bluuebunny/crossref_metadata_2025 huggingface dataset:
C1q Regulatory Region Polymorphism Down-Regulating Murine C1q Protein Levels with Linkage to Lupus Nephritis
Authors: Miura-Shimura Yuko , Nakamura Kazuhiro , Ohtsuji Mareki , Tomita Hideaki , Jiang Yi , Abe Masaaki , Zhang Danqing , Hamano Yoshitomo , Tsuda Hiroshi , Hashimoto Hiroshi , Nishimura Hiroyuki , Taki Shinsuke , Shirai Toshikazu , Hirose Sachiko
Published: April 2014

Much of the pathology of systemic lupus erythematosus (SLE) is caused by deposition of immune complexes (ICs) into various tissues, including renal glomeruli. Because clearance of ICs depends largely on early complement component C1q, homozygous C1q deficiency is a strong genetic risk factor in SLE, although it is rare in SLE patients overall. In this work we addressed the issue of whether genetic polymorphisms affecting C1q levels may predispose to SLE, using the (NZB × NZW)F1 model. C1q genes are composed of three genes, C1qa, C1qc, and C1qb, arranged in this order, and each gene consists of two exons separated by one intron. Sequence analysis of the C1q gene in New Zealand Black (NZB), New Zealand White (NZW), and BALB/c mice showed no polymorphisms in exons and introns of three genes. However, Southern blot analysis revealed unique insertion polymorphism of a total of ∼3.5 kb in the C1qa upstream region of NZB mice. C1q levels in sera and culture supernatants of LPS-stimulated peritoneal macrophages and C1q messages in spleen cells were all lower in disease-free young NZB and (NZB × NZW)F1 mice than in age-matched non-autoimmune NZW and BALB/c mice. Quantitative trait loci analysis using (NZB × NZW)F1 × NZW backcrosses showed that NZB microsatellites in the vicinity of the C1q allele on chromosome 4 were significantly linked to low serum C1q levels and the development of nephritis. These data imply that not only C1q deficiency but also regulatory region polymorphisms down-regulating C1q levels may confer the risk for lupus nephritis by reducing IC clearance and thus promoting IC deposition in glomeruli.
The Vα14 NKT Cell TCR Exhibits High-Affinity Binding to a Glycolipid/CD1d Complex
Authors: Sidobre Stéphane , Naidenko Olga V. , Sim Bee-Cheng , Gascoigne Nicholas R. J. , Garcia K. Christopher , Kronenberg Mitchell
Published: April 2014

Most CD1d-dependent NKT cells in mice have a canonical Vα14Jα18 TCR rearrangement. However, relatively little is known concerning the molecular basis for their reactivity to glycolipid Ags presented by CD1d. Using glycolipid Ags, soluble forms of a Vα14 NKT cell-derived TCR, and mutant and wild-type CD1d molecules, we probed the TCR/CD1d interaction by surface plasmon resonance, tetramer equilibrium staining, and tetramer staining decay experiments. By these methods, several CD1d α-helical amino acids could be defined that do not greatly alter lipid binding, but that affect the interaction with the TCR. Binding of the Vα14+ TCR to CD1d requires the agonist α-galactosylceramide (α-GalCer), as opposed to the nonantigenic β-galactosylceramide, although both Ags bind to CD1d, indicating that the carbohydrate moiety of the CD1d-bound Ag plays a major role in the TCR interaction. The TCR has a relatively high-affinity binding to the α-GalCer/CD1d complex, with a particularly slow off rate. These unique properties are consistent with the coreceptor-independent action of the Vα14 TCR and may be related to the intense response to α-GalCer by NKT cells in vivo.
Diversification of Ig Heavy Chain Genes in Human Preterm Neonates Prematurely Exposed to Environmental Antigens
Authors: Bauer Karl , Zemlin Michael , Hummel Michael , Pfeiffer Sabine , Karstaedt Julia , Steinhauser Gudrun , Xiao Xin , Versmold Hans , Berek Claudia
Published: April 2014

Preterm neonates are exposed to extrauterine environmental Ags during the time period that corresponds to the last trimester of normal intrauterine development. To study whether this precocious exposure to Ags accelerates the Ig repertoire diversification, we compared IgH chain genes of preterm neonates (gestational age, 25–29 wk) during their first postnatal months with those of term neonates. Preterm infants approaching their expected date of delivery after 8–13 wk of extrauterine life used a similar VH, DH, and JH gene segment repertoire as term neonates born after intrauterine development. Furthermore, the length increase of the NDN region between VH and JH by 0.25 nt per gestational week (r = 0.556, p < 0.0001) was not accelerated. Thus, the generation of the VH region gene repertoire is developmentally controlled and independent of environmental influences. However, exposure to extrauterine Ags induced class switch and somatic mutations in IgH chain genes within 2 wk after premature birth and IgG transcript diversity and mutational frequency increased with the duration of extrauterine life. Three-month-old preterm infants expressed a heterogeneous IgG repertoire at their expected date of delivery with VH region genes carrying significant numbers of somatic mutations with evidence for Ag selection. Term neonates, however, had no such IgG repertoire. We conclude that restrictions in the neonatal Ig VH region gene repertoire persist until term despite exposure to environmental Ags. Yet, many weeks before term the immune system of the preterm neonate can already support germinal center reactions in response to environmental Ags.
The Avian Chb6 Alloantigen Triggers Apoptosis in a Mammalian Cell Line
Authors: Pifer Jeannette , Robison Donald , Funk Phillip E.
Published: April 2014

