bluuebunny/crossref_metadata_2025 huggingface dataset:
Different NK Cell Surface Phenotypes Defined by the DX9 Antibody Are Due toKIR3DL1Gene Polymorphism
Authors: Gardiner Clair M. , Guethlein Lisbeth A. , Shilling Heather G. , Pando Marcelo , Carr William H. , Rajalingam Raja , Vilches Carlos , Parham Peter
Published: April 2014

KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic genes, with KIR3DS1 segregating like an allele of KIR3DL1. A KIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Consequently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded protein: 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail.
Potent Cytolytic Response by a CD8+ CTL Clone to Multiple Peptides from the Same Protein in Association with an Allogeneic Class I MHC Molecule
Authors: Kageyama Shigeki , Tsomides Theodore J. , Fukusen Naomi , Papayannopoulos Ioannis A. , Eisen Herman N. , Sykulev Yuri
Published: April 2014

CTL clone 2C recognizes the allogeneic class I MHC molecule Ld in association with peptides derived from α-ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface Ld than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with Ld as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.
Expression of a V Region-Less B Cell Receptor Confers a Tolerance-Like Phenotype on Transgenic B Cells
Authors: Corcos Daniel , Grandien Alf , Vazquez Aimé , Dunda Olga , Lorès Patrick , Bucchini Danielle
Published: April 2014

Neoplastic B cells from H chain disease patients express a truncated B cell receptor (BCR), comprising a membrane Ig that lacks part of its extracellular domain. It has been speculated that deletion of the Ag binding domain would confer a constitutive activity on the BCR, as it has been shown for oncogenic growth factor receptors. A V region-less BCR has constitutive activity, because in transgenic mice it causes inhibition of endogenous H chain gene rearrangements and relieves the requirement for surrogate L chain in pre-B cell development. However, it has been speculated that normal Ag receptors also display constitutive activity. Here we show that transgenic B cells expressing a membrane H chain disease protein on their surface are phenotypically and functionally similar to B cells developing in the presence of their cognate Ag and that cells with normal levels of mutant BCR are eliminated in spleen via a bcl-2 sensitive pathway while progressing toward the mature stage. In contrast, cells with lower levels of mutant receptors develop as mature B cells. These findings support the view that the truncated BCR has a constitutive activity that mimics ligand binding, in analogy to what has been shown for oncogenic growth factor receptors.
Involvement of CD1 in Peripheral Deletion of T Lymphocytes Is Independent of NK T Cells
Authors: Dao Tao , Exley Mark , Mehal Wajahat Z. , Tahir Syed Muhammad Ali , Snapper Scott , Taniguchi Masaru , Balk Steven P. , Crispe I. Nicholas
Published: April 2014

During peripheral T cell deletion, lymphocytes accumulate in nonlymphoid organs including the liver, a tissue that expresses the nonclassical, MHC-like molecule, CD1. Injection of anti-CD3 Ab results in T cell activation, which in normal mice is followed by peripheral T cell deletion. However, in CD1-deficient mice, the deletion of the activated T cells from the lymph nodes was impaired. This defect in peripheral T cell deletion was accompanied by attenuated accumulation of CD8+ T cells in the liver. In tetra-parental bone marrow chimeras, expression of CD1 on the T cells themselves was not required for T cell deletion, suggesting a role for CD1 on other cells with which the T cells interact. We tested whether this role was dependent on the Ag receptor-invariant, CD1-reactive subset of NK T cells using two other mutant mouse lines that lack most NK T cells, due to deletion of the genes encoding either β2-microglobulin or the TCR element Jα281. However, these mice had no abnormality of peripheral T cell deletion. These findings indicate a novel role for CD1 in T cell deletion, and show that CD1 functions in this process through mechanisms that does not involve the major, TCR-invariant set of NK T cells.
The Identification and Characterization of a Ligand for Bovine CD5
Authors: Haas Karen M. , Estes D. Mark
Published: April 2014

