bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Regulation of Fcgr2b Gene by Polymorphic Promoter Region and Its Contribution to Humoral Immune Responses
Authors: Xiu Yan , Nakamura Kazuhiro , Abe Masaaki , Li Na , Wen Xiang Shu , Jiang Yi , Zhang Danqing , Tsurui Hiromichi , Matsuoka Shuji , Hamano Yoshitomo , Fujii Hiroyuki , Ono Masao , Takai Toshiyuki , Shimokawa Toshibumi , Ra Chisei , Shirai Toshikazu , Hirose Sachiko
Published: April 2014

FcγRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcγRIIB1 function may possibly be related to aberrant B cell activation. We earlier found deletion polymorphism in the Fcgr2b promoter region among mouse strains in which systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune diabetes-prone nonobese diabetic, but not NZW, BALB/c, and C57BL/6 mice have two identical deletion sites, consisting of 13 and 3 nucleotides. In this study, we established congenic C57BL/6 mice for NZB-type Fcgr2b allele and found that NZB-type allele down-regulates FcγRIIB1 expression levels in germinal center B cells and up-regulates IgG Ab responses. We did luciferase reporter assays to determine whether NZB-type deletion polymorphism affects transcriptional regulation of Fcgr2b gene. Although NZW- and BALB/c-derived segments from position −302 to +585 of Fcgr2b upstream region produced significant levels of luciferase activities, only a limited activity was detected in the NZB-derived sequence. EMSA and Southwestern analysis revealed that defect in transcription activity in the NZB-derived segment is likely due to absence of transactivation by AP-4, which binds to the polymorphic 13 nucleotide deletion site. Our data imply that because of the deficient AP-4 binding, the NZB-type Fcgr2b allele polymorphism results in up-regulation of IgG Ab responses through down-regulation of FcγRIIB1 expression levels in germinal center B cells, and that such polymorphism may possibly form the basis of autoimmune susceptibility in combination with other background contributing genes.
Distinct Roles for c-Myb and Core Binding Factor/Polyoma Enhancer-Binding Protein 2 in the Assembly and Function of a Multiprotein Complex on the TCR δ Enhancer In Vivo
Authors: Hernández-Munain Cristina , Krangel Michael S.
Published: April 2014

Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCRαδ locus, the TCR α enhancer and the TCR δ enhancer (Eδ), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR α and δ genes, respectively. Eδ function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin-integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, we used in vivo footprinting to study the requirements for protein binding to Eδ within wild-type and mutant versions of a human TCR δ minilocus in stably transfected Jurkat cells. Our data indicate that CBF/PEBP2 plays primarily a structural role as it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, our data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an Eδ enhanceosome in the context of chromatin in vivo.
Uniquely Conformed Peptide-Containing β2-Microglobulin- Free Heavy Chains of HLA-B2705 on the Cell Surface
Authors: Malik Pratap , Klimovitsky Polina , Deng Lih-Wen , Boyson Jonathan E. , Strominger Jack L.
Published: April 2014

The human class I MHC molecules are known to generally exist on the cell surface either as peptide-containing complexes of H chain (α-chain) and β2-microglobulin (β2m) or as β2m-free H chains incapable of binding peptides. In this study, a uniquely conformed peptide-containing β2m-free HLA-B2705 H chain has been isolated using the recently described highly efficient perfusion-affinity chromatography system for purification of class I MHC protein molecules. This form recognized by the mAb MARB4 is very closely associated with the remainder of the peptide containing HLA-B2705/β2m complex reactive with mAb ME1 and is present to ∼1–10% of mAb ME1 reactive forms on the cell surface. Also, HLA-B2705 purified using the mAb ME1 affinity column includes this unique mAb MARB4-reactive, unusually stable peptide-containing β2m-free form. A peptide nonamer GRWRGWYTY was isolated and identified from this β2m-free HLA-B2705 H chain and was used to assemble the mAb MARB4 reactive form efficiently on the surface of cells expressing HLA-B2705. The discovery of this form opens new avenues for further investigation of the role of HLA-B27 in spondyloarthropathies.
Modification of a Tumor-Derived Peptide at an HLA-A2 Anchor Residue Can Alter the Conformation of the MHC-Peptide Complex: Probing with TCR-Like Recombinant Antibodies
Authors: Denkberg Galit , Klechevsky Eynav , Reiter Yoram
Published: April 2014