Many developing B lymphocytes are deleted by apoptosis. However, the mechanism signaling their demise remains poorly understood. Like mammals, chicken B cells are selected during their development; >95% of the cells in the bursa of Fabricius die without entering the secondary immune system. The molecule chB6 (Bu-1) has been used as a marker to identify B cells in the chicken. ChB6 is a type I transmembrane glycoprotein whose function is enigmatic. We have provided evidence that chB6 can induce a rapid form of cell death exhibiting characteristics of apoptosis. Here we further examine cell death induced by chB6 in a transfected mouse cell line. ChB6 is shown to cause apoptosis in this cell line as detected by a TUNEL assay for DNA fragmentation. This apoptosis is subject to regulation by signals from growth factor or by Bcl-xL. Furthermore, we show that Ab binding to chB6 leads to cleavage of caspase 8, caspase 3, and poly(ADP ribose) polymerase. Overall, these data support the hypothesis that chB6 is a novel death receptor on avian B cells.
Isotype Can Affect the Fine Specificity of an Antibody for a Polysaccharide Antigen
Authors: McLean Gary R. , Torres Marcela , Elguezabal Natalia , Nakouzi Antonio , Casadevall Arturo
Published: April 2014

Ab specificity is determined by V region sequence. The murine Mab 18B7 (IgG1) binds to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan and produces annular immunofluorescence (IF) on yeast cells. The heavy and light V regions of 18B7 were expressed with the human C regions μ, γ1, γ2, γ3, γ4, and α1, and the specificity and binding properties of these mouse-human chimeric (ch) Abs was determined. The chIgG1, chIgG2, chIgG4, and the chIgA produced annular IF, whereas the IgM and IgG3 produced punctate IF, despite identical V region sequences. Competition experiments with murine Abs that competed with mAb 18B7 and binding assays to peptide mimetics of glucuronoxylomannan provided additional evidence for altered specificity in some of the ch Abs. Expression of 18B7 heavy V region with murine μ C region produced IgM with a punctate IF, indicating that a change in fine specificity also accompanied the change from murine IgG1 to IgM. Our results show that Ab fine specificity can be a function of isotype. This phenomenon may be most apparent for Abs that bind to Ag with repeating epitopes, such as polysaccharides, where the quarternary structure of the Ag-Ab complex may be influenced by such constraints as Fab-Fab angles, Fc-Fc interactions, Ab size, and solvent accessibility to exposed surfaces. Alterations in Ab fine specificity following isotype change could have important implications for current concepts on the generation of secondary Ab responses to certain Ags and for the isotype preference observed in Abs to polysaccharides.
The Chemokine ESkine/CCL27 Displays Novel Modes of Intracrine and Paracrine Function
Authors: Gortz Andreas , Nibbs Robert J. B. , McLean Pauline , Jarmin David , Lambie Wendy , Baird Janet W. , Graham Gerard J.
Published: April 2014

We have previously shown that the β-chemokine ESkine/CCL27 is differentially spliced to produce two alternative forms. One is a secreted chemokine (ESkine), whereas the other (PESKY) lacks a signal peptide and is translocated to the nucleus. The role of this nuclear-targeted chemokine has not so far been defined, and it was the purpose of this study to examine this chemokine variant in more depth. To identify the region of PESKY involved in the nuclear translocation we tagged fragments with enhanced green fluorescent protein and expressed them in Chinese hamster ovary cells. We show PESKY nuclear translocation to be dependent on C-terminal residues that are shared with the signal peptide-bearing variant ESkine. Indeed we further demonstrate that ESkine can also use these C-terminal residues to enter the nucleus of cells following receptor (CCR10)-mediated internalization. To examine biological roles for PESKY we have overexpressed it in 3T3 cells. Such overexpression results in marked cytoskeletal rearrangements that are coincident with a radical reorganization of the cellular actin cytoskeleton. Microarray analyses and Ab neutralization studies indicate that these changes are mediated in part by insulin-like growth factor-1. Furthermore, monolayer wounding assays indicate that PESKY expression correlates with markedly increased migratory capacity. Thus, it is our contention that nuclear PESKY and ESkine both enter the nucleus by either intracrine or paracrine mechanisms and may facilitate cellular migration by inducing actin cytoskeletal relaxation. Therefore, nuclear ESkine/PESKY represents a novel paradigm for chemokine function.
Structural Studies of Allelic Diversity of the MHC Class I Homolog MIC-B, a Stress-Inducible Ligand for the Activating Immunoreceptor NKG2D
Authors: Holmes Margaret A. , Li Pingwei , Petersdorf Effie W. , Strong Roland K.
Published: April 2014