CD5, a type I glycoprotein expressed by T cells and a subset of B cells, is thought to play a significant role in modulating Ag receptor signaling. Previously, our laboratory has shown that bovine B cells are induced to express this key regulatory molecule upon Ag receptor cross-linking. To date, a ligand has not been described for bovine CD5. Given the importance ligand binding presumably plays in the functioning of CD5 on this B cell subset and on T cells, we sought to characterize the ligand for this protein using a bovine CD5-human IgG1 (CD5Ig) fusion protein produced by both mammalian and yeast cells. As determined by CD5Ig binding, expression of this ligand is negative to low on freshly isolated lymphocytes, with low-density expression being limited to activated B cells. Activation with LPS, PMA, and calcium ionophore, or ligation of CD40 alone or in combination with anti-IgM, resulted in B cell-specific expression of this ligand. Interestingly, activation through B cell Ag receptor cross-linking alone, although able to induce CD5 expression, did not result in expression of CD5 ligand (CD5L). In addition, we demonstrate a functional role for CD5L as a costimulatory molecule that augments CD40L-stimulated B cell proliferation. Finally, immunoprecipitation with CD5Ig suggests that the ligand characterized in this study has a molecular mass of ∼200 kDa. The data reported herein, as well as future studies aimed at further characterizing this newly identified bovine CD5L, will undoubtedly aid in understanding the role that the CD5-CD5L interaction plays in immune responses.
Activation of the EBV/C3d Receptor (CR2, CD21) on Human B Lymphocyte Surface Triggers Tyrosine Phosphorylation of the 95-kDa Nucleolin and Its Interaction with Phosphatidylinositol 3 Kinase
Authors: Barel Monique , Le Romancer Muriel , Frade Raymond
Published: April 2014

We previously demonstrated that CR2 activation on human B lymphocyte surface triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3′ (PI 3) kinase. Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1. To identify this tyrosine phosphorylated p95 component, we first purified it by affinity chromatography on anti-phosphotyrosine mAb covalently linked to Sepharose 4B, followed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa tyrosine phosphorylated band was submitted to amino acid analysis by mass spectrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, localized between aa 411–420 and 611–624. Anti-nucleolin mAb was used to confirm the antigenic properties of this p95 component. Functional studies demonstrated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domains of the p85 subunit of PI 3 kinase and of 3BP2 and Grb2, but not with Src homology 2 domains of Fyn and Gap. These properties of nucleolin were identical with those of the p95 previously described and induced by CR2 activation. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activation. These data support that tyrosine phosphorylation of nucleolin and its interaction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.
Uncoupling p70s6 Kinase Activation and Proliferation: Rapamycin-Resistant Proliferation of Human CD8+ T Lymphocytes
Authors: Slavik Jacqueline M. , Lim Dong-Gyun , Burakoff Steven J. , Hafler David A.
Published: April 2014

Rapamycin is a fungal macrolide that inhibits the proliferation of T cells. Studies in both animals and humans have found that rapamycin significantly reduces graft rejection. However, though CD8+ T cells are involved in graft infiltration and rejection, little is known regarding the effects of rapamycin on CD8+ human T cell responses. In this study, we examined the mechanism of rapamycin-induced inhibition of Ag-driven activation of CD8+ T cells. Surprisingly, a heterogeneous proliferative response in the presence of rapamycin was observed among different Ag-specific CD8+ T cell clones; this was also observed in CD8+ peripheral blood T cells activated with TCR cross-linking ex vivo. Inhibition of T cell proliferation by rapamycin was controlled by both the strength of signal delivered through the Ag receptor as well as the specific costimulatory signals received by the T cell. Rapamycin-resistant proliferation occurred despite inhibition of p70s6 kinase activity. Moreover, rapamycin-resistant proliferation of the CD8+ T cell clones was blocked by anti-IL-2 Abs, suggesting that while some of the parallel pathways triggered by IL-2R signaling are sensitive to the effects of rapamycin, others account for the Ag-driven rapamycin resistance. These data provide a new framework for examining the specific mechanism of action of rapamycin in human disease.
In Vivo CD40-CD154 (CD40 Ligand) Interaction Induces Integrated HIV Expression by APC in an HIV-1-Transgenic Mouse Model
Authors: Chougnet Claire , Freitag Corona , Schito Marco , Thomas Elaine K. , Sher Alan , Shearer Gene M.
Published: April 2014

Because of their relative resistance to viral cytopathic effects, APC can provide an alternative reservoir for latently integrated HIV. We used an HIV-transgenic mouse model in which APC serve as the major source of inducible HIV expression to study mechanisms by which integrated virus can be activated in these cells. When admixed with transgenic APC, activated T lymphocytes provided a major contact-dependent stimulus for viral protein expression in vitro. Using blocking anti-CD154 mAb as well as CD154-deficient T cells, the HIV response induced by activated T lymphocytes was demonstrated to require CD40-CD154 interaction. The role of this pathway in the induction of HIV expression from APC in vivo was further studied in an experimental model involving infection of the HIV-transgenic mice with Plasmodium chabaudi parasites. Enhanced viral production by dendritic cells and macrophages in infected mice was associated with up-regulated CD40 expression. More importantly, in vivo treatment with blocking anti-CD154 mAb markedly reduced viral expression in P. chabaudi-infected animals. Together, these findings indicate that immune activation of integrated HIV can be driven by the costimulatory interaction of activated T cells with APC. Because chronic T cell activation driven by coinfections as well as HIV-1 itself is a characteristic of HIV disease, this pathway may be important in sustaining viral expression from APC reservoirs.
The T Cell-Dependent B Cell Immune Response and Germinal Center Reaction Are Intact in A-myb-Deficient Mice
Authors: Vora Kalpit A. , Lentz Vicky M. , Monsell William , Rao Sambasiva P. , Mettus Richard , Toscani Antonio , Reddy E. Premkumar , Manser Tim
Published: April 2014