A common assumption about peptide binding to the class I MHC complex is that each residue in the peptide binds independently. Based on this assumption, modifications in class I MHC anchor positions were used to improve the binding properties of low-affinity peptides (termed altered peptide ligands), especially in the case when tumor-associated peptides are used for immunotherapy. Using a new molecular tool in the form of recombinant Abs endowed with Ag-specific MHC-restricted specificity of T cells, we show that changes in the identity of anchor residues may have significant effects, such as altering the conformation of the peptide-MHC complex, and as a consequence, may affect the TCR-contacting residues. We herein demonstrate that the binding of TCR-like recombinant Abs, specific for the melanoma differentiation Ag gp100 T cell epitope G9-209, is entirely dependent on the identity of a single peptide anchor residue at position 2. An example is shown in which TCR-like Abs can recognize the specific complex only when a modified peptide, G9-209-2 M, with improved affinity to HLA-A2 was used, but not with the unmodified natural peptide. Importantly, these results demonstrate, using a novel molecular tool, that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Moreover, these results may have important implications in designing modifications in peptides for cancer immunotherapy, because most such peptides studied are of low affinity.
Role of STAT6 and Mast Cells in IL-4- and IL-13-Induced Alterations in Murine Intestinal Epithelial Cell Function
Authors: Madden Kathleen B. , Whitman Lucia , Sullivan Carolyn , Gause William C. , Urban Joseph F. , Katona Ildy M. , Finkelman Fred D. , Shea-Donohue Terez
Published: April 2014

Gastrointestinal nematode infections generally invoke a type 2 cytokine response, characterized by the production of IL-4, IL-5, IL-9, and IL-13. Among these cytokines, IL-4 and IL-13 exhibit a functional overlap that can be explained by the sharing of a common receptor or receptor component (IL-4Rα). Binding of IL-4 by either the type 1 or 2 IL-4R, or of IL-13 by the type 2 IL-4R, initiates Jak-dependent tyrosine phosphorylation of the IL-4Rα-chain and the transcription factor, STAT6. In the present study, we investigated: 1) whether IL-13 has effects on intestinal epithelial cells similar to those observed with IL-4, and 2) whether the effects of IL-4 and IL-13 depend on STAT6 signaling and/or mast cells. BALB/c, STAT6−/−, and mast cell-deficient W/Wv mice or their +/+ littermates were treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or with IL-13 for seven days. Segments of jejunum were mounted in Ussing chambers to measure mucosal permeability; chloride secretion in response to PGE2, histamine, 5-hydroxytryptamine, or acetylcholine; and Na+-linked glucose absorption. IL-4C and IL-13 increased mucosal permeability, decreased glucose absorption, and decreased chloride secretion in response to 5-hydroxytryptamine. These effects were dependent on STAT6 signaling. Responses to PGE2 and histamine, which were dependent on mast cells and STAT6, were enhanced by IL-4C, but not by IL-13. The effects of IL-4 and IL-13 on intestinal epithelial cell function may play a critical role in host protection against gastrointestinal nematodes.
An Increase in the Susceptibility of Burned Patients to Infectious Complications Due to Impaired Production of Macrophage Inflammatory Protein 1α
Authors: Kobayashi Makiko , Takahashi Hitoshi , Sanford Arthur P. , Herndon David N. , Pollard Richard B. , Suzuki Fujio
Published: April 2014

Sepsis is a major mortality concern with burned patients, who have an increased susceptibility to infectious complications. PBMC from 41 of 45 severely burned patients (91%) failed to produce macrophage inflammatory protein 1α (MIP-1α) in cultures, while 2355–6900 pg/ml MIP-1α were produced by healthy donor PBMC, stimulation with anti-human CD3 mAb. Healthy chimeras (SCID mice inoculated with healthy donor PBMC) treated with anti-human MIP-1α mAb and patient chimeras (SCID mice reconstituted with burned patient PBMC) were susceptible (0% survival) to infectious complications induced by well-controlled cecal ligation and puncture. In contrast, patient chimeras treated with human recombinant MIP-1α and healthy chimeras were resistant (∼77–81% survival). Similarly, after anti-mouse CD3 mAb stimulation, splenic mononuclear cells from burned mice (6 h to 3 days after thermal injury) did not produce significant amounts of MIP-1α in their culture fluids. Normal mice treated with anti-murine MIP-1α mAb and burned mice were susceptible to cecal ligation- and puncture-induced infectious complications, while burned mice treated with murine recombinant MIP-1α and normal mice were resistant. Burned patients seemed to be more susceptible to infectious complications when the production of MIP-1α was impaired.
Thrombin Induces Mast Cell Adhesion to Fibronectin: Evidence for Involvement of Protease-Activated Receptor-1
Authors: Vliagoftis Harissios
Published: April 2014