MIC-A and MIC-B are distant MHC class I homologs that serve as stress-inducible Ags on epithelial and epithelially derived cells. They are ligands for the widely expressed activating immunoreceptor NKG2D. To define the structural and functional consequences of sequence differences between MIC-A and MIC-B and between alleles of MIC-A and alleles of MIC-B, we determined the crystal structure of one allele of human MIC-B. Comparisons between the two previously reported MIC-A crystal structures and the MIC-B crystal structure show that, as expected, MIC-B is very similar in structure to MIC-A and likely interacts with NKG2D in an analogous manner. The interdomain flexibility observed in the MIC-A structures, a feature unique to MIC proteins among MHC class I proteins and homologs, is also displayed by MIC-B, with an interdomain relationship intermediate between the two examples of MIC-A structures. Mapping sequence variations onto the structures of MIC-A and MIC-B reveals patterns completely distinct from those displayed by classical MHC class I proteins, with a number of substitutions falling on positions likely to affect interactions with NKG2D, but with other positions lying distant from the NKG2D binding sites or buried within the core of the proteins.
Cross-Presentation ofListeria monocytogenes-Derived CD4 T Cell Epitopes
Authors: Škoberne Mojca , Schenk Simone , Hof Herbert , Geginat Gernot
Published: April 2014

Listeriolysin O (LLO) mediates the evasion of Listeria monocytogenes from the phagolysosome into the cytoplasm of the host cell. The recognition of infected cells by CD4 T cells is thought to be limited by the evasion of bacteria from the phagolysosome and also by the direct LLO-mediated inhibition of CD4 T cell activation. To analyze the influence of these immunoevasive mechanisms on the antilisterial CD4 T cell response, the expansion of L. monocytogenes-specific CD4 and CD8 T cells was monitored in infected mice. It was found that expansion of L. monocytogenes-specific CD4 T cells occurred synchronously with CD8 T cell expansion. The analysis of Ag presentation by macrophages and dendritic cells isolated from spleens of infected mice revealed efficient presentation of L. monocytogenes-derived CD4 T cell epitopes that was not dependent on the actA-mediated intercellular spread of bacteria. The further in vitro Ag presentation analysis revealed that although L. monocytogenes-infected macrophages and dendritic cells were poor presenters of CD4 T cell epitopes, more efficient presentation occurred after cocultivation of noninfected dendritic cells or macrophages with infected cells. These data indicate that the suppressive effect of LLO on the antilisterial CD4 T cell response is maintained only in infected APC and support the hypothesis that cross-priming plays a role in the induction of the strong CD4 T cell response in Listeria-infected mice.
The CXC Chemokine Murine Monokine Induced by IFN-γ (CXC Chemokine Ligand 9) Is Made by APCs, Targets Lymphocytes Including Activated B Cells, and Supports Antibody Responses to a Bacterial Pathogen In Vivo
Authors: Park Matthew K. , Amichay Doron , Love Paul , Wick Elizabeth , Liao Fang , Grinberg Alex , Rabin Ronald L. , Zhang Hongwei H. , Gebeyehu Senkuta , Wright Timothy M. , Iwasaki Akiko , Weng Youmin , DeMartino Julie A. , Elkins Karen L. , Farber Joshua M.
Published: April 2014

Monokine induced by IFN-γ (Mig; CXC chemokine ligand 9) is an IFN-γ-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4+ and CD8+, and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig−/− mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig−/− mice showed reductions of 50–75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-γ induced the expression of mumig in APCs, including CD8α+ and CD8α− dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.
Marginal Zone Macrophages and Immune Responses Against Viruses
Authors: Oehen Stephan , Odermatt Bernhard , Karrer Urs , Hengartner Hans , Zinkernagel Rolf , López-Macías Constantino
Published: April 2014

The effective establishment of antiviral protection requires a coordinated interplay between the innate and adaptive immune system. Using osteopetrotic (op−/−) mice, this study investigated the influence of marginal zone macrophages in controlling and initiating a protective immune response against a cytopathic vs a non- or low-cytopathic virus. Despite the generation of potent adaptive immune responses, antiviral protection against cytopathic vesicular stomatitis virus critically depended on the presence of marginal zone macrophages. Infection with low doses (100 PFU) of non- or low-cytopathic lymphocytic choriomeningitis virus was rarely cleared and usually resulted in a carrier state in the majority of mice. This shows that the early innate immune system provides an important preparatory phase to the adaptive immune system and is particularly important for antiviral protection.
Effects of Rhinovirus Infection on Histamine and Cytokine Production by Cell Lines from Human Mast Cells and Basophils
Authors: Hosoda Masayoshi , Yamaya Mutsuo , Suzuki Tomoko , Yamada Norihiro , Kamanaka Masato , Sekizawa Kiyohisa , Butterfield Joseph H. , Watanabe Takehiko , Nishimura Hidekazu , Sasaki Hidetada
Published: April 2014