Expression of the protooncogene A-myb is restricted to the developing CNS, adult testes, breasts in late pregnancy, and germinal centers of secondary B cell follicles. The functional relevance of A-myb expression at three of these sites has been demonstrated previously via the generation and analysis of A-myb-deficient mice, which display behavioral abnormalities, male sterility, and perturbed breast development during pregnancy. In contrast, here we show that the germinal center response driven by T cell-dependent Ag immunization and the associated processes of Ab V gene somatic hypermutation, affinity maturation, and heavy chain class switching are overtly normal in A-myb-deficient mice. Nonetheless, these mice display mild splenic white pulp hypoplasia and blunted primary serum Ab responses, suggesting that although A-myb is not directly involved in the regulation of the memory B cell response, it may play a role in enhancing peripheral B cell survival or proliferative capacity.
The Globular Heads of C1q Specifically Recognize Surface Blebs of Apoptotic Vascular Endothelial Cells
Authors: Navratil Jeannine S. , Watkins Simon C. , Wisnieski Jeffrey J. , Ahearn Joseph M.
Published: April 2014

Complement protein C1q is required to maintain immune tolerance. The molecular mechanism responsible for this link has not been determined. We have previously demonstrated that C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes, suggesting that it may participate in clearance of self Ags generated during programmed cell death. Here, we demonstrate that C1q also binds directly to apoptotic blebs of vascular endothelial cells and PBMC. These apoptotic cells are recognized by the globular heads of C1q, which bind specifically to the surface blebs, and deposition increases as the blebs mature on the cell surface. These observations suggest that C1q may participate in the clearance of apoptotic cells from the circulation and from the walls of the vascular lumen. The interaction of surface blebs with the globular heads of C1q suggests that surface blebs may be capable of directly activating the classical pathway of complement under certain circumstances, generating C4- and C3-derived ligands for receptors such as CR1, CR2, CR3, and CR4. Appropriate recognition of apoptotic cells by C1q and targeted clearance of the molecular contents of surface blebs to complement receptors may be critical for the maintenance of immune tolerance.
Notch Signaling Suppresses IgH Gene Expression in Chicken B Cells: Implication in Spatially Restricted Expression of Serrate2/Notch1 in the Bursa of Fabricius
Authors: Morimura Toshifumi , Miyatani Seiji , Kitamura Daisuke , Goitsuka Ryo
Published: April 2014

The bursa of Fabricius is a central organ for chicken B cell development and provides an essential microenvironment for expansion of the B cell pool and for generation of a diversified B cell repertoire. We report here that genes encoding the Notch family of transmembrane proteins, key regulators of cell fate determination in development, are differentially expressed in the bursa of Fabricius: Notch1 is expressed in medullary B cells located close to the basement membrane-associated epithelium (BMAE). In contrast, a Notch ligand, Serrate2, is expressed exclusively in the BMAE, which surrounds bursal medulla. A basic helix-loop-helix-type transcription factor, Hairy1, a downstream target of Notch signaling, is expressed in the bursa coordinately with Notch1 and Serrate2 and an immature B cell line, TLT1, which expresses both Notch1 and Serrate2. Furthermore, stable expression of a constitutively active form of chicken Notch1 or Notch2 in a B cell line results in a down-regulation of surface IgM expression, which is accompanied by the reduction of IgH gene transcripts. Transient reporter assay with the human IgH gene intronic enhancer reveals that an active form of Notch1 inhibits the IgH enhancer activity in chicken B cells, suggesting that Notch-mediated signals suppress the IgH gene expression via influencing the IgH intronic enhancer. These findings raise the possibility that the local activation of Notch1 in a subset of B cells by Serrate2 expressed in BMAE may influence the cell fate decision that is involved in B cell differentiation and selection inside the bursa.
The Dendritic Cell-Specific CC-Chemokine DC-CK1 Is Expressed by Germinal Center Dendritic Cells and Attracts CD38-Negative Mantle Zone B Lymphocytes
Authors: Lindhout Ernst , Vissers Joost L. M. , Hartgers Franca C. , Huijbens Richard J. F. , Scharenborg Nicole M. , Figdor Carl G. , Adema Gosse J.
Published: April 2014