Thrombin activates mast cells to release inflammatory mediators through a mechanism involving protease-activated receptor-1 (PAR-1). We hypothesized that PAR-1 activation would induce mast cell adhesion to fibronectin (FN). Fluorescent adhesion assay was performed in 96-well plates coated with FN (20 μg/ml). Murine bone marrow cultured mast cells (BMCMC) were used after 3–5 wk of culture (>98% mast cells by flow cytometry for c-Kit expression). Thrombin induced β-hexosaminidase, IL-6, and matrix metalloproteinase-9 release from BMCMC. Thrombin and the PAR-1-activating peptide AparafluoroFRCyclohexylACitY-NH2 (cit) induced BMCMC adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive peptide FSLLRY-NH2 had no effect. Thrombin and cit induced also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was inhibited by anti-α5 integrin Ab (51.1 ± 6.7%; n = 5). The combination of anti-α5 and anti-α4 Abs induced higher inhibition (65.7 ± 7.1%; n = 5). Unlike what is known for FcεRI-mediated adhesion, PAR-1-mediated adhesion to FN did not increase mediator release. We then explored the signaling pathways involved in PAR-1-mediated mast cell adhesion. Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 ± 7.3% (n = 4), indicating that Gi proteins are involved. Wortmannin and calphostin almost completely inhibited PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and protein kinase C are involved. Adhesion was partially inhibited by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5 ± 3.3%; n = 3) and the p38 inhibitor SB203580 (25.1 ± 10.4%; n = 3). The two inhibitors had additive effects. Therefore, thrombin mediates mast cell adhesion through the activation of Gi proteins, phosphoinositol 3-kinase, protein kinase C, and mitogen-activated protein kinase pathways.
P-Selectin, and Not E-Selectin, Negatively Regulates Murine Megakaryocytopoiesis
Authors: Banu Naheed , Avraham Shalom , Avraham Hava Karsenty
Published: April 2014

To assess the role of P-selectin and E-selectin in megakaryocytopoiesis, in vitro assays were performed in animal models deficient in both adhesion receptors. There was a significantly greater number of IL-3-responsive megakaryocyte progenitors CFU (CFU-MK) and an increase in immature megakaryoblasts in response to IL-6 in the P-selectin-null mice compared with the wild-type controls. Furthermore, P-selectin-null mice showed a greater number of CFU-MK colonies derived from CD34+ cells in response to IL-3 or IL-3 plus stem cell factor. A significant shift in baseline ploidy with a reduction in 8N cells and an increase in 32N cells was also observed in the P-selectin-null mice. Secretion of the inhibitory growth factor TGF-β1 and not TGF-β2 was significantly lower in the supernatants of cultures containing bone marrow cells from P-selectin-deficient mice as compared with those from the wild-type control bone marrow cells. No differences in the responsiveness of murine CFU-MK, immature megakaryocytes, or 5-fluorouracil-selected stem cells to cytokines were observed in E-selectin-null mice as compared with the control mice. These studies indicate that the absence of P-selectin, and not E-selectin, resulted in an altered adhesion environment with subsequent expansion of megakaryocyte progenitors and immature megakaryoblasts, enhanced secretion of TGF-β1, and apparent increased responsiveness to inflammatory cytokines.
Cutting Edge: Localization of the Host Recognition Functions of Complement Factor H at the Carboxyl-Terminal: Implications for Hemolytic Uremic Syndrome
Authors: Pangburn Michael K.
Published: April 2014

Incidents of hemolytic uremic syndrome (HUS) include a subset of patients that exhibit mutations in C factor H. These mutations cluster in the C-terminal domains of factor H where previous reports have identified polyanion and C3b-binding sites. In this study, we show that recombinant human factor H with deletions at the C-terminal end of the protein loses the ability to control the spontaneous activation of the alternative C pathway on host-like surfaces. For the pathology of HUS, the findings imply that mutations that disrupt the normal functions of the C-terminal domains prevent host polyanion recognition. The resulting uncontrolled activation of complement on susceptible host tissues appears to be the initiating event behind the acute renal failure of familial HUS patients.
Effect of IL-12 on TNF-α-Mediated Osteoclast Formation in Bone Marrow Cells: Apoptosis Mediated by Fas/Fas Ligand Interaction
Authors: Kitaura Hideki , Nagata Noriko , Fujimura Yuji , Hotokezaka Hitoshi , Yoshida Noriaki , Nakayama Koji
Published: April 2014

