bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation and Function of Reactive Oxygen Species in Dendritic Cells During Antigen Presentation
Authors: Matsue Hiroyuki , Edelbaum Dale , Shalhevet David , Mizumoto Norikatsu , Yang Chendong , Mummert Mark E. , Oeda Junichi , Masayasu Hiroyuki , Takashima Akira
Published: April 2014

Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5–20 μM inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4+ T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5–20 μM inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.
TCR-Mediated Notch Signaling Regulates Proliferation and IFN-γ Production in Peripheral T Cells
Authors: Palaga Tanapat , Miele Lucio , Golde Todd E. , Osborne Barbara A.
Published: April 2014

Notch genes encode membrane receptors that regulate cell fate decisions in metazoa. Notch receptors and ligands are expressed in developing lymphoid tissue and mature lymphocytes and the role of Notch signaling in early T and B cell development has been studied extensively. However, its contribution to mature T cell function is unknown. TCR-mediated T cell activation is a fundamental process of the adaptive immune system that has been studied for decades; however, the details of this process are incompletely understood. In this study, we present evidence that Notch is required for TCR-mediated activation of peripheral T cells. Inhibition of Notch activation dramatically decreases T cell proliferation in both CD4 and CD8 cells and blocks both NF-κB activity and IFN-γ production in peripheral T cells. Our data reveal a new, nondevelopmental function of Notch as a previously unknown key link in peripheral T cell activation and cytokine secretion.
Differential Recognition of Altered Peptide Ligands Distinguishes Two Functionally Discordant (Arthritogenic and Nonarthritogenic) Autoreactive T Cell Hybridoma Clones
Authors: Buzás Edit I. , Hanyecz Anita , Murad Yanal , Hudecz Ferenc , Rajnavölgyi Eva , Mikecz Katalin , Glant Tibor T.
Published: April 2014

Intravenous injection of a cartilage proteoglycan (aggrecan)-specific Th1 hybridoma clone 5/4E8 induced joint lesions similar to those seen in either primary or adoptively transferred arthritis in BALB/c mice. A sister clone, TA20, recognizing the same peptide epitope of human aggrecan and using the same Vβ4 and Vα1 segments, failed to induce joint inflammation. This study examines the fine epitope specificities of these two clones. Both 5/4E8 and TA20 hybridomas were generated using T cells from the same arthritic animal that has been immunized with human aggrecan, and both clones recognized peptides containing a consensus GRVRVNSAY sequence. However, flanking regions outside this nonapeptide sequence region had differential impact on peptide recognition by the two clones. Similarly, when single amino acid substitutions were introduced to the consensus sequence, significant differences were detected in the epitope recognition patterns of the T cell hybridomas. The 5/4E8 hybridoma showed greater flexibility in recognition, including a higher responsiveness to the corresponding self (mouse) aggrecan peptide, and produced more inflammatory cytokines (IFN-γ and TNF-α), whereas hybridoma TA20 produced IL-5 in response to either human or mouse self peptide stimulation. These results demonstrate that, within the pool of immunodominant (foreign) peptide-activated lymphocytes, marked individual differences of degeneracy exist in T cell recognition, with possible implications to autopathogenic T cell functions.
Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD200
Authors: Wright Gavin J. , Cherwinski Holly , Foster-Cuevas Mildred , Brooke Gary , Puklavec Michael J. , Bigler Mike , Song Yaoli , Jenmalm Maria , Gorman Dan , McClanahan Terri , Liu Man-Ru , Brown Marion H. , Sedgwick Jonathon D. , Phillips Joseph H. , Barclay A. Neil
Published: April 2014

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.
Candida albicans Induces Selectively Transcriptional Activation of Cyclooxygenase-2 in HeLa Cells: Pivotal Roles of Toll-Like Receptors, p38 Mitogen-Activated Protein Kinase, and NF-κB
Authors: Deva Rupal , Shankaranarayanan Pattabhiraman , Ciccoli Roberto , Nigam Santosh
Published: April 2014