DC-CK1 (CCL18) is a dendritic cell (DC)-specific chemokine expressed in both T and B cell areas of secondary lymphoid organs that preferentially attracts CD45RA+ T cells. In this study, we further explored the nature of DC-CK1 expressing cells in germinal centers (GCs) of secondary lymphoid organs using a newly developed anti-DC-CK1 mAb. Immunohistochemical analysis demonstrated a remarkable difference in the number of DC-CK1 expressing cells in adjacent GCs within one tonsil, implicating that the expression of DC-CK1 in GCs depends on the activation and/or progression stage of the GC reaction. Using immunohistology and RNA analysis, we demonstrated that GCDC are the source of DC-CK1 production in the GCs. Considering the recently described function of GCDC in (naive) B cell proliferation, isotype switching and Ab production, we investigated the ability of DC-CK1 to attract B lymphocytes. Here we demonstrate that DC-CK1 is a pertussis toxin-dependent chemoattractant for B lymphocytes with a preference in attracting mantle zone (CD38−) B cells. The findings that GCDC produce DC-CK1 and attract mantle zone B cells support a key role for GCDC in the development of GCs and memory B cell formation.
Immature Multipotent Hemopoietic Progenitors Lacking Long-Term Bone Marrow-Reconstituting Activity in the Aorta-Gonad-Mesonephros Region of Murine Day 10 Fetuses
Authors: Ohmura Koichiro , Kawamoto Hiroshi , Lu Min , Ikawa Tomokatsu , Ozaki Shoichi , Nakao Kazuwa , Katsura Yoshimoto
Published: April 2014

Previous studies indicated that multipotent progenitors exist in early fetuses that do not contain long-term reconstituting (LTR) activity. However, it remained unclear whether these multipotent progenitors are committed to the hemopoietic lineage or are immature mesodermal cells or hemangioblasts. In this study, we have succeeded in enriching the multipotent progenitors that are capable of generating myeloid, T, and B cells in the LFA-1− subpopulation of TER-119−c-kit+CD45+ cells from the aorta-gonad-mesonephros (AGM) region of day 10 fetuses. We found that these day 10 AGM LFA-1− cells do not show the LTR activity, whereas day 11 AGM LFA-1− cells do have such an activity. These results strongly suggest that multipotent progenitors lacking LTR activity emerge as CD45+ hemopoietic progenitor cells in the AGM region on the 10th day of gestation, and such p-Multi mature into hemopoietic stem cells by acquiring LTR activity.
At Least One Class I Gene in Restriction Fragment Pattern-Y (Rfp-Y), the SecondMHCGene Cluster in the Chicken, Is Transcribed, Polymorphic, and Shows Divergent Specialization in Antigen Binding Region
Authors: Afanassieff Marielle , Goto Ronald M. , Ha Jennifer , Sherman Mark A. , Zhong Lingwen , Auffray Charles , Coudert Françoise , Zoorob Rima , Miller Marcia M.
Published: April 2014

MHC genes in the chicken are arranged into two genetically independent clusters located on the same chromosome. These are the classical B system and restriction fragment pattern-Y (Rfp-Y), a second cluster of MHC genes identified recently through DNA hybridization. Because small numbers of MHC class I and class II genes are present in both B and Rfp-Y, the two clusters might be the result of duplication of an entire chromosomal segment. We subcloned, sequenced, and analyzed the expression of two class I loci mapping to Rfp-Y to determine whether Rfp-Y should be considered either as a second, classical MHC or as a region containing specialized MHC-like genes, such as class Ib genes. The Rfp-Y genes are highly similar to each other (93%) and to classical class Ia genes (73% with chicken B class I; 49% with HLA-A). One locus is disrupted and unexpressed. The other, YFV, is widely transcribed and polymorphic. Mature YFV protein associated with β2m arrives on the surface of chicken B (RP9) lymphoma cells expressing YFV as an epitope-tagged transgene. Substitutions in the YFV Ag-binding region (ABR) occur at four of the eight highly conserved residues that are essential for binding of peptide-Ag in the class Ia molecules. Therefore, it is unlikely that Ag is bound in the YFV ABR in the manner typical of class Ia molecules. This ABR specialization indicates that even though YFV is polymorphic and widely transcribed, it is, in fact, a class Ib gene, and Rfp-Y is a region containing MHC genes of specialized function.
Impaired Pulmonary Host Defense in Mice Lacking Expression of the CXC Chemokine Lungkine
Authors: Chen Shu-Cheng , Mehrad Borna , Deng Jane C. , Vassileva Galya , Manfra Denise J. , Cook Donald N. , Wiekowski Maria T. , Zlotnik Albert , Standiford Theodore J. , Lira Sergio A.
Published: April 2014