Recently, it has been found that differentiation into osteoclasts is induced by TNF-α. In this study, we investigated the effect of IL-12 on TNF-α-mediated osteoclastogenesis. When mouse bone marrow cells were cultured with TNF-α, osteoclast-like cells were formed. When they were cultured with both TNF-α and IL-12, the number of adherent cells in the bone marrow cells decreased in an IL-12 dose-dependent manner. A combination of IL-12 and TNF-α was necessary to induce death of the adherent cells in this culture system. Apoptotic alterations, which were indicated by morphological changes such as cellular atrophy, nuclear and cellular fragmentation, and biochemical changes such as DNA fragmentation, were observed in the adherent cells. Apoptosis of the adherent cells was markedly inhibited by anti-Fas ligand (FasL) Ab. RT-PCR and FACS analyses revealed that TNF-α up-regulated Fas transcription to lead to Fas expression on the surfaces of the adherent cells, whereas IL-12 could not induce Fas on the cells. In contrast, IL-12 induced FasL transcription to lead to FasL expression on the surfaces of nonadherent bone marrow cells, whereas TNF-α could not induce FasL on the cells. These results implied that apoptosis of the adherent cells in bone marrow cells might be caused by interaction between TNF-α-induced Fas on the adherent cells and IL-12-induced FasL on the nonadherent cells.
Monocyte Chemoattractant Protein-1 Selectively Inhibits the Acquisition of CD40 Ligand-Dependent IL-12-Producing Capacity of Monocyte-Derived Dendritic Cells and Modulates Th1 Immune Response
Authors: Omata Nemuko , Yasutomi Motoko , Yamada Akiko , Iwasaki Hiromichi , Mayumi Mitsufumi , Ohshima Yusei
Published: April 2014

Accumulating evidence indicates that monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, also displays immunoregulatory functions and may be involved in Th subset differentiation. In this study, we examined the effects of MCP-1 on the cytokine-driven differentiation of monocytes into dendritic cells (DCs), the most potent APCs for naive T cells. We found that DCs generated in the presence of MCP-1 displayed a markedly reduced production of IL-12 in response to CD40 ligand but not in response to Staphylococcus aureus stimulation in the presence or absence of IFN-γ. The production of IL-10, a potent endogenous IL-12 inhibitor, was not affected by MCP-1. Whereas the inhibitory activity of MCP-1 on IL-12 production by monocytes was sensitive to pertussis toxin, its effects on DC differentiation were pertussis toxin resistant. MCP-1 did not affect the surface phenotype and T cell-stimulating activity of DCs, but most interestingly, naive T cells stimulated with MCP-1-primed DCs produced much less IFN-γ but the same levels of IL-13. Taken together, our results indicated that MCP-1 modulates the differentiation of monocytes into DCs and may thereby inhibit Th1 cell development.
Defective T Cell Priming Associated with Aging Can Be Rescued by Signaling Through 4-1BB (CD137)
Authors: Bansal-Pakala Pratima , Croft Michael
Published: April 2014

Aging is associated with an increased susceptibility to infectious agents and correlates with a decreased ability to mount an immune response. It has been postulated that the major defect is related to a reduced capacity of an aged T cell to proliferate and to survive after encounter with Ag. This is similar to the phenotype associated with T cell tolerance in young adults. In this study, we determined whether targeting 4-1BB (CD137), a member of the TNFR family implicated in providing expansion and survival signals to T cells, can rescue defective priming in aged and tolerized animals. Agonist Abs to 4-1BB injected in vivo were capable of preventing CD4 T cell tolerance to soluble peptide in young mice. Moreover, anti-4-1BB rescued defective priming of aged TCR transgenic CD4 T cells responding to peptide Ag in a young host, and as importantly, anti-4-1BB completely restored T cell priming to protein Ag in nontransgenic aged mice. These studies demonstrate that 4-1BB, and potentially other costimulatory members of the TNFR family, are targets for therapies aimed at augmenting weak T cell responses in elderly immunocompromised individuals.
Chemoattractant-Stimulated Rac Activation in Wild-Type and Rac2-Deficient Murine Neutrophils: Preferential Activation of Rac2 and Rac2 Gene Dosage Effect on Neutrophil Functions
Authors: Li Shijun , Yamauchi Akira , Marchal Christophe C. , Molitoris Jason K. , Quilliam Lawrence A. , Dinauer Mary C.
Published: April 2014