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-κB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.
Long-Term Protective and Antigen-Specific Effect of Heat-Killed Mycobacterium vaccae in a Murine Model of Allergic Pulmonary Inflammation
Authors: Zuany-Amorim Claudia , Manlius Corinne , Trifilieff Alexandre , Brunet Laura R. , Rook Graham , Bowen Gareth , Pay Graham , Walker Christoph
Published: April 2014

This report examines the effect of heat-killed Mycobacterium vaccae in a mouse model of allergic pulmonary inflammation. The s.c. administration of M. vaccae 3 wk before the immunization significantly reduced Ag-induced airway hyperreactivity and the increase in the numbers of eosinophils observed in the bronchoalveolar lavage fluid, blood, and bone marrow, even though no detectable changes in either cytokine (IL-4, IL-13, IL-5, and IFN-γ) or total IgE levels were observed. Furthermore, transfer of splenocytes from OVA-immunized and M. vaccae-treated mice into recipient, OVA-immunized mice significantly reduced the allergen-induced eosinophilia by an IFN-γ-independent mechanism, clearly indicating that the mechanism by which M. vaccae induces its inhibitory effect is not due to a redirection from a predominantly Th2 to a Th1-dominated immune response. The protective effect of M. vaccae on the allergen-induced eosinophilia lasted for at least 12 wk after its administration, and the treatment was also effective in presensitized mice. Moreover, the allergen specificity of the inhibitory effect could be demonstrated using a double-immunization protocol, where M. vaccae treatment before OVA immunization had no effect on the eosinophilic inflammation induced by later immunization and challenge with cockroach extract Ag. Taken together, these results clearly demonstrate that M. vaccae is effective in blocking allergic inflammation by a mechanism independent of IFN-γ, induces long term and Ag-specific protection, and therefore has both prophylactic and therapeutic potential for the treatment of allergic diseases.
Stimulation by Toll-Like Receptors Inhibits Osteoclast Differentiation
Authors: Takami Masamichi , Kim Nacksung , Rho Jaerang , Choi Yongwon
Published: April 2014

Osteoclasts, the cells capable of resorbing bone, are derived from hemopoietic precursor cells of monocyte-macrophage lineage. The same precursor cells can also give rise to macrophages and dendritic cells, which are essential for proper immune responses to various pathogens. Immune responses to microbial pathogens are often triggered because various microbial components induce the maturation and activation of immunoregulatory cells such as macrophages or dendritic cells by stimulating Toll-like receptors (TLRs). Since osteoclasts arise from the same precursors as macrophages, we tested whether TLRs play any role during osteoclast differentiation. We showed here that osteoclast precursors prepared from mouse bone marrow cells expressed all known murine TLRs (TLR1-TLR9). Moreover, various TLR ligands (e.g., peptidoglycan, poly(I:C) dsRNA, LPS, and CpG motif of unmethylated DNA, which act as ligands for TLR2, 3, 4, and 9, respectively) induced NF-κB activation and up-regulated TNF-α production in osteoclast precursor cells. Unexpectedly, however, TLR stimulation of osteoclast precursors by these microbial products strongly inhibited their differentiation into multinucleated, mature osteoclasts induced by TNF-related activation-induced cytokine. Rather, TLR stimulation maintained the phagocytic activity of osteoclast precursors in the presence of osteoclastogenic stimuli M-CSF and TNF-related activation-induced cytokine. Taken together, these results suggest that TLR stimulation of osteoclast precursors inhibits their differentiation into noninflammatory mature osteoclasts during microbial infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone.
Monocyte-Derived Dendritic Cells Induce a House Dust Mite-Specific Th2 Allergic Inflammation in the Lung of Humanized SCID Mice: Involvement of CCR7
Authors: Hammad Hamida , Lambrecht Bart N. , Pochard Pierre , Gosset Philippe , Marquillies Philippe , Tonnel André-Bernard , Pestel Joël
Published: April 2014