Candidiasis, in its mucocutaneous form as well as in an invasive form, is frequently associated with high morbidity. PGE2, which is generated by enzymatic activity of cyclooxygenases (COXs) 1 and 2, has been shown to trigger morphogenesis in Candida albicans. In the present study, we investigated whether C. albicans altered COX-2 expression in HeLa cells. RT-PCR and Western blot analyses revealed a time-dependent biphasic behavior of COX-2 mRNA expression and COX-2 protein level. COX-1 protein remained unaffected. Neutralization with Abs against Toll-like receptors (TLR) 2 and 4 inhibited the Candida-induced production of PGE2, suggesting a vital role for TLRs in the recognition and signaling in mammalian cells upon infection with C. albicans. Transient transfections with COX-2 promoter-luciferase construct and various inhibitors of mitogen-activated protein kinases (MAPK), such as protein kinase C (PKC) inhibitor GF203190X, p38MAPK inhibitor SB203109, and extracellular-regulated kinases 1 and 2 inhibitor PD98509 showed that C. albicans up-regulates selectively COX-2, but not COX-1, through p38MAPK and PKC pathways. No involvement of other stress kinases, e.g., c-Jun NH2-terminal kinase and extracellular-regulated kinases 1 and 2, was observed. Transient transfection of NF-κB promoter construct and dominant negative plasmid of IκBβ kinase showed that COX-2 transcription is mediated through p38MAPK and NF-κB pathways. That NF-κB up-regulates p38MAPK is novel and is in contradiction to earlier reports in which NF-κB was shown to inhibit p38MAPK. In conclusion, multiple converging signaling pathways, involving TLRs followed by PKC, p38MAPK, and/or NF-κB, are triggered by C. albicans in activation of COX-2 gene.
Killer Cell Ig-Like Receptor and Leukocyte Ig-Like Receptor Transgenic Mice Exhibit Tissue- and Cell-Specific Transgene Expression
Authors: Belkin Danny , Torkar Michaela , Chang Chiwen , Barten Roland , Tolaini Mauro , Haude Anja , Allen Rachel , Wilson Michael J. , Kioussis Dimitris , Trowsdale John
Published: April 2014

To generate an experimental model for exploring the function, expression pattern, and developmental regulation of human Ig-like activating and inhibitory receptors, we have generated transgenic mice using two human genomic clones: 52N12 (a 150-Kb clone encompassing the leukocyte Ig-like receptor (LILR)B1 (ILT2), LILRB4 (ILT3), and LILRA1 (LIR6) genes) and 1060P11 (a 160-Kb clone that contains ten killer cell Ig-like receptor (KIR) genes). Both the KIR and LILR families are encoded within the leukocyte receptor complex, and are involved in immune modulation. We have also produced a novel mAb to LILRA1 to facilitate expression studies. The LILR transgenes were expressed in a similar, but not identical, pattern to that observed in humans: LILRB1 was expressed in B cells, most NK cells, and a small number of T cells; LILRB4 was expressed in a B cell subset; and LILRA1 was found on a ring of cells surrounding B cell areas on spleen sections, consistent with other data showing monocyte/macrophage expression. KIR transgenic mice showed KIR2DL2 expression on a subset of NK cells and T cells, similar to the pattern seen in humans, and expression of KIR2DL4, KIR3DS1, and KIR2DL5 by splenic NK cells. These observations indicate that linked regulatory elements within the genomic clones are sufficient to allow appropriate expression of KIRs in mice, and illustrate that the presence of the natural ligands for these receptors, in the form of human MHC class I proteins, is not necessary for the expression of the KIRs observed in these mice.
Tax and M1 Peptide/HLA-A2-Specific Fabs and T Cell Receptors Recognize Nonidentical Structural Features on Peptide/HLA-A2 Complexes
Authors: Biddison William E. , Turner Richard V. , Gagnon Susan J. , Lev Avital , Cohen Cyril J. , Reiter Yoram
Published: April 2014

Both TCRs and Ab molecules are capable of MHC-restricted recognition of peptide/MHC complexes. However, such MHC restriction is the predominant mode of recognition by T cells, but is extremely rare for B cells. The present study asks whether the dichotomy in Ag recognition modes of T and B cells could be due to fundamental differences in the methods by which TCRs and Abs recognize peptide/MHC complexes. We have compared MHC and peptide recognition by panels of CTL lines specific for the Tax and M1 peptides presented by HLA-A2 plus Tax and M1 peptide/HLA-A2-specific human Fabs that were selected from a naive phage display library. Collectively, the results indicate both striking similarities and important differences between Fab and TCR recognition of MHC and peptide components of the Tax and M1/HLA-A2 complexes. These findings suggest that these two classes of immunoreceptors have solved the problem of specific recognition of peptide/MHC complexes by nonidentical mechanisms. This conclusion is important in part because it indicates that Ab engineering approaches could produce second-generation Ab molecules that more closely mimic TCR fine specificity. Such efforts may produce more efficacious diagnostic and therapeutic agents.
Herpes Simplex Virus Type 1 Targets the MHC Class II Processing Pathway for Immune Evasion
Authors: Neumann Jürgen , Eis-Hübinger Anna Maria , Koch Norbert
Published: April 2014