The hemopoietic-specific Rho family GTPase Rac2 shares 92% amino acid identity with ubiquitously expressed Rac1. Neutrophils from rac2−/− mice have multiple defects, including chemoattractant-stimulated NADPH oxidase activity and chemotaxis, which may result from an overall reduction in cellular Rac or mechanisms that discriminate Rac1 and Rac2. We show that murine neutrophils have similar amounts of Rac1 and Rac2, unlike human neutrophils, which express predominantly Rac2. An affinity precipitation assay for Rac-GTP showed that although FMLP-induced activation of both isoforms in wild-type neutrophils, ≈4-fold more Rac2-GTP was detected than Rac1-GTP. Wild-type and Rac2-deficient neutrophils have similar levels of total Rac1. FMLP-induced Rac1-GTP in rac2−/− neutrophils was ≈3-fold greater than in wild-type cells, which have similar levels of total Rac1, yet FMLP-stimulated F-actin, chemotaxis, and superoxide production are markedly impaired in rac2−/− neutrophils. Heterozygous rac2+/− neutrophils, which had intermediate levels of total and FMLP-induced activated Rac2, exhibited intermediate functional responses to FMLP, suggesting that Rac2 was rate limiting for these functions. Thus, phenotypic defects in FMLP-stimulated Rac2-deficient neutrophils appear to reflect distinct activation and signaling profiles of Rac 1 and Rac2, rather than a reduction in the total cellular level of Rac.
δ Tryptase Is Expressed in Multiple Human Tissues, and a Recombinant Form Has Proteolytic Activity
Authors: Wang Hong-Wei , McNeil H. Patrick , Husain Ahsan , Liu Ke , Tedla Nicodemus , Thomas Paul S. , Raftery Mark , King Garry C. , Cai Zhao Yan , Hunt John E.
Published: April 2014

Tryptases are neutral serine proteases selectively expressed in mast cells and have been implicated in the development of a number of inflammatory diseases including asthma. It has recently been established that the number of genes encoding human mast cell tryptases is much larger than originally believed, but it is not clear how many of these genes are expressed. A recent report suggested that the transcript for at least one of these genes, originally named mMCP-7-like tryptase, is not expressed. To further address this question, we screened tissue-specific RNA samples by RT-PCR, using primers designed to match the putative exonic sequence of this gene. We successfully generated and cloned the correctly sized RT-PCR product from mRNA isolated from the human mast cell-I cell line. Two distinct clones were identified whose nucleotide sequence matched the published sequence of the mMCP-7-like I and mMCP-7-like II genes. Transcripts were detected in a wide variety of human tissues including lung, heart, stomach, spleen, skin, and colon. A polyclonal antipeptide Ab that specifically recognizes the translated product of this transcript was used to demonstrate its expression in mast cells that reside in the colon, lung, and inflamed synovium. A recombinant form of this protein expressed in bacterial cells was able to cleave a synthetic trypsin-sensitive substrate, d-Ile-Phe-Lys pNA. These results suggest that the range of functional tryptases is larger than previously recognized. For simplicity, we suggest that the gene, transcripts, and corresponding protein product be named δ tryptase.
Interaction with Factor Associated with Neutral Sphingomyelinase Activation, a WD Motif-Containing Protein, Identifies Receptor for Activated C-Kinase 1 as a Novel Component of the Signaling Pathways of the p55 TNF Receptor
Authors: Tcherkasowa Anna Ewgenjewna , Adam-Klages Sabine , Kruse Marie-Luise , Wiegmann Katja , Mathieu Sabine , Kolanus Waldemar , Krönke Martin , Adam Dieter
Published: April 2014

Factor associated with neutral sphingomyelinase activation (FAN) represents a p55 TNFR (TNF-R55)-associated protein essential for the activation of neutral sphingomyelinase. By means of the yeast interaction trap system, we have identified the scaffolding protein receptor for activated C-kinase (RACK)1 as an interaction partner of FAN. Mapping studies in yeast revealed that RACK1 is recruited to the C-terminal WD-repeat region of FAN and binds to FAN through a domain located within WD repeats V to VII of RACK1. Our data indicate that binding of both proteins is not mediated by linear motifs but requires folding into a secondary structure, such as the multibladed propeller characteristic of WD-repeat proteins. The interaction of FAN and RACK1 was verified in vitro by glutathione S-transferase-based coprecipitation assays as well as in eukaryotic cells by coimmunoprecipitation experiments. Colocalization studies in transfected cells suggest that TNF-R55 forms a complex with FAN and that this complex recruits RACK1 to the plasma membrane. Furthermore, activation of N-SMase by TNF was strongly enhanced when RACK1, FAN, and a noncytotoxic TNF-R55 mutant were expressed concurrently, suggesting RACK1 as a modulator of N-SMase activation. Together, these findings implicate RACK1 as a novel component of the signaling pathways of TNF-R55.
CD11b/CD18-Dependent Interactions of Neutrophils with Intestinal Epithelium Are Mediated by Fucosylated Proteoglycans
Authors: Zen Ke , Liu Yuan , Cairo Dana , Parkos Charles A.
Published: April 2014

