS TLR complex RP105/MD-1 and respond to much lower concentrations of LPS than do FOB cells. Furthermore, increasing doses of LPS do not accelerate the kinetics by which FOB cells transition into Ab secretion. Ultrastructural analysis of resting cells demonstrated that rough endoplasmic reticulum is more abundant in MZB cells than in FOB cells. Additionally, RT-PCR and immunoblot analyses revealed that numerous endoplasmic reticulum resident chaperones and folding enzymes are expressed at greater levels in resting MZB cells than in resting FOB cells. Although both LPS-stimulated MZB and FOB cells increase expression of these factors, MZB cells exhibit a more rapid increase that correlates with accelerated kinetics of Ab secretion and higher per cell output of secreted IgM. These data indicate that MZB cells are equipped for exquisite sensitivity to bacterial components like LPS and poised for rapid, robust Ab production, making MZB cells ideally suited as frontline defenders in humoral immunity.
Transgenic Expression of the Viral FLIP MC159 Causes lpr/gld-Like Lymphoproliferation and Autoimmunity
Authors: Woelfel Melissa , Bixby Jacqueline , Brehm Michael A. , Chan Francis Ka-Ming
Published: April 2014

Death receptor-induced programmed cell death (PCD) is crucial for the maintenance of immune homeostasis. However, interference of downstream death receptor signaling by genetic ablation or transgenic (Tg) expression of different apoptosis inhibitors often impairs lymphocyte activation. The viral FLICE (caspase-8)-like inhibitor proteins (v-FLIPs) are potent inhibitors of death receptor-induced apoptosis and programmed necrosis. We generated Tg mice expressing the v-FLIP MC159 from Molluscum contagiosum virus under the control of the H2Kb class I MHC promoter to examine the role of death receptor-induced PCD in the control of immune functions and homeostasis. We found that expression of MC159 led to lymphoproliferation and autoimmunity as exemplified by T and B lymphocyte expansion, accumulation of TCRαβ+CD3+B220+CD4−CD8− lymphocytes in secondary lymphoid organs, elevated serum Ig levels, and increased anti-dsDNA Ab titers. These phenotypes were caused by defective death receptor-induced apoptosis, but not by defective passive cell death in the absence of mitogenic stimulation. Lymphocyte activation was normal, as demonstrated by normal thymidine incorporation and CSFE dilution of T cells stimulated with anti-CD3 and anti-CD28 Abs. In addition, effector CD8+ T cell responses to acute and memory lymphocytic choriomeningitis virus infections were unaffected in the Tg mice. These phenotypes are reminiscent of the lpr and gld mice, and show that the v-FLIP MC159 is a bona fide PCD inhibitor that does not interfere with other essential lymphocyte functions. Thus, the MC159-Tg mice provide a model to study the effects of PCD in immune responses without hampering other important lymphocyte functions.
Evolution of the CD4 Family: Teleost Fish Possess Two Divergent Forms of CD4 in Addition to Lymphocyte Activation Gene-3
Authors: Laing Kerry J. , Zou Jun J. , Purcell Maureen K. , Phillips Ruth , Secombes Christopher J. , Hansen John D.
Published: April 2014

The T cell coreceptor CD4 is a transmembrane glycoprotein belonging to the Ig superfamily and is essential for cell-mediated immunity. Two different genes were identified in rainbow trout that resemble mammalian CD4. One (trout CD4) encodes four extracellular Ig domains reminiscent of mammalian CD4, whereas the other (CD4REL) codes for two Ig domains. Structural motifs within the amino acid sequences suggest that the two Ig domains of CD4REL duplicated to generate the four-domain molecule of CD4 and the related gene, lymphocyte activation gene-3. Here we present evidence that both of these molecules in trout are homologous to mammalian CD4 and that teleosts encode an additional CD4 family member, lymphocyte activation gene-3, which is a marker for activated T cells. The syntenic relationships of similar genes in other teleost and non-fish genomes provide evidence for the likely evolution of CD4-related molecules in vertebrates, with CD4REL likely representing the primordial form in fish. Expression of both CD4 genes is highest in the thymus and spleen, and mRNA expression of these genes is limited to surface IgM− lymphocytes. consistent with a role for T cell functionality. Finally, the intracellular regions of both CD4 and CD4REL possess the canonical CXC motif involved in the interaction of CD4 with p56LCK, implying that similar mechanisms for CD4+ T cell activation are present in all vertebrates. Our results therefore raise new questions about T cell development and functionality in lower vertebrates that cannot be answered by current mammalian models and, thus, is of fundamental importance for understanding the evolution of cell-mediated immunity in gnathosomes.
Requirements for Apoptotic Cell Contact in Regulation of Macrophage Responses
Authors: Lucas Mark , Stuart Lynda M. , Zhang Ailiang , Hodivala-Dilke Kairbaan , Febbraio Maria , Silverstein Roy , Savill John , Lacy-Hulbert Adam
Published: April 2014