HSV type 1 (HSV-1) has evolved numerous strategies for modifying immune responses that protect against infection. Important targets of HSV-1 infection are the MHC-encoded peptide receptors. Previous studies have shown that a helper T cell response and Ab production play important roles in controlling HSV-1 infection. The reduced capacity of infected B cells to stimulate CD4+ T cells is beneficial for HSV-1 to evade immune defenses. We investigated the impact of HSV-1 infection on the MHCII processing pathway, which is critical to generate CD4+ T cell help. HSV-1 infection targets the molecular coplayers of MHC class II processing, HLA-DR (DR), HLA-DM (DM), and invariant chain (Ii). HSV-1 infection strongly reduces expression of Ii, which impairs formation of SDS-resistant DR-peptide complexes. Residual activity of the MHC class II processing pathway is diminished by viral envelope glycoprotein B (gB). Binding of gB to DR competes with binding to Ii. In addition, we found gB associated with DM molecules. Both, gB-associated DR and DM heterodimers are exported from the endoplasmic reticulum, as indicated by carbohydrate maturation. Evaluation of DR, DM, and gB subcellular localization revealed abundant changes in intracellular distribution. DR-gB complexes are localized in subcellular vesicles and restrained from cell surface expression.
Dominating IgE-Binding Epitope of Bet v 1, the Major Allergen of Birch Pollen, Characterized by X-ray Crystallography and Site-Directed Mutagenesis
Authors: Spangfort Michael D. , Mirza Osman , Ipsen Henrik , van Neerven R. J. Joost , Gajhede Michael , Larsen Jørgen N.
Published: April 2014

Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab′ fragment. The epitope occupies ∼10% of the molecular surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid substitution, Glu45-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45 is a critical amino acid also in a major human IgE-binding epitope. By solving the three-dimensional crystal structure of the Bet v 1 Glu45-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu45-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.
Immunoglobulin μ Heavy Chains Do Not Mediate Tyrosine Phosphorylation of Igα from the ER-cis-Golgi
Authors: Mielenz Dirk , Ruschel Anja , Vettermann Christian , Jäck Hans-Martin
Published: April 2014

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early μ heavy chain (μHC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a μHC, a surrogate L chain, and the signal dimer Igαβ. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a μHC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Igα in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing μHC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing μHC assembled with the Igαβ dimer; however, in contrast to a pairing μHC, the nonpairing μHC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Igα after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of μHC, Igαβ, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.
Measles Virus Infection Induces Chemokine Synthesis by Neurons
Authors: Patterson Catherine E. , Daley John K. , Echols Lisa A. , Lane Thomas E. , Rall Glenn F.
Published: April 2014

The role that neurons play in the induction of the immune response following CNS viral infection is poorly understood, largely owing to the belief that these cells are immunologically quiescent. In this report, we show that virus infection of neurons results in the synthesis of proinflammatory chemokines, which are early and important mediators of leukocyte recruitment to sites of viral infection. For these studies, a transgenic mouse model of neuron-restricted measles virus (MV) infection was used. Inoculation of immunocompetent and immunodeficient transgenic adult mice resulted in CNS induction of the mRNAs encoding IFN-γ inducible protein of 10 kD, monokine inducible by γ and RANTES. Colocalization of chemokine proteins with MV-infected neurons was detected by immunofluorescence in infected brain sections. Both IFN-γ inducible protein 10 kD and RANTES were also induced in MV-infected primary hippocampal neurons cultured from transgenic embryos, as shown by RNase protection assay, confocal microscopy, and ELISA. Interestingly, neuronal infection with another RNA virus (lymphocytic choriomeningitis virus) was not associated with induction of these chemokines. In immunocompetent mice, chemokine synthesis preceded the infiltration of T lymphocytes, and chemokine ablation by neutralizing Abs resulted in a 20–50% reduction in the number of infiltrating lymphocytes. Collectively, these data indicate that neurons play an important role in the recruitment of a protective antiviral response to the CNS following viral infection, although such a role may be virus type-dependent.
Reactivation of Latent Tuberculosis Infection in TNF-Deficient Mice
Authors: Botha Tania , Ryffel Bernhard
Published: April 2014