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these findings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace.
Depletion of CD4+ T Cells Precipitates Immunopathology in Immunodeficient Mice Infected with a Noncytocidal Virus
Authors: Christensen Jan Pravsgaard , Bartholdy Christina , Wodarz Dominik , Thomsen Allan Randrup
Published: April 2014

IFN-γ-deficient (IFN-γ−/−) mice inoculated with intermediate doses of a slowly replicating strain of lymphocytic choriomeningitis virus become chronically infected. In such mice a hypercompensated CTL response is observed that partially controls virus replication. Here we have investigated whether CD4+ Th cells are required to establish and maintain this new equilibrium. The absence of IFN-γ does not impair the generation of IL-2-producing CD4+ cells, and depletion of these cells precipitates severe CD8+ T cell-mediated immunopathology in IFN-γ−/− mice, indicating an important role of CD4+ T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-γ−/− mice following CD4+ cell depletion. Initially the antiviral CTL response did not require CD4+ cells, but with time an impaired reactivity toward especially the glycoprotein 33–41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33–41 and nucleoprotein 396–404) CD8+ T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4+-depleted mice, especially in mice also deficient in IFN-γ. Thus, our findings disclose that T cell help is required to sustain the expanded CTL precursor pool required in IFN-γ−/− mice. This interpretation is supported by mathematical modeling that predicts an increased requirement for help in IFN-γ−/− hosts similar to what is found with fast replicating virus strains in normal hosts. Thus, the functional integrity of CD8+ effector T cells is one important factor influencing the requirement for T cell help during viral infection.
Mycobacterium tuberculosis Phagosomes Exhibit Altered Calmodulin-Dependent Signal Transduction: Contribution to Inhibition of Phagosome-Lysosome Fusion and Intracellular Survival in Human Macrophages
Authors: Malik Zulfiqar A. , Iyer Shankar S. , Kusner David J.
Published: April 2014

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca2+ ([Ca2+]c), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca2+]c that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca2+-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca2+]c resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca2+ ionophore-induced phagosomal maturation and enhanced the bacilli’s intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca2+-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.
Organ-Specific Regulation of the CD8 T Cell Response toListeria monocytogenesInfection
Authors: Pope Constance , Kim Sung-Kwon , Marzo Amanda , Williams Kristina , Jiang Jiu , Shen Hao , Lefrançois Leo
Published: April 2014

The intestinal mucosal CD8 T cell response to infection with Listeria monocytogenes was measured using MHC class I tetramers and was compared with the response in peripheral blood, secondary lymphoid tissue, and liver. To assess the vaccination potential of Listeria and to analyze responses in C57BL/6 mouse strains, a recombinant Listeria expressing OVA (rLM-ova) was generated. The response peaked at 9 days postinfection with a much larger fraction of the intestinal mucosa and liver CD8 T cell pool OVA specific, as compared with the spleen. However, these differences were not linked to bacterial titers in each site. The higher responses in lamina propria and liver resulted in a larger CD8 memory population in these tissues. Furthermore, the level of memory induced was dependent on infectious dose and inversely correlated with the magnitude of the recall response after oral challenge. Recall responses in the tissues were most robust in the lamina propria and liver, and reactivated Ag-specific T cells produced IFN-γ. Infection of CD40- or MHC class II-deficient mice induced poor CD8 T cell responses in the intestinal mucosa, but only partially reduced responses in the spleen and liver. Overall, the results point to novel pathways of tissue-specific regulation of primary and memory antimicrobial CD8 T cell responses.
General Suppression of Macrophage Gene Expression DuringLeishmania donovaniInfection
Authors: Buates Sureemas , Matlashewski Greg
Published: April 2014