CD11b/CD18-mediated adhesive interactions play a key role in regulating polymorphonuclear leukocytes (PMN)) migration across intestinal epithelium. However, the identity of epithelial ligands for migrating PMN remains obscure. In this study we investigated the role of carbohydrates in mediating adhesive interactions between T84 intestinal epithelial cells and CD11b/CD18 purified from PMN. Fucoidin, heparin/heparin sulfate, N-acetyl-d-glucosamine, mannose-6-phosphate, and laminarin were found to inhibit adhesion of T84 cells to CD11b/CD18. The most potent inhibitory effects were observed with fucoidin (50% inhibition at 1–5 × 10−8 M). Binding assays demonstrated that fucoidin directly bound to CD11b/CD18 in a divalent cation- and sulfation-dependent fashion that was blocked by anti-CD11b mAbs. Experiments employing CD11b/CD18 as a probe to blot T84 cell fucosylated proteins purified via fucose-specific lectin column revealed several candidate CD11b/CD18 binding proteins with molecular masses of 95, 50, 30, 25, and 20 kDa. Fucosidase treatment of T84 cells resulted in significantly reduced cell adhesion to CD11b/CD18, while no inhibition was observed after neuraminidase treatment. Finally, significant inhibition of T84 cell adhesion to CD11b/CD18 was observed after blocking cell proteoglycan synthesis with p-nitrophenyl-β-d-xylopyranoside. These findings implicate epithelial cell surface proteoglycans decorated with sulfated fucose moieties as ligands for CD11b/CD18 during PMN migration across mucosal surfaces.
Effect of Microbial Heat Shock Proteins on Airway Inflammation and Hyperresponsiveness
Authors: Rha Yeong-Ho , Taube Christian , Haczku Angela , Joetham Anthony , Takeda Katsuyuki , Duez Catherine , Siegel Marvin , Aydintug M. Kemal , Born Willi K. , Dakhama Azzeddine , Gelfand Erwin W.
Published: April 2014

Microbial heat shock proteins (hsp) have been associated with the generation and induction of Th1-type immune responses. We tested the effects of treatment with five different microbial hsp (Mycobacterium leprae, Streptococcus pneumoniae, Helicobacter pylori, bacillus Calmette-Guérin, and Mycobacterium tuberculosis) in a murine model of allergic airway inflammation and airway hyperresponsiveness (AHR). Mice were sensitized to OVA by i.p. injection and then challenged by OVA inhalation. Hsp were administered to each group by i.p. injection before sensitization and challenge. Sensitized and challenged mice developed increased serum levels of OVA-specific IgE with significant airway eosinophilia and heightened responsiveness to methacholine when compared with nonsensitized animals. Administration of M. leprae hsp prevented both development of AHR as well as bronchoalveolar lavage fluid eosinophilia in a dose-dependent manner. Treatment with M. leprae hsp also resulted in suppression of IL-4 and IL-5 production in bronchoalveolar lavage fluid, while IL-10 and IFN-γ production were increased. Furthermore, M. leprae hsp treatment significantly suppressed OVA-specific IgE production and goblet cell hyperplasia/mucin hyperproduction. In contrast, treatment with the other hsp failed to prevent changes in airway responsiveness, lung eosinophilia, or cytokine production. Depletion of γ/δ T lymphocytes before sensitization and challenge abolished the effect of M. leprae hsp treatment on AHR. These results indicate selective and distinctive properties among the hsp, and that M. leprae hsp may have a potential therapeutic role in the treatment of allergic airway inflammation and altered airway function.
Vaccination of Macaques with Long-Standing SIVmac251 Infection Lowers the Viral Set Point After Cessation of Antiretroviral Therapy
Authors: Tryniszewska Elzbieta , Nacsa Janos , Lewis Mark G. , Silvera Peter , Montefiori David , Venzon David , Hel Zdeněk , Parks Robyn Washington , Moniuszko Marcin , Tartaglia Jim , Smith Kendall A. , Franchini Genoveffa
Published: April 2014

A cohort of rhesus macaques with long-standing SIVmac251 infection (≥5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4+ and CD8+ T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4+ and CD8+ T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.
Cutting Edge: Monarch-1: A Pyrin/Nucleotide-Binding Domain/Leucine-Rich Repeat Protein That Controls Classical and Nonclassical MHC Class I Genes
Authors: Williams Kristi L. , Taxman Debra J. , Linhoff Michael W. , Reed William , Ting Jenny P.-Y.
Published: April 2014