TNF-deficient mice are highly susceptible to Mycobacterium tuberculosis H37Rv infection. Here we asked whether TNF is required for postinfectious immunity in aerosol-infected mice. Chemotherapy for 4 wk commencing 2 wk postinfection reduced CFU to undetectable levels. While wild-type mice had a slight rise in CFU, but controlled infection upon cessation of chemotherapy, TNF-deficient mice developed reactivation of infection with high bacterial loads in lungs, spleen, and liver, which was fatal within 13–18 wk. The increased susceptibility of TNF-deficient mice was accompanied by diminished recruitment and activation of T cells and macrophages into the lung, with defective granuloma formation and reduced inducible NO synthase expression. Reduced chemokine production in the lung might explain suboptimal recruitment and activation of T cells and uncontrolled infection. Therefore, despite a massive reduction of the mycobacterial load by chemotherapy, TNF-deficient mice were unable to compensate and mount a protective immune response. In conclusion, endogenous TNF is critical to maintain latent tuberculosis infection, and in its absence no specific immunity is generated.
Toll-Like Receptor 4-Mediated Innate IL-10 Activates Antigen-Specific Regulatory T Cells and Confers Resistance toBordetella pertussisby Inhibiting Inflammatory Pathology
Authors: Higgins Sarah C. , Lavelle Ed C. , McCann Chantelle , Keogh Brian , McNeela Edel , Byrne Patricia , O’Gorman Brian , Jarnicki Andrew , McGuirk Peter , Mills Kingston H. G.
Published: April 2014

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-γ responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.
Dynamics of Macrophage Cell Populations During Murine Pulmonary Tuberculosis
Authors: Gonzalez-Juarrero Mercedes , Shim Tae Sun , Kipnis Andre , Junqueira-Kipnis Ana Paula , Orme Ian M.
Published: April 2014

The influx of macrophages into the lungs is the major component of the granulomatous response to infection with Mycobacterium tuberculosis. In this investigation we used flow cytometric analysis to define macrophage populations entering the airways and lung tissues of infected mice. We demonstrate that by the judicious use of cell surface markers, especially CD11b and CD11c, several cell populations can be distinguished, allowing cell sorting and morphological definition. Primary populations of CD11b−/CD11c+/high were defined as alveolar macrophages, CD11bhigh/CD11c+/high as dendritic cells, and CD11b+/mid/CD11c+/mid as small macrophages or monocytes, and changes in the activation phenotype of these populations were followed over the early course of the infection. In further studies, these cell populations were compared with cells harvested during the chronic stage of the disease. During the chronic stage of infection, Ag-presenting class II molecules and activation markers were poorly expressed on dendritic, small macrophage, and monocyte cell populations, which may have important implications for the breakdown of the lesions during reactivation disease. This analytical approach may facilitate the further characterization of macrophage populations entering into the lung tissues and their relative contributions to host resistance to tuberculosis infection.
Over-Expression of Bcl-2 Provides Protection in Septic Mice by a trans Effect
Authors: Iwata Akiko , Stevenson Vicki Morgan , Minard Annie , Tasch Michael , Tupper Joan , Lagasse Eric , Weissman Irving , Harlan John M. , Winn Robert K.
Published: April 2014

Transgenic mice that over-express B cell leukemia/lymphomas (Bcl)-2 in myeloid cells under control of the human MRP8 promoter (hMRP8-Bcl-2) or in T lymphocytes under the Eμ promoter (Eμ-Bcl-2) were compared with C57BL/6 control mice following cecal ligation and puncture (CLP). There was a significant difference in outcome between the hMRP8-Bcl-2 and control mice with 100% survival in the hMRP8-Bcl-2 mice vs 25% survival in the control mice. In separate experiments there was a significant difference between Eμ-Bcl-2 and control mice with 87.5 and 22.2% survival, respectively. Adoptive transfer of CD11b-positive bone marrow cells from hMRP8-Bcl-2 or C57BL/6 mice to C57BL/6 mice subjected to CLP resulted in 100 and 0% survival, respectively. Adoptive transfer of CD11b-positive cells from either hMRP8-Bcl-2 or C57BL/6 mice to Rag-1−/− mice (no mature T or B cells) subjected to CLP resulted in survival of 87.5 and 12.5%, respectively. The hMRP8-Bcl-2 mice had significantly more neutrophils and fewer bacteria in the peritoneum compared with C57BL/6 mice 24 h after CLP. These experiments show that Bcl-2 over-expression is protective in CLP and that protection is independent of lymphocytes. We propose that over-expression of Bcl-2 in T cells or myeloid cells induce release of a molecule(s) that protects against death following CLP.
IL-10 Prevents Liver Necrosis During Murine Infection withTrichinella spiralis
Authors: Bliss Susan K. , Alcaraz Ana , Appleton Judith A.
Published: April 2014