In rodents, airway dendritic cells (DCs) capture inhaled Ag, undergo maturation, and migrate to the draining mediastinal lymph nodes (MLN) to initiate the Ag-specific T cell response. However, the role of human DCs in the pathogenesis of the Th2 cell-mediated disease asthma remains to be clarified. Here, by using SCID mice engrafted with T cells from either house dust mite (HDM)-allergic patients or healthy donors, we show that DCs pulsed with Der p 1, one of the major allergens of HDM, and injected intratracheally into naive animals migrated into the MLN. In the MLN, Der p 1-pulsed DCs from allergic patients induced the proliferation of IL-4-producing CD4+ T cells, whereas those from healthy donors induced IFN-γ-secreting cells. In reconstituted human PBMC-reconstituted SCID mice primed with pulsed DCs from allergic patients, repeated exposure to aerosols of HDM induced 1) a strong pulmonary inflammatory reaction rich in T cells and eosinophils, 2) an increase in IL-4 and IL-5 production in the lung lavage fluid, and 3) increased IgE production compared with that in mice primed with unpulsed DCs. All these effects were reduced following in vivo neutralization of the CCR7 ligand secondary lymphoid tissue chemokine. These data in human PBMC-reconstituted SCID mice show that monocyte-derived DCs might play a key role in the pathogenesis of the pulmonary allergic response by inducing Th2 effector function following migration to the MLN.
Activation of Toll-Like Receptor 2 in Acne Triggers Inflammatory Cytokine Responses
Authors: Kim Jenny , Ochoa Maria-Teresa , Krutzik Stephan R. , Takeuchi Osamu , Uematsu Satoshi , Legaspi Annaliza J. , Brightbill Hans D. , Holland Diana , Cunliffe William J. , Akira Shizuo , Sieling Peter A. , Godowski Paul J. , Modlin Robert L.
Published: April 2014

One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Transfection of TLR2 into a nonresponsive cell line was sufficient for NF-κB activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes. P. acnes also induced activation of IL-12 p40 promoter activity via TLR2. Furthermore, P. acnes induced IL-12 and IL-8 protein production by primary human monocytes and this cytokine production was inhibited by anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for treatment of this common skin disease.
Bystander CD8 T Cell-Mediated Demyelination After Viral Infection of the Central Nervous System
Authors: Haring Jodie S. , Pewe Lecia L. , Perlman Stanley
Published: April 2014

Multiple sclerosis, a chronic inflammatory disease of the CNS, is characterized by immune-mediated demyelination. Many patients have a remitting-relapsing course of disease with exacerbations often following unrelated microbial illnesses. The relationship between the two events remains obscure. One possibility is that T cells specific for the inciting microbial pathogen are able to effect demyelination at a site of ongoing inflammation within the CNS. This possibility was examined in mice infected with mouse hepatitis virus, a well-described model of virus-induced demyelination. Using transgenic TCR/recombination activation gene 2−/− mice with only non-mouse hepatitis virus-specific T cells, we show that CD8 T cells are able to cause demyelination in the absence of cognate Ag in the CNS, but only if specifically activated. These findings demonstrate a novel mechanism for immune-mediated neuropathology and show that activated CD8 T cells may serve as important mediators of bystander demyelination during times of infection, including in patients with multiple sclerosis.
V75R576 IL-4 Receptor α Is Associated with Allergic Asthma and Enhanced IL-4 Receptor Function
Authors: Risma Kimberly A. , Wang Ning , Andrews Ryan P. , Cunningham Christie M. , Ericksen Mark B. , Bernstein Jonathan A. , Chakraborty Ranajit , Hershey Gurjit K. Khurana
Published: April 2014

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4Rα in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4Rα allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4Rα with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4Rα allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4Rα haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4Rα requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.
Detection and Induction of CTLs Specific for SYT-SSX-Derived Peptides in HLA-A24+ Patients with Synovial Sarcoma
Authors: Sato Yuriko , Nabeta Yuki , Tsukahara Tomohide , Hirohashi Yoshihiko , Syunsui Rong , Maeda Akiko , Sahara Hiroeki , Ikeda Hideyuki , Torigoe Toshihiko , Ichimiya Shingo , Wada Takuro , Yamashita Toshihiko , Hiraga Hiroaki , Kawai Akira , Ishii Takeshi , Araki Nobuhito , Myoui Akira , Matsumoto Seiichi , Umeda Tohru , Ishii Seiichi , Kawaguchi Satoshi , Sato Noriyuki
Published: April 2014

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24+ synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24+ synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24+ patients with synovial sarcoma.
Role of the Passive Apoptotic Pathway in Graft-Versus-Host Disease
Authors: Drobyski William R. , Komorowski Richard , Logan Brent , Gendelman Maria
Published: April 2014