Proteins containing a limited number of N-terminal motifs followed by nucleotide-binding domain and leucine-rich repeat regions are emerging as important regulators for immunity. A search of human genome scaffold databases has identified a large family of known and unknown genes, which we have recently called the CATERPILLER (caspase recruitment domain, transcription enhancer, r(purine)-binding, pyrin, lots of leucine repeats) gene family. This work describes the characterization of a new member, Monarch-1. Monarch-1 has four different splice forms due to the differential splicing of leucine-rich repeat motifs. It is expressed in cells of myeloid-monocytic origin. Affymetrix microarrays and small interfering RNA were used to elucidate the downstream effects of Monarch-1 expression in cells including those of myeloid-monocytic origin. These analyses show that Monarch-1 enhances nonclassical and classical MHC class I expression at the level of the promoter, RNA, and protein expression.
Mature But Not Immature Fas Ligand (CD95L)-Transduced Human Monocyte-Derived Dendritic Cells Are Protected from Fas-Mediated Apoptosis and Can Be Used as Killer APC
Authors: Hoves Sabine , Krause Stefan W. , Halbritter Dagmar , Zhang Huang-Ge , Mountz John D. , Schölmerich Jürgen , Fleck Martin
Published: April 2014

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas+ Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas+ target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas+ target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.
Stimulation of Toll-Like Receptor 4 by Lipopolysaccharide During Cellular Invasion by Live Salmonella typhimurium Is a Critical But Not Exclusive Event Leading to Macrophage Responses
Authors: Royle Matthew C. J. , Tötemeyer Sabine , Alldridge Louise C. , Maskell Duncan J. , Bryant Clare E.
Published: April 2014

Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of pathogen-associated molecular patterns. LPS is one of the prime candidates, but its precise role in the process, especially when presented as a component of live infecting bacteria, is unclear. We thus investigated this question using the lipid A antagonist E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4−/− (TLR-4−/−) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-α. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-κB is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS or live salmonellae. Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531. Both purified LPS and bacterial invasion caused translocation of NF-κB, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial invasion was only inhibited at later time points. In conclusion, we show for the first time that production of NO and TNF-α is critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae.
Absence of Allograft ICAM-1 Attenuates Alloantigen-Specific T Cell Priming, But Not Primed T Cell Trafficking into the Graft, to Mediate Acute Rejection
Authors: Zhang Qi-Wei , Kish Danielle D. , Fairchild Robert L.
Published: April 2014

The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1−/− donors were tested. Wild-type C57BL/6 (H-2b) heart allografts were rejected by A/J (H-2a) recipients on days 7–9, whereas B6.ICAM-1−/− allografts survived until days 18–23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4+ and CD8+ T cells in the ICAM-1−/− allografts were 20–30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-γ from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1−/− mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1−/− allografts on days 5–6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.
Normal Somatic Hypermutation of Ig Genes in the Absence of 8-Hydroxyguanine-DNA Glycosylase
Authors: Winter David B. , Phung Quy H. , Zeng Xianmin , Seeberg Erling , Barnes Deborah E. , Lindahl Tomas , Gearhart Patricia J.
Published: April 2014

The hypermutation cascade in Ig V genes can be initiated by deamination of cytosine in DNA to uracil by activation-induced cytosine deaminase and its removal by uracil-DNA glycosylase. To determine whether damage to guanine also contributes to hypermutation, we examined the glycosylase that removes oxidized guanine from DNA, 8-hydroxyguanine-DNA glycosylase (OGG1). OGG1 has been reported to be overexpressed in human B cells from germinal centers, where mutation occurs, and could be involved in initiating Ab diversity by removing modified guanines. In this study, mice deficient in Ogg1 were immunized, and V genes from the H and κ L chain loci were sequenced. Both the frequency of mutation and the spectra of nucleotide substitutions were similar in ogg1−/− and Ogg1+/+ clones. More importantly, there was no significant increase in G:C to T:A transversions in the ogg1−/− clones, which would be expected if 8-hydroxyguanine remained in the DNA. Furthermore, Ogg1 was not up-regulated in murine B cells from germinal centers. These findings show that hypermutation is unaffected in the absence of Ogg1 activity and indicate that 8-hydroxyguanine lesions most likely do not cause V gene mutations.
Tryptase Precursors Are Preferentially and Spontaneously Released, Whereas Mature Tryptase Is Retained by HMC-1 Cells, Mono-Mac-6 Cells, and Human Skin-Derived Mast Cells
Authors: Schwartz Lawrence B. , Min Hae-Ki , Ren Shunlin , Xia Han-Zhang , Hu Jiang , Zhao Wei , Moxley George , Fukuoka Yoshihiro
Published: April 2014