Infection with Trichinella spiralis rarely leads to significant morbidity. In this study, we show that IL-10 knockout mice infected with this parasite develop extensive areas of coagulative necrosis in the liver, and newborn larvae are required for lesion formation. Histopathological examination revealed that the hepatic inflammatory infiltrate was mixed but dominated by eosinophils. Accordingly, infected IL-10 knockout mice displayed a marked eosinophilia. IL-10 was expressed during infection in mesenteric lymph node populations and liver tissue. Analysis of cytokine profiles revealed a codominant expression of type 1 and 2 mediators that was enhanced in the absence of IL-10. Additionally, CD11c+ MHC class II+ cells were increased in mesenteric lymph nodes of IL-10 knockout mice, suggesting a possible link between IL-10 and dendritic cell trafficking. Nevertheless, there were no significant differences in mortality or parasite burdens between the strains of mice, indicating that IL-10 is necessary to control the host’s inflammatory response but does not impact establishment of the parasite. Expression of IL-10 appears to be an adaptation used by the liver to protect itself from damage caused by migrating newborn larvae.
Successful Induction of CD8 T Cell-Dependent Protection Against Malaria by Sequential Immunization with DNA and Recombinant Poxvirus of Neonatal Mice Born to Immune Mothers
Authors: Sedegah Martha , Belmonte Maria , Epstein Judith E. , Siegrist Claire-Anne , Weiss Walter R. , Jones Trevor R. , Lu Minh , Carucci Daniel J. , Hoffman Stephen L.
Published: April 2014

In some parts of Africa, 50% of deaths attributed to malaria occur in infants less than 8 mo. Thus, immunization against malaria may have to begin in the neonatal period, when neonates have maternally acquired Abs against malaria parasite proteins. Many malaria vaccines in development rely upon CD8 cells as immune effectors. Some studies indicate that neonates do not mount optimal CD8 cell responses. We report that BALB/c mice first immunized as neonates (7 days) with a Plasmodium yoelii circumsporozoite protein (PyCSP) DNA vaccine mixed with a plasmid expressing murine GM-CSF (DG) and boosted at 28 days with poxvirus expressing PyCSP were protected (93%) as well as mice immunized entirely as adults (70%). Protection was dependent on CD8 cells, and mice had excellent anti-PyCSP IFN-γ and cytotoxic T lymphocyte responses. Mice born of mothers previously exposed to P. yoelii parasites or immunized with the vaccine were protected and had excellent T cell responses. These data support assessment of this immunization strategy in neonates/young infants in areas in which malaria exacts its greatest toll.
Subcellular Localization of Toll-Like Receptor 3 in Human Dendritic Cells
Authors: Matsumoto Misako , Funami Kenji , Tanabe Masako , Oshiumi Hiroyuki , Shingai Masashi , Seto Yoshiyuki , Yamamoto Akitsugu , Seya Tsukasa
Published: April 2014

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-κB and IFN-β promoter. Type I IFNs (IFN-α/β) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-β secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-α and -β in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c+ blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.
HLA Class II-Restricted CD4+ T Cell Responses Directed Against Influenza Viral Antigens Postinfluenza Vaccination
Authors: Danke Nancy A. , Kwok William W.
Published: April 2014