Within the mammalian host, Leishmania donovani is an obligatory intracellular protozoan that resides and multiplies exclusively in the phagolysosomes of macrophages. The outcome of this infection is governed by the interaction between Leishmania and macrophage molecules that ultimately effect the expression of genes within both cells. To explore the effect of this intracellular infection on macrophage gene expression, a cDNA expression array analysis was performed to compare gene expression profiles in noninfected and L. donovani-infected macrophages. In this manner, it was possible to examine the effect of infection on the expression of several hundred well-characterized host cell genes in an unbiased manner. Interestingly, ∼40% of the genes whose expression was detected in macrophages were down-regulated during infection with L. donovani. However, several genes were also induced during the infection process, some of which could play a role in recruitment of additional macrophages to the site of infection. Taken together, the general suppression of gene expression in addition to the selective induction of key genes is likely to play an important role in allowing the parasite to survive and proliferate within its host macrophage cell.
Requirement of Mitogen-Activated Protein Kinases and IκB Phosphorylation for Induction of Proinflammatory Cytokines Synthesis by Macrophages Indicates Functional Similarity of Receptors Triggered by Glycosylphosphatidylinositol Anchors from Parasitic Protozoa and Bacterial Lipopolysaccharide
Authors: Ropert Catherine , Almeida Igor C. , Closel Meire , Travassos Luiz R. , Ferguson Michael A. J. , Cohen Philip , Gazzinelli Ricardo T.
Published: April 2014

In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypomastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related transcription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF-α and IL-12 synthesis by IFN-γ-primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of IκB, and the use of SN50 peptide, an inhibitor of NF-κB translocation, resulted in 70% of TNF-α synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and IκB phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates.
Intranasal Immunization with CpG Oligodeoxynucleotides as an Adjuvant Dramatically Increases IgA and Protection Against Herpes Simplex Virus-2 in the Genital Tract
Authors: Gallichan W. Scott , Woolstencroft Robert N. , Guarasci Tina , McCluskie Michael J. , Davis Heather L. , Rosenthal Kenneth L.
Published: April 2014

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.
Fas/Fas Ligand Pathway, Apoptosis, and Clonal Anergy Involved in Systemic Acetylcholine Receptor T Cell Epitope Tolerance
Authors: Deng Caishu , Goluszko Elzbieta , Christadoss Premkumar
Published: April 2014

The cellular mechanisms of high dose systemic acetylcholine receptor (AChR) T cell epitope, α146–162 peptide-induced tolerance in experimental myasthenia gravis were examined. CD4 cells are the prime target for α146–162 peptide-induced tolerance. The expression of CD69, Fas, and B7.2 molecules on AChR-immune lymphocytes was enhanced within 4–12 h after tolerance induction. A high dose of α146–162 peptide in IFA failed to suppress T cell proliferation and/or clinical myasthenia gravis in lpr and gld mice deficient in Fas and Fas ligand, respectively. A high dose of α146–162 peptide in IFA in AChR-immunized mice induced apoptosis of BV6 cells. Further, reconstitution of IL-2 in vitro-recovered α146–162 peptide tolerized T cell proliferation, IFN-γ, and IL-10 production. The findings implicate the possible role of Fas-/Fas ligand-mediated apoptosis and the resulting clonal anergy as the mechanisms of high dose AChR α146–162 peptide-induced tolerance on CD4 cells.
Promotion of Neutrophil Apoptosis by TNF-α
Authors: Salamone Gabriela , Giordano Mirta , Trevani Analía S. , Gamberale Romina , Vermeulen Mónica , Schettinni Jorge , Geffner Jorge R.
Published: April 2014

We examined the ability of TNF-α to modulate human neutrophil apoptosis. Neutrophils cultured with TNF-α alone undergo a low but significant increase in the number of apoptotic cells. More interestingly, when neutrophils were pretreated with TNF-α for 1–2 min at 37°C and then were exposed to a variety of agents such as immobilized IgG, IgG-coated erythrocytes, complement-treated erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP, Escherichia coli, and GM-CSF for 3 h at 37°C, a marked stimulation of apoptosis was observed. Similar results were obtained in neutrophils pretreated with TNF-α for 30 min, 1 h, 3 h, and 18 h. Dose-dependent studies showed that TNF-α enhances neutrophil apoptosis at concentrations ranging from 1 to 100 ng/ml. In contrast to the observations made in neutrophils pretreated with TNF-α, there was no stimulation of apoptosis when TNF-α was added to neutrophils previously activated by conventional agonists. Experiments performed to establish the mechanism through which TNF-α promotes neutrophil apoptosis showed that neither reactive oxygen intermediates nor the Fas/Fas ligand system appear to be involved. Our results suggest that TNF-α plays a critical role in the control of neutrophil survival by virtue of its ability to induce an apoptotic death program which could be triggered by a variety of conventional agonists.
Differential Effects of CD18, CD29, and CD49 Integrin Subunit Inhibition on Neutrophil Migration in Pulmonary Inflammation
Authors: Ridger Victoria C. , Wagner Bart E. , Wallace William A. H. , Hellewell Paul G.
Published: April 2014