Tryptase (α and β) levels in serum are used to assess mast cell involvement in human disease. Using cultured cells, the current study examines the hypothesis that protryptase(s) are spontaneously secreted by mast cells at rest, whereas mature tryptase(s) are stored in secretory granules until their release by activated cells. HMC-1 cells have only β-tryptase genes and the corresponding mRNA. Mono-Mac-6 cells have both α- and β-tryptase genes but preferentially express α-tryptase. Mono-Mac-6 cells spontaneously secrete most of their tryptase, which consists of α-protryptase, whereas mature tryptase is retained inside these cells. HMC-1 cells also spontaneously secrete most of their tryptase, identified as β-protryptase, and retain mature tryptase. Skin-derived mast cells retain most of their tryptase, which is mature, and spontaneously secrete protryptase(s). Total tryptase levels in plasma are detectable but no different in healthy subjects with and without the gene for α-tryptase, consistent with pro forms of both α- and β-tryptase being spontaneously secreted. Thus, protryptase(s) are spontaneously secreted by resting mast cells, whereas mature tryptase is retained by mast cells until they are activated to degranulate.
Cutting Edge: Influence of the TCR Vβ Domain on the Avidity of CD1d:α-Galactosylceramide Binding by Invariant Vα14 NKT Cells
Authors: Schümann Jens , Voyle Roger B. , Wei Bing-Yuan , MacDonald H. Robson
Published: April 2014

CD1d tetramers loaded with α-galactosylceramide (α-GalCer) bind selectively to mouse invariant Vα14 (Vα14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Vα14-Jα18 chain in murine NKT cells, the associated β-chain (typically expressing Vβ8.2 or Vβ7) appears not to influence tetramer binding. In this study, we describe novel α-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-β chain on the avidity of CD1d:α-GalCer binding. A subset of Vα14i NKT cells clearly discriminated α-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vβ domain of the TCR-β chain, with Vβ8.2 conferring higher avidity binding than Vβ7.
Tapasin−/− and TAP1−/− Macrophages Are Deficient in Vacuolar Alternate Class I MHC (MHC-I) Processing due to Decreased MHC-I Stability at Phagolysosomal pH
Authors: Chefalo Peter J. , Grandea Andres G. , Van Kaer Luc , Harding Clifford V.
Published: April 2014

Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin−/− cells and experiments with tapasin−/− and TAP1−/− macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin−/− cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin−/− cells might be peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both tapasin−/− and TAP1−/− macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin−/− and TAP1−/− macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin−/− or TAP1−/− cells at 26°C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments.
Paxillin Binding to the α4 Integrin Subunit Stimulates LFA-1 (Integrin αLβ2)-Dependent T Cell Migration by Augmenting the Activation of Focal Adhesion Kinase/Proline-Rich Tyrosine Kinase-2
Authors: Rose David M. , Liu Shouchun , Woodside Darren G. , Han Jaewon , Schlaepfer David D. , Ginsberg Mark H.
Published: April 2014

Engagement of very late Ag-4 (integrin α4β1) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin αLβ2). This form of integrin trans-regulation in T cells requires the binding of paxillin to the α4 integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an α4 mutation (α4(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an α4-binding fragment of paxillin that blocks the α4-paxillin interaction, selectively blocked VCAM-1 stimulation of αLβ2-dependent cell migration. The α4-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the α4 tail resulted in much less α4β1-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked α4β1 stimulation of αLβ2-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin αLβ2 by α4β1.
Deleterious Effect of HIV-1 Plasma Viremia on B Cell Costimulatory Function
Authors: Malaspina Angela , Moir Susan , Kottilil Shyamasundaran , Hallahan Claire W. , Ehler Linda A. , Liu Shuying , Planta Marie A. , Chun Tae-Wook , Fauci Anthony S.
Published: April 2014

HIV infection leads to numerous immunologic defects, including impaired B cell function. An effective humoral response requires bidirectional interactions between B cells and CD4+ T cells, critical of which are interactions between CD80/CD86 expressed on activated B cells and CD28 expressed on responder CD4+ T cells. In the present study, we examined the effect of active HIV replication on B cell costimulatory function. Induction of CD80/CD86 on B cells following B cell receptor and CD40 triggering and responsiveness of CD4+ T cells to activated B cells were investigated in a system where B cells of HIV-infected patients were compared concurrently to B cells of HIV-negative donors. In contrast to HIV-aviremic patients, B cells of HIV-viremic patients were ineffective at stimulating CD4+ T cells, as measured by the induction of activation markers and proliferation. The importance of interactions of CD80/CD86 and CD28 in activating CD4+ T cells was clear; the ablation of a normal response following the addition of neutralizing anti-CD86/CD80 Abs mirrored the response of CD4+ T cells to B cells of HIV-viremic patients, while the addition of exogenous CD28 ligands partially restored the poor CD4+ T cell response to the B cells of HIV-viremic patients. Ineffective B cell costimulatory function in HIV-viremic patients was associated with low induction of CD80/CD86 expression on B cells. Our findings further delineate the scope of defects associated with cognate B cell-CD4+ T cell interactions in HIV infection and suggest that therapeutic interventions designed to enhance CD28-dependent costimulatory pathways may help restore immune functions.