Donor T cells have been shown to undergo apoptosis during graft-vs-host disease (GVHD). Although active apoptosis mediated through Fas/Fas ligand interactions has been implicated in GVHD, little is known about the role of the passive apoptotic pathway. To examine this question, we compared the ability of normal donor T cells and T cells overexpressing the antiapoptotic protein, Bcl-xL, to mediate alloreactive responses in vitro and lethal GVHD in vivo. In standard MLCs, T cells that overexpressed Bcl-xL had significantly higher proliferative responses but no difference in cytokine phenotype. Overexpression of Bcl-xL prolonged survival of both resting and alloactivated CD4+ and CD8+ T cells as assessed by quantitative flow cytometry, accounting for the higher proliferative responses. Analysis of engraftment in murine transplantation experiments demonstrated an increase in donor T cell chimerism in animals transplanted with Bcl-xL T cells, suggesting that overexpression of Bcl-xL prolonged T cell survival in vivo as well. Notably, transplantation of Bcl-xL T cells into nonirradiated F1 recipients also significantly exacerbated GVHD as assessed by mortality and pathological damage in the gastrointestinal tract. However, when mice were irradiated no difference in GVHD mortality was observed between animals transplanted with wild-type and Bcl-xL T cells. These data demonstrate that the passive apoptotic pathway plays a role in the homeostatic survival of transplanted donor T cells. Moreover, the susceptibility of donor T cells to undergo passive apoptosis is a significant factor in determining GVHD severity under noninflammatory but not inflammatory conditions.
Transfection of Macrophage Inflammatory Protein 1α into B16 F10 Melanoma Cells Inhibits Growth of Pulmonary Metastases But Not Subcutaneous Tumors
Authors: van Deventer Hendrik W. , Serody Jonathon S. , McKinnon Karen P. , Clements Casey , Brickey W. June , Ting Jenny P.-Y.
Published: April 2014

Macrophage inflammatory protein 1α (MIP-1α), a CC chemokine, is a chemoattractant for T cells and immature dendritic cells. Plasmacytoma cells expressing MIP-1α generate a cytotoxic T cell response without affecting tumor growth. To understand this discrepancy, we compared a local tumor model with a metastatic one using MIP-1α-transfected B16 F10 melanoma cells. Clonal idiosyncrasies were controlled by selecting three lipotransfected tumor clones and two pcDNA vector transfected control clones with equivalent in vitro proliferative capacities. No significant differences were seen between the MIP-1α-producing and control melanoma cells after s.c. injection in the hind leg. All animals had a leg diameter of 10 cm in 18.5–21.5 days. However, after i.v. injection the number of pulmonary foci was significantly reduced in the MIP-1α-producing clones. Injection of 106 control transfected cells resulted in a median of 98.5 tumor foci in 2 wk, whereas the injection of the MIP-1α-producing clones resulted in 89.5, 26.5, and 0 foci. The number of metastatic foci was inversely proportional to the amount of MIP-1α produced by the clone in vitro. Flow cytometry showed a significant increase in CD8+ cells in lungs of mice with MIP-1α-transfected tumors 3 days after injection. This increase was not maintained 10 days later despite continued production of MIP-1α. The protection offered by transfection with MIP-1α was significantly impaired in β2-microglobulin−/− mice. Our findings suggest that MIP-1α is effective in preventing the initiation of metastasis, but not at sustaining an effective antitumor response.
Cutting Edge: A Novel Role for Fas Ligand in Facilitating Antigen Acquisition by Dendritic Cells
Authors: Tada Yuji , O-Wang Jiyang , Takiguchi Yuichi , Tatsumi Koichiro , Kuriyama Takayuki , Okada Seiji , Tokuhisa Takeshi , Sakiyama Shigeru , Tagawa Masatoshi
Published: April 2014

Fas ligand (FasL)-expressing tumor cells are found to effectively mediate rejection of the coinoculated FasL negative parental cells while having no effect on the growth of histologically distinct tumor cells. These observations indicate that FasL induces a specific immune response against Ag derived from FasL-bearing tumors and suggest a possible role for FasL in tumor Ag presentation. Indeed, tumor cells expressing FasL can efficiently interact with dendritic cells (DCs) and this interaction requires the expression of membrane-bound FasL on tumors and Fas on DCs. Moreover, DCs cocultured with FasL-expressing tumors are able to elicit a tumor-specific immune response in vivo, suggesting that DCs acquire tumor Ag during the Fas/FasL-mediated DC-tumor contact. These results identify a novel role for FasL in augmenting tumor-DC interactions and subsequent tumor Ag acquisition by DCs, and suggest that FasL-expressing tumor cells could be used to generate tumor-specific DC vaccines.
Shaping Gene Expression in Activated and Resting Primary Macrophages by IL-10
Authors: Lang Roland , Patel Divyen , Morris John J. , Rutschman Robert L. , Murray Peter J.
Published: April 2014

IL-10 regulates inflammation by reducing cytokine and chemokine production from activated macrophages. We performed microarray experiments to identify possible effector molecules of IL-10 and to investigate the global effect of IL-10 on the transcriptional response induced in LPS-activated macrophages. To exclude background effects of endogenous IL-10, macrophages from IL-10-deficient mice were used. IL-10 up-regulated expression of a small number of genes (26 and 37 after 45 min and 3 h, respectively), including newly identified and previously documented targets such as suppressor of cytokine signaling-3 and IL-1 receptor antagonist. However, the activation program triggered by LPS was profoundly affected by IL-10. IL-10 repressed 62 and further increased 15 of 259 LPS-induced genes. For all genes examined, the effects of IL-10 were determined to be STAT3-dependent. These results suggest that IL-10 regulates STAT3-dependent pathways that selectively target a broad component of LPS-induced genes at the mRNA level.
Follicular Dendritic Cell-Mediated Up-Regulation of CXCR4 Expression on CD4 T Cells and HIV Pathogenesis
Authors: Estes Jacob D. , Keele Brandon F. , Tenner-Racz Klara , Racz Paul , Redd Michael A. , Thacker Tyler C. , Jiang Yongjun , Lloyd Michael J. , Gartner Suzanne , Burton Gregory F.
Published: April 2014