Neutrophil migration to lung alveoli is a characteristic of lung diseases and is thought to occur primarily via capillaries rather than postcapillary venules. The role of adhesion molecules CD18 and CD29 on this migration in a mouse model of lung inflammation has been investigated. The number of neutrophils present in bronchoalveolar lavage fluid was determined 4 h after intratracheal instillation of LPS (0.1–1 μg) or murine recombinant KC (CXC chemokine, 0.03–0.3 μg). Both stimuli produced a dose-related increase in neutrophil accumulation. Intravenous anti-mouse CD18 mAb, 2E6 (0.5 mg/mouse), significantly (p < 0.001) attenuated LPS (0.3 μg)- but not KC (0.3 μg)-induced neutrophil accumulation. The anti-mouse CD29 mAb, HMβ1-1 (0.02 mg/mouse), significantly (p < 0.05) inhibited both LPS (0.3 μg)- and KC (0.3 μg)-induced neutrophil migration. A second mAb to CD18 (GAME-46) and both F(ab′)2 and Fab of HMβ1-1 produced similar results to those above, while coadministration of mAbs did not result in greater inhibition. Electron microscopy studies showed that CD29 was involved in the movement of neutrophils from the interstitium into alveoli. The effect of mAbs to CD49 (α integrin) subunits of CD29 was also examined. mAbs to CD49e and CD49f inhibited both responses, while anti-CD49b and CD49d significantly inhibited responses to KC only. These data suggest that CD29 plays a critical role in neutrophil migration in pulmonary inflammation and that CD49b and CD49d mediate CD18-independent neutrophil accumulation.
Effective Treatment of Established Murine Collagen-Induced Arthritis by Systemic Administration of Dendritic Cells Genetically Modified to Express IL-4
Authors: Kim Seon Hee , Kim Sunyoung , Evans Christopher H. , Ghivizzani Steven C. , Oligino Thomas , Robbins Paul D.
Published: April 2014

Dendritic cells (DC) are APCs that are able to stimulate or inhibit immune responses, depending on levels of expression of MHC class I and II costimulatory molecules and cytokines. Our previous studies have suggested that the observed contralateral effect, where injection of a vector carrying certain immunomodulatory genes into one joint resulted in inhibition of arthritis in untreated joints, is mediated by in vivo modification of DC. Therefore, we have examined the ability of genetically modified DC to suppress established murine collagen-induced arthritis (CIA) after i.v. delivery. IL-4 has been shown to partially reduce the severity of CIA after repeated injection of recombinant protein or by injection of an adenoviral vector expressing IL-4. Here we demonstrate that i.v. injection of immature DC, infected with an adenoviral vector expressing IL-4, into mice with established CIA resulted in almost complete suppression of disease, with no recurrence for up to 4 wk posttreatment. Injection i.v. of fluorescently labeled DC demonstrated that the cells rapidly migrated to the liver and spleen after 6 h and to the lymph nodes by 24 h. In culture, spleen cells from DC/IL-4-treated mice produced less IFN-γ after stimulation by collagen than did control groups. In addition, DC/IL-4 administration decreased the level of specific Abs against type II collagen, in particular the IgG2 Th1 isotype 14 days posttreatment. These results demonstrate the ability to treat effectively established murine arthritis by systemic administration of DC expressing IL-4.
Extracellular Signal-Regulated Kinase 1/2-Mediated Phosphorylation of Cytosolic Phospholipase A2 Is Essential for Human Eosinophil Adhesion to Fibronectin
Authors: Sano Hiroyuki , Zhu Xiangdong , Sano Akiko , Boetticher Evan E. , Shioya Takanobu , Jacobs Benjamin , Munoz Nilda M. , Leff Alan R.
Published: April 2014

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A2 (cPLA2) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 μg/ml FN at 30 min, and decreased after 60–90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA2 were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA2 inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA2 phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA2 phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA2 activation is essential for eosinophil adhesion to FN.
Oral Administration of Recombinant Cholera Toxin Subunit B Inhibits IL-12-Mediated Murine Experimental (Trinitrobenzene Sulfonic Acid) Colitis
Authors: Boirivant Monica , Fuss Ivan J. , Ferroni Lucietta , De Pascale Mariateresa , Strober Warren
Published: April 2014