The memory T cell response is polyclonal, with the magnitude and specificity of the response controlled in part by the burst size of T cells expanded from effector/memory precursors. Sensitive assays using HLA class II multimers were used to detect low-frequency Ag-specific T cells directed against influenza viral Ags in subjects immunized with the influenza vaccine. Direct ex vivo tetramer staining of PBMC from five individuals identified frequencies of hemagglutinin (HA) 306–318 tetramer binding CD4+ T cells in the peripheral blood ranging from 1 in 600 to 1 in 30,000 CD4+ T cells. These frequencies were validated by counting CFSElow, tetramer-positive T cells after in vitro expansion. Low frequency of T cells directed to other influenza epitopes, including DRA1*0101/DRB1*0401-restricted matrix protein 60–73, DRA1*0101/DRB1*0101-restricted matrix protein 18–29, DRA1*0101/DRB1*0701-restricted HA 232–244 and DRA1*0101/DRB1*0101-restricted nucleoprotein 206–217 were also determined. T cells which occurred at a frequency as low as 1 in 350,000 could be ascertained by in vitro expansion of precursors. Peripheral HA306–318-responsive T cells expanded 2- to 5-fold following influenza vaccination. Examination of phenotypic markers of the HA306–318-responsive T cells in the peripheral blood indicated that the majority were CD45RA−, CD27+, CD25−, CD28+, and CD62L−, while T cell clones derived from this population were CD45RA−, CD27−, CD25+, CD28+, and CD62L−.
Vγ4+ γδ T Cells Regulate Airway Hyperreactivity to Methacholine in Ovalbumin-Sensitized and Challenged Mice
Authors: Hahn Youn-Soo , Taube Christian , Jin Niyun , Takeda Katsuyuki , Park Jung-Won , Wands J. M. , Aydintug M. Kemal , Roark Christina L. , Lahn Michael , O’Brien Rebecca L. , Gelfand Erwin W. , Born Willi K.
Published: April 2014

The Vγ4+ pulmonary subset of γδ T cells regulates innate airway responsiveness in the absence of αβ T cells. We now have examined the same subset in a model of allergic airway disease, OVA-sensitized and challenged mice that exhibit Th2 responses, pulmonary inflammation, and airway hyperreactivity (AHR). In sensitized mice, Vγ4+ cells preferentially increased in number following airway challenge. Depletion of Vγ4+ cells before the challenge substantially increased AHR in these mice, but had no effect on airway responsiveness in normal, nonchallenged mice. Depletion of Vγ1+ cells had no effect on AHR, and depletion of all TCR-δ+ cells was no more effective than depletion of Vγ4+ cells alone. Adoptively transferred pulmonary lymphocytes containing Vγ4+ cells inhibited AHR, but lost this ability when Vγ4+ cells were depleted, indicating that these cells actively suppress AHR. Eosinophilic infiltration of the lung and airways, or goblet cell hyperplasia, was not affected by depletion of Vγ4+ cells, although cytokine-producing αβ T cells in the lung increased. These findings establish Vγ4+ γδ T cells as negative regulators of AHR and show that their regulatory effect bypasses much of the allergic inflammatory response coincident with AHR.
Identification of Tissue Transglutaminase as a Novel Molecule Involved In Human CD8+ T Cell Transendothelial Migration
Authors: Mohan Karkada , Pinto Devanand , Issekutz Thomas B.
Published: April 2014

During inflammation, T lymphocytes migrate out of the blood across the vascular endothelium in a multistep process. The receptors mediating T cell adhesion to endothelium are well characterized; however, the molecules involved in T cell transendothelial migration (TEM) subsequent to lymphocyte adhesion to the endothelium are less clear. To identify receptors mediating TEM, mAbs were produced against human blood T cells adhering to IFN-γ-activated HUVEC in mice and tested for inhibition of lymphocyte TEM across cytokine-activated HUVEC. Most of the mAbs were against β1 and β2 integrins, but one mAb, 6B9, significantly inhibited T cell TEM across IFN-γ, TNF-α, and IFN-γ plus TNF-α-stimulated HUVEC, and did not react with an integrin. 6B9 mAb did not inhibit T cell adhesion to HUVEC, suggesting that 6B9 blocked a novel pathway in T cell TEM. The 6B9 Ag was 80 kDa on SDS-PAGE, and was expressed by both blood leukocytes and HUVEC. Immunoaffinity purification and mass spectrometry identified this Ag as tissue transglutaminase (tTG), a molecule not known to mediate T cell TEM. Treatment of HUVEC with 6B9 was more effective than treatment of T cells. 6B9 blockade selectively inhibited CD4−, but not CD4+, T cell TEM, suggesting a role for tTG in recruitment of CD8+ T lymphocytes. Thus, 6B9 is a new blocking mAb to human tTG, which demonstrates that tTG may have a novel role in mediating CD8+ T cell migration across cytokine-activated endothelium and infiltration of tissues during inflammation.
Experimental Evidence for Lack of Homodimerization of the G Protein-Coupled Human N-Formyl Peptide Receptor
Authors: Gripentrog Jeannie M. , Kantele Katrin P. , Jesaitis Algirdas J. , Miettinen Heini M.
Published: April 2014