An important consequence of macrophage engulfment of apoptotic cells is suppression of inflammatory responses, which was first defined by assay of TNF-α release stimulated by LPS. These effects are apparently mediated in part by paracrine effects of TGF-β released by the subset of stimulated macrophages that ingest apoptotic cells, which suppresses neighboring cells. However, the apoptotic cell-derived signal that stimulates TGF-β release, and the nature of any additional signals required for the anti-inflammatory response remain poorly defined. In this study, we investigate the requirements for apoptotic cell engagement of macrophage surface receptors in these responses. We show that the apoptotic cell receptors CD36 and αvβ3 contribute to apoptotic cell phagocytosis by mouse macrophages, but are not essential for anti-inflammatory responses, suggesting that the mechanisms of response and phagocytosis are separate. In further defining requirements for response, we confirm the importance of TGF-β in suppression by apoptotic cells, and identify an additional level of control of these effects. We show that LPS-stimulated mouse macrophage TNF-α release is only suppressed if macrophages have first contacted apoptotic cells, and hence, bystander macrophages are refractory to TGF-β released by phagocytosing macrophages. We conclude that the profound suppression of LPS-driven TNF-α release by macrophage populations requires hitherto obscure contact-dependent licensing of macrophage responsiveness to TGF-β by apoptotic cells.
Induction of RelB Participates in Endotoxin Tolerance
Authors: Yoza Barbara K. , Hu Jean Y.-Q. , Cousart Sue L. , Forrest Lolita M. , McCall Charles E.
Published: April 2014

Using a THP-1 human promonocyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype, we previously showed that tolerant cells remain responsive to LPS endotoxin with degradation of IκB in the cytosol and nuclear translocation and accumulation of p50 and p65 NF-κB transcription factors. Despite this, endotoxin-inducible NF-κB-dependent innate immunity genes, like IL-1β, remained transcriptionally unresponsive in the tolerant phenotype, similar to the endotoxin tolerance observed in sepsis patients. In this study, we examined this paradox and found that RelB, another member of the NF-κB family, is induced during the establishment of tolerance. RelB expression correlated with IL-1β repression, and sepsis patients showed increased RelB when compared with normal controls. Transient expression of RelB inhibited IL-1β in endotoxin-responsive cells. In the inverse experiment, small inhibitory RNAs decreased RelB expression in tolerant cells and restored endotoxin induction of IL-1β. When we examined tolerant cell extracts, we found transcriptionally inactive NF-κB p65/RelB heterodimers. Taken together, our findings demonstrate that RelB can repress proinflammatory gene expression, and suggest that RelB expression in sepsis patient blood leukocytes may play a role in the endotoxin-tolerant phenotype.
MaxiK Blockade Selectively Inhibits the Lipopolysaccharide-Induced IκB-α/NF-κB Signaling Pathway in Macrophages
Authors: Papavlassopoulos Martin , Stamme Cordula , Thon Lutz , Adam Dieter , Hillemann Doris , Seydel Ulrich , Schromm Andra B.
Published: April 2014

Macrophages have a pivotal function in innate immunity against bacterial infections. They are present in all body compartments and able to detect invading microorganisms with high sensitivity. LPS (endotoxin) of Gram-negative bacteria is among the most potent stimuli for macrophages and initiates a wide panel of cellular activation responses. The release of mediators such as TNF-α and ILs is essential for the initiation of a proinflammatory antibacterial response. Here, we show that blockade of the large-conductance Ca2+-activated potassium channel MaxiK (BK) inhibited cytokine production from LPS-stimulated macrophages at the transcriptional level. This inhibitory effect of channel blockade was specific to stimulation with LPS and affected neither stimulation of macrophages with the cytokine TNF-α nor LPS-induced activation of cells that do not express MaxiK. Investigation of the upstream intracellular signaling pathways induced by LPS revealed that the blockade of MaxiK selectively inhibited signaling pathways leading to the activation of the transcription factor NF-κB and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor IκB-α is critically involved in the observed inhibition of NF-κB translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-κB and subsequent cytokine production is not restricted to in vitro-generated monocyte-derived macrophages but also can be observed in primary cells. Thus, MaxiK appears to be a central molecule in the NF-κB-dependent inflammatory response of macrophages to bacterial LPS.