Trinitrobenzene sulfonic acid (TNBS)-induced colitis is an IL-12-driven, Th1 T cell-mediated colitis that resembles human Crohn’s disease. In the present study, we showed initially that the oral administration of recombinant subunit B of cholera toxin (rCT-B) at the time of TNBS-induced colitis by intrarectal TNBS instillation inhibits the development of colitis or, at later time when TNBS-induced colitis is well established, brings about resolution of the colitis. Dose-response studies showed that a majority of mice (68%) treated with rCT-B at a dose of 100 μg (times four daily doses) exhibited complete inhibition of the development of colitis, whereas a minority (30%) treated with rCT-B at a dose of 10 μg (times four daily doses) exhibited complete inhibition; in both cases, however, the remaining mice exhibited some reduction in the severity of inflammation. In further studies, we showed that rCT-B administration is accompanied by prevention/reversal of increased IFN-γ secretion (the hallmark of a Th1 response) without at the same time causing an increase in IL-4 secretion. This decreased IFN-γ secretion was not associated with the up-regulation of the secretion of counterregulatory cytokines (IL-10 or TGF-β), but was associated with a marked inhibition of IL-12 secretion, i.e., the secretion of the cytokine driving the Th1 response. Finally, we showed that rCT-B administration results in increased apoptosis of lamina propria cells, an effect previously shown to be indicative of IL-12 deprivation. From these studies, rCT-B emerges as a powerful inhibitor of Th1 T cell-driven inflammation that can conceivably be applied to the treatment of Crohn’s disease.
Role of Nitric Oxide Synthase Type 2 in Acute Infection with Murine Cytomegalovirus
Authors: Noda Satoshi , Tanaka Kazuo , Sawamura Sada-aki , Sasaki Masafumi , Matsumoto Takako , Mikami Katsunaka , Aiba Yuji , Hasegawa Hideaki , Kawabe Noboru , Koga Yasuhiro
Published: April 2014

Whether or not NO plays a critical role in murine CMV (MCMV) infection has yet to be elucidated. In this study, we examined the role of NO in acute infection with MCMV using NO synthase type 2 (NOS2)-deficient mice. NOS2−/− mice were more susceptible to lethal infection with MCMV than NOS2+/+ mice and generated a much higher peak virus titer in the salivary gland after acute infection. A moderate increase in the MCMV titer was also observed in other organs of NOS2−/− mice such as the spleen, lung, and liver. The immune responses to MCMV infection including NK cell cytotoxicity and CTL response in NOS2−/− mice were comparable with those of NOS2+/+ mice. Moreover, the ability to produce IFN-γ is not impaired in NOS2−/− mice after MCMV infection. The peritoneal macrophages from NOS2−/− mice, however, exhibited a lower antiviral activity than those from NOS2+/+ mice, resulting in an enhanced viral replication in macrophages themselves. Treatment of these cells from NOS2+/+ mice with a selective NOS2 inhibitor decreased the antiviral activity to a level below that obtained with NOS2−/− mice. In addition, the absence of NOS2 and NOS2-mediated antiviral activity of macrophages resulted in not only an enhanced MCMV replication and a high mortality but also a consequent risk of the latency. It was thus concluded that the NOS2-mediated antiviral activity of macrophages via NO plays a protective role against MCMV infection at an early and late stage of the infection.
IL-13-Induced Airway Hyperreactivity During Respiratory Syncytial Virus Infection Is STAT6 Dependent
Authors: Tekkanat Kim K. , Maassab Hunein F. , Cho David S. , Lai Joyce J. , John Alison , Berlin Aaron , Kaplan Mark H. , Lukacs Nicholas W.
Published: April 2014

Airway damage and hyperreactivity induced during respiratory syncytial virus (RSV) infection can have a prolonged effect in infants and young children. These infections can alter the long-term function of the lung and may lead to severe asthma-like responses. In these studies, the role of IL-13 in inducing and maintaining a prolonged airway hyperreactivity response was examined using a mouse model of primary RSV infection. Using this model, there was evidence of significant airway epithelial cell damage and sloughing, along with mucus production. The airway hyperreactivity response was significantly increased by 8 days postinfection, peaked during days 10–12, and began to resolve by day 14. When the local production of Th1- and Th2-associated cytokines was examined, there was a significant increase, primarily in IL-13, as the viral response progressed. Treatment of RSV-infected mice with anti-IL-13 substantially inhibited airway hyperreactivity. Anti-IL-4 treatment had no effect on the RSV-induced responses. Interestingly, when IL-13 was neutralized, an early increase in IL-12 production was observed within the lungs, as was a significantly lower level of viral Ags, suggesting that IL-13 may be regulating an important antiviral pathway. The examination of RSV-induced airway hyperreactivity in STAT6−/− mice demonstrated a significant attenuation of the response, similar to the anti-IL-13 treatment. In addition, STAT6−/− mice had a significant alteration of mucus-producing cells in the airway. Altogether, these studies suggest that a primary factor leading to chronic RSV-induced airway dysfunction may be the inappropriate production of IL-13.