A large number of G protein-coupled receptors have been shown to form homodimers based on a number of different techniques such as receptor coimmunoprecipitation, cross-linking, and fluorescence resonance energy transfer. In addition, functional assays of cells coexpressing a mutant receptor with a wild-type receptor have shown receptor phenotypes that can best be explained through dimerization. We asked whether the human neutrophil N-formyl peptide receptor (FPR) forms dimers in Chinese hamster ovary cells by coexpressing wild-type FPR with one of two mutants: D71A, which is uncoupled from G protein, and N297A, which has a defect in receptor phosphorylation and endocytosis. Experiments measuring chemotaxis, ligand-induced release of intracellular calcium, and p42/44 mitogen-activated protein kinase activation did not show an inhibitory effect of the coexpressed FPR D71A mutant. Coexpressed wild-type receptor was efficiently internalized, but failed to correct the endocytosis defects of the D71A and the N297A mutants. To explore the possibility that the mutations themselves prevented dimerization, we examined the coimmunoprecipitation of differentially epitope-tagged FPR. Immunoprecipitation of hemagglutinin-tagged FPR failed to coimmunoprecipitate coexpressed c-myc-tagged FPR and vice versa. Together, these data suggest that, unlike many other G protein-coupled receptors, FPR does not form homodimers.
IL-6 Induces NF-κB Activation in the Intestinal Epithelia
Authors: Wang Lixin , Walia Baljit , Evans John , Gewirtz Andrew T. , Merlin Didier , Sitaraman Shanthi V.
Published: April 2014

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-κB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-κB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-κB activation and NF-κB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-κB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-κB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-κB by IL-6 in intestinal epithelia and the down-regulation of NF-κB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.
Soluble IL-6 Receptor Governs IL-6 Activity in Experimental Arthritis: Blockade of Arthritis Severity by Soluble Glycoprotein 130
Authors: Nowell Mari A. , Richards Peter J. , Horiuchi Sankichi , Yamamoto Naoki , Rose-John Stefan , Topley Nicholas , Williams Anwen S. , Jones Simon A.
Published: April 2014

Studies in IL-6-deficient (IL-6−/−) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6−/− mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis.
B Cell Deficiency Confers Protection from Renal Ischemia Reperfusion Injury
Authors: Burne-Taney Melissa J. , Ascon Dolores B. , Daniels Frank , Racusen Lorraine , Baldwin William , Rabb Hamid
Published: April 2014

Recent data have demonstrated a role for CD4+ cells in the pathogenesis of renal ischemia reperfusion injury (IRI). Identifying engagement of adaptive immune cells in IRI suggests that the other major cell of the adaptive immune response, B cells, may also mediate renal IRI. An established model of renal IRI was used: 30 min of renal pedicle clamping was followed by reperfusion in B cell-deficient (μMT) and wild-type mice. Renal function was significantly improved in μMT mice compared with wild-type mice at 24, 48, and 72 h postischemia. μMT mice also had significantly reduced tubular injury. Both groups of mice had similar renal phagocyte infiltration postischemia assessed by myeloperoxidase levels and similar levels of CD4+ T cell infiltration postischemia. Peritubular complement C3d staining was also similar in both groups. To identify the contribution of cellular vs soluble mechanism of action, serum transfer into μMT mice partially restored ischemic phenotype, but B cell transfers did not. These data are the first demonstration of a pathogenic role for B cells in ischemic acute renal failure, with a serum factor as a potential underlying mechanism of action.
Lipid Raft Localization of Cell Surface E-Selectin Is Required for Ligation-Induced Activation of Phospholipase Cγ
Authors: Kiely Jeanne-Marie , Hu Yenya , García-Cardeña Guillermo , Gimbrone Michael A.
Published: April 2014