Follicular dendritic cells (FDCs) represent a major reservoir of HIV, and active infection occurs surrounding these cells, suggesting that this microenvironment is highly conducive to virus transmission. Because CD4 T cells around FDCs in germinal centers express the HIV coreceptor, CXCR4, whereas CD4 lymphocytes in many other sites do not, it prompted the hypothesis that FDCs may increase CXCR4 expression on CD4 T cells, thereby facilitating infection. To test this, HIV receptor/coreceptor expression was determined on CD4 T cells cultured with or without FDCs, and its consequence to infection was assessed by measuring virus binding and entry. FDCs had little effect on CCR5 or CD4 expression but increased CXCR4 expression on CD4 T cells. FDC-mediated up-regulation of CXCR4 on CD4 T cells occurred by 24 h and was sustained for at least 96 h in vitro, and FDC-CD4 T cell contact was necessary. Importantly, increased CXCR4 expression directly correlated with increased binding and entry of HIV-1 X4 isolates. Furthermore, CD4+CD57+ germinal center T cells expressed high levels of CXCR4 and supported enhanced entry of X4 HIV compared with other CD4 T cells from the same tissue. Thus, in addition to serving as a reservoir of infectious virus, FDCs render surrounding germinal center T cells highly susceptible to infection with X4 isolates of HIV-1.
Murine Malaria Is Exacerbated by CTLA-4 Blockade
Authors: Jacobs Thomas , Graefe Sebastian E. B. , Niknafs Sonja , Gaworski Iris , Fleischer Bernhard
Published: April 2014

Cytolytic T lymphocyte-associated Ag-4 (CD152) is a negatively regulating molecule, which is primarily expressed on T cells following their activation. In this study, we have examined the role of CTLA-4 expression in experimental blood-stage malaria. Similar to human malaria, CTLA-4 is expressed on CD4+ T cells of C57BL/6 mice after infection with Plasmodium berghei. A kinetic analysis revealed that CTLA-4 expression was increased on day 5 postinfection and reached a peak on day 9 postinfection, when almost 10% of splenic CD4+ T cells expressed CTLA-4. Blockade of CTLA-4 in vivo by a specific mAb and subsequent challenge with P. berghei caused neurological signs reminiscent of murine cerebral malaria and earlier death. Histologic examination of brain sections from anti-CTLA-4-treated mice revealed pathologic changes such as hemorrhages and edema, which were absent in control mice. Furthermore, treatment with anti-CTLA-4 also reversed the extensive loss of CD4+ T cells and the suppressed T cell response occurring during blood-stage malaria. Our data suggest that CTLA-4 expression prevents immune pathology by restricting T cell activation during malaria. They also indicate that the development of cerebral malaria is mediated by a failure to down-regulate T cell activation.
Requirement for RhoA Kinase Activation in Leukocyte De-Adhesion
Authors: Liu Li , Schwartz Barbara R. , Lin Nancy , Winn Robert K. , Harlan John M.
Published: April 2014

Leukocyte migration from bloodstream to tissue requires rapid coordinated regulation of integrin-dependent adhesion and de-adhesion. Whether de-adhesion is an active process mediated by a distinct signaling pathway(s) or a passive decay of initial adhesion remains undetermined. We found that blockade of RhoA with C3 exoenzyme or inhibition of RhoA kinase by the specific inhibitor Y-27632 enhanced phorbol ester-stimulated α4β1-dependent adhesion of Jurkat cells at 30 min. Similarly, Y-27632 treatment increased stimulated β2 integrin-dependent neutrophil adhesion at 30 min but not at 5 min. Because reduced de-adhesion could mimic augmentation of adhesion at later time points, we developed an assay to measure de-adhesion specifically. Treatment of phorbol ester—or bacterial chemoattractant peptide—but not Mn2+-stimulated neutrophils adherent to serum-coated plastic or endothelial cells with Y-27632 or C3 exoenzyme markedly reduced the rate of de-adhesion, while markedly increasing their spreading. RhoA kinase inhibitor effects on de-adhesion and spreading were reversed by treatment with the cytoskeletal-disrupting agent cytochalasin D. Treatment with Y-27632 influenced neither integrin activation epitope nor integrin clustering. We conclude that activation of RhoA kinase promotes leukocyte de-adhesion by inhibiting cytoskeletal-dependent spreading, and that these effects of RhoA kinase constitute a new mechanism for regulation of integrin receptor avidity.
CCR6 Colocalizes with CD18 and Enhances Adhesion to Activated Endothelial Cells in CCR6-Transduced Jurkat T Cells
Authors: Maki Wusi , Morales Romeo E. , Carroll Virginia A. , Telford William G. , Knibbs Randall N. , Stoolman Lloyd M. , Hwang Sam T.
Published: April 2014

CCR6 is expressed by memory T cells (mTC) and is a requirement for efficient arrest of a subset of mTC to activated human dermal microvascular endothelial cells (HDMEC) under physiologic shear stress. We now address whether CCR6 alone is sufficient to induce arrest of a model T cell line (Jurkat) that shows low expression of all CCRs tested (CCR1–10). Herein, we transduced Jurkat (JK) T cells expressing fucosyltransferase VII with a chimeric chemokine receptor consisting of CCR6 fused to enhanced green fluorescent protein. In contrast to the starting JK lines, the resulting cell line (JK fucosyltransferase VII-CCR6) migrated 6-fold better to CCL20 in chemotaxis assays, arrested in response to CCL20 that was immobilized to plastic, and demonstrated a 2.5-fold increase in adhesion to activated HDMEC (p = 0.001). Adhesion was blocked by anti-CD18 mAb (p = 0.005) but not by anti-CD49d mAb (p = 0.3). After arrest on recombinant substrates, CCR6 clustered on the surface as detected by real-time observation of enhanced green fluorescent protein fluorescence. Dual-label confocal microscopy revealed that LFA-1 (CD18 and CD11a), but not CXCR4, colocalized with clustered CCR6 in the presence of immobilized CCL20. Thus, the functional expression of CCR6 is sufficient to provide the chemokine signaling necessary to induce arrest of a JK T cell line to activated HDMEC. Clustering of CCR6 and coassociation with critical integrins may serve to strengthen adhesion between T cells and activated endothelial cells.
Endogenous H/K ATPase β-Subunit Promotes T Cell Tolerance to the Immunodominant Gastritogenic Determinant
Authors: Laurie Karen L. , van Driel Ian R. , Zwar Tricia D. , Barrett Simon P. , Gleeson Paul A.
Published: April 2014

A CD4+ T cell response to the gastric H/K ATPase β-subunit (H/Kβ) is required for the onset of experimental autoimmune gastritis in BALB/c mice. The extent to which endogenous H/Kβ contributes toward the tolerance of the H/Kβ-specific T cell repertoire in normal individuals is not known. By comparison of T cell responses in H/Kβ-deficient (o/o) and H/Kβ-expressing BALB/c mice, in this work we show that the endogenous H/Kβ autoantigen plays a major role in the tolerance of pathogenic H/Kβ-specific T cells. First, T cell-dependent Ab responses to the H/Kβ Ag were enhanced in H/K ATPase-immunized H/Kβ-deficient mice compared with wild-type mice. Second, peptide immunization experiments indicated that immune responses to the major gastritogenic epitope of the H/K ATPase, namely H/Kβ253–277, were significantly more vigorous in H/Kβ-deficient mice compared with wild-type mice. Third, unfractionated splenocytes from H/Kβ-deficient mice, but not H/Kβ-expressing mice, induced autoimmune gastritis after adoptive transfer to BALB/c nude mice. The enhanced responses to H/Kβ in H/Kβ-deficient mice were shown to be intrinsic to CD4+CD25− T cells rather than a change in status of CD4+CD25+ regulatory T cells. We conclude from these studies that the H/Kβ-specific T cells in wild-type mice represent the residue of a T cell repertoire, directed toward a single determinant, that has been subjected to partial tolerance induction.
Distinct Response of Human B Cell Subpopulations in Recognition of an Innate Immune Signal, CpG DNA
Authors: Jung Jaeho , Yi Ae-Kyung , Zhang Xin , Choe Jongseon , Li Li , Choi Yong Sung
Published: April 2014

Innate immunity has recently gained renewed interest in its ability to regulate adaptive immunity. Among the innate immune signals, CpG DNA has revealed its potential as a vaccine adjuvant. However, the cellular mechanism for the effect of CpG DNA on the humoral immune response is not well understood. Here, we investigated the effects of CpG DNA on human B cell differentiation using highly purified B cell subsets: naive, germinal center (GC), and memory B cells. In the in vitro culture system that mimics the primary or secondary immune response in vivo, CpG DNA markedly augmented the proliferation and generation of plasma cells from naive and memory B cells. CpG DNA dramatically increased plasma cell generation from GC B cells. However, CpG DNA did not have effect on memory B cell generation from GC B cells. These results suggest that CpG DNA potentiates the B cell adaptive immune response by enhancing terminal differentiation, but does not affect the generation of memory B cells.
Promotion of Skin Graft Tolerance Across MHC Barriers by Mobilization of Dendritic Cells in Donor Hemopoietic Cell Infusions
Authors: Eto Masatoshi , Hackstein Holger , Kaneko Katsuhiko , Nomoto Kikuo , Thomson Angus W.
Published: April 2014

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2b, IE−) mice were given 108 spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2d, IE+) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 107 T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vβ11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.