E-selectin, an endothelial cell surface adhesion receptor for leukocytes, also acts as a signaling receptor. Upon multivalent ligation, E-selectin transduces outside-in signals into the endothelium leading to changes in intracellular Ca2+ concentration and activation of the mitogen-activated protein kinase signaling pathway. In addition, following leukocyte engagement, E-selectin associates via its cytoplasmic domain with components of the actin cytoskeleton and undergoes alterations in phosphorylation state that result in changes in gene expression. In this study, we show that E-selectin is localized in cholesterol-rich lipid rafts at the cell surface, and that upon ligation E-selectin clusters and redistributes in the plasma membrane colocalizing with a fraction of caveolin-1-containing rafts. In addition, we demonstrate that leukocyte adhesion via E-selectin results in association with and activation of phospholipase Cγ (PLCγ). Moreover, we show that disruption of lipid rafts with the cholesterol-depleting drug methyl-β-cyclodextrin disrupts the raft localization of E-selectin as well as the ligation-induced association of E-selectin with PLCγ, and subsequent tyrosine phosphorylation of PLCγ. In contrast, cholesterol depletion has no effect on E-selectin-dependent mitogen-activated protein kinase activation. Thus, these findings demonstrate that the presence of E-selectin in lipid rafts is necessary for its association with, and activation of, PLCγ, and suggest that this subcellular localization of E-selectin is related to its signaling function(s) during leukocyte-endothelial interactions.
Hypoxia Inactivates Inducible Nitric Oxide Synthase in Mouse Macrophages by Disrupting Its Interaction with α-Actinin 4
Authors: Daniliuc Sharon , Bitterman Haim , Rahat Michal A. , Kinarty Amalia , Rosenzweig Doron , Nitza Lahat
Published: April 2014

Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-γ induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 ± 14 μM, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 ± 1.7 μM, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and α-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.
Crucial Role of IL-4/STAT6 in T Cell-Mediated Hepatitis: Up-Regulating Eotaxins and IL-5 and Recruiting Leukocytes
Authors: Jaruga Barbara , Hong Feng , Sun Rui , Radaeva Svetlana , Gao Bin
Published: April 2014

T cell-mediated immune responses are implicated in the pathogenesis of a variety of liver disorders; however, the underlying mechanism remains obscure. Con A injection is a widely accepted mouse model to study T cell-mediated liver injury, in which STAT6 is rapidly activated. Disruption of the IL-4 and STAT6 gene by way of genetic knockout abolishes Con A-mediated liver injury without affecting IFN-γ/STAT1, IL-6/STAT3, or TNF-α/NF-κB signaling or affecting NKT cell activation. Infiltration of neutrophils and eosinophils in Con A-induced hepatitis is markedly suppressed in IL-4 −/− and STAT6−/− mice compared with wild-type mice. IL-4 treatment induces expression of eotaxins in hepatocytes and sinusoidal endothelial cells isolated from wild-type mice but not from STAT6−/− mice. Con A injection induces expression of eotaxins in the liver and elevates serum levels of IL-5 and eotaxins; such induction is markedly attenuated in IL-4−/− and STAT6−/− mice. Finally, eotaxin blockade attenuates Con A-induced liver injury and leukocyte infiltration. Taken together, these findings suggest that IL-4/STAT6 plays a critical role in Con A-induced hepatitis, via enhancing expression of eotaxins in hepatocytes and sinusoidal endothelial cells, and induces IL-5 expression, thereby facilitating recruitment of eosinophils and neutrophils into the liver and resulting in hepatitis.
Glucocorticoid Receptor Nitration Leads to Enhanced Anti-Inflammatory Effects of Novel Steroid Ligands
Authors: Paul-Clark Mark J. , Roviezzo Fiorentina , Flower Roderick J. , Cirino Giuseppe , Soldato Piero Del , Adcock Ian M. , Perretti Mauro
Published: April 2014

It has recently emerged that posttranslational modification of proteins via nitration of tyrosine residues can alter their function. In this study, we describe that specific nitration of the glucocorticoid receptor (GR) by NCX-1015, a novel NO-donating prednisolone derivative (prednisolone 21-[4′-(nitrooxymethyl)benzoate), results in an enhancement of GR-mediated events. Incubation of PBMC and U937 cells with 1–10 μM NCX-1015 caused faster activation of GR as assessed by augmented 1) binding to [3H]dexamethasone, 2) dissociation from heat shock protein 90, and 3) nuclear translocation. PBMCs treated with NCX-1015 contained GR that had undergone tyrosine nitration. The chemistry facilitating the increase in steroid binding capacity observed with NCX-1015 is specific, because changing the position of the NO-donating group or ubiquitous nitration by addition of an NO donor was unable to mimic this event. In vivo treatment with NCX-1015 provoked GR nitration and faster heat shock protein 90 dissociation as assessed in peritoneal cells. Accordingly, NCX-1015, but not prednisolone or other derivatives, produced a rapid inhibition of the early neutrophil recruitment and mediator generation in a model of peritonitis. In conclusion, we report here for the first time that posttranslational modification of GR by this novel nitrosteroid is associated with its enhanced anti-inflammatory activity.