bluuebunny/crossref_metadata_2025 huggingface dataset:
Serine 6 of Lck Tyrosine Kinase: A Critical Site for Lck Myristoylation, Membrane Localization, and Function in T Lymphocytes
Authors: Yasuda Koubun , Kosugi Atsushi , Hayashi Fumie , Saitoh Shin-ichiroh , Nagafuku Masakazu , Mori Yoshiko , Ogata Masato , Hamaoka Toshiyuki
Published: April 2014

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.
An Intron Transcriptional Enhancer Element Regulates IL-4 Gene Locus Accessibility in Mast Cells
Authors: Hural John A. , Kwan Millie , Henkel Greg , Hock M. Benjamin , Brown Melissa A.
Published: April 2014

The cell type-specific expression of a gene is dependent on developmentally regulated modifications in chromatin structure that allow accessibility of basal and inducible transcription factors. In this study, we demonstrate that a cis-acting element in the second intron of the murine IL-4 gene has a dual function in regulating transcription in mast cells as well as chromatin accessibility of the IL-4 gene locus through its influence on the methylation state of the gene. Previous studies have shown that mast cell-restricted transcription factors GATA-1/2 and PU.1 associate with the intron element and regulate its activity. In this study, we use DNase I footprinting and mutational analyses to identify two additional sites that contribute to the element’s ability to enhance transcription. One of these sites associates preferentially with STAT5a and STAT5b. We also demonstrate that deletion of the element or mutation of the GATA binding site in the context of a stably integrated IL-4 genomic construct prevents maintenance of a demethylated locus in IL-4-producing mast cells. These data indicate that, analogous to Ig and TCR intron regulatory elements, the intron enhancer has an essential role in maintaining developmentally regulated demethylation at the IL-4 gene locus. In addition, they indicate that members of the GATA family of transcription factors likely play an important role in these processes.
The Primate Lentiviral Receptor Bonzo/STRL33 Is Coordinately Regulated with CCR5 and Its Expression Pattern Is Conserved Between Human and Mouse
Authors: Unutmaz Derya , Xiang Wenkai , Sunshine Mary Jean , Campbell Jim , Butcher Eugene , Littman Dan R.
Published: April 2014

Chemokines play necessary and important roles in regulating the trafficking of lymphocytes to intra- or interlymphoid tissues as well as to sites of inflammation. The complex migratory patterns of lymphoid lineage cells is governed by subset-specific expression of chemokine receptors and their access to specific ligands. Several chemokine receptors and chemokine receptor-like orphan receptors also serve, in conjunction with CD4, as coreceptors for infection by human and simian immunodeficiency viruses (HIV and SIV). Here we show that the expression pattern of Bonzo/STRL33, an orphan SIV/HIV coreceptor, is highly restricted to the memory subset of T cells and is up-regulated upon stimulation of these cells with IL-2 or IL-15. Both the pattern and the regulation of Bonzo expression closely paralleled that of CC family chemokine receptors CCR5 or CCR6 and inversely correlated with CXCR4 expression. However, in striking contrast to CCR5, Bonzo expression was not down-modulated by PMA or mitogen stimulation of T cells. Targeted replacement of the Bonzo gene with a gene encoding green fluorescent protein in mice revealed that the expression and cytokine regulation of mouse Bonzo are comparable to those of its human counterpart. The similar expression and regulation patterns of Bonzo and the HIV coreceptor CCR5 may have implications for understanding the role of HIV/SIV receptors in viral evolution and pathogenesis.
The Phagocytosis-Associated Respiratory Burst in Human Monocytes Is Associated with Increased Uptake of Glutathione
Authors: Seres Tamas , Knickelbein Roy G. , Warshaw Joseph B. , Johnston Richard B.
Published: April 2014

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20–30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by γ-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 μM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.
ICAM-1-Coupled Cytoskeletal Rearrangements and Transendothelial Lymphocyte Migration Involve Intracellular Calcium Signaling in Brain Endothelial Cell Lines
Authors: Etienne-Manneville Sandrine , Manneville Jean-Baptiste , Adamson Peter , Wilbourn Barry , Greenwood John , Couraud Pierre-Olivier
Published: April 2014

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)γ1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC’s activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.
Induction of Heme Oxygenase-1 Expression in Macrophages by Diesel Exhaust Particle Chemicals and Quinones via the Antioxidant-Responsive Element
Authors: Li Ning , Venkatesan M. Indira , Miguel Antonio , Kaplan Roman , Gujuluva Chandrasekhar , Alam Jawed , Nel Andre
Published: April 2014

Diesel exhaust particles (DEP) contain organic chemicals that contribute to the adverse health effects of inhaled particulate matter. Because DEP induce oxidative stress in the lung and in macrophages, effective antioxidant defenses are required. One type of defense is through the expression of the antioxidant enzyme, heme oxygenase I (HO-1). HO-1 as well as phase II detoxifying enzymes are induced via antioxidant response elements (ARE) in their promoters of that gene. We show that a crude DEP total extract, aromatic and polar DEP fractions, a benzo(a)pyrene quinone, and a phenolic antioxidant induce HO-1 expression in RAW264.7 cells in an ARE-dependent manner. N-acetyl cysteine and the flavonoid, luteolin, inhibited HO-1 protein expression. We also demonstrate that the same stimuli induce HO-1 mRNA expression in parallel with the activation of the SX2 enhancer of that gene. Mutation of the ARE core, but not the overlapping AP-1 binding sequence, disrupted SX2 activation. Finally, we show that biological agents, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, could also induce HO-1 expression via an ARE-dependent mechanism. Prior induction of HO-1 expression, using cobalt-protoporphyrin, protected RAW264.7 cells against DEP-induced toxicity. Taken together, these data show that HO-1 plays an important role in cytoprotection against redox-active DEP chemicals, including quinones.
A SAF Binding Site in the Promoter Region of Human γ-Fibrinogen Gene Functions as an IL-6 Response Element
Authors: Ray Alpana
Published: April 2014

Expression of fibrinogen is highly induced during inflammation, and such abnormal expression of this protein is considered as a major cardiovascular risk factor. IL-6 is one of the main mediators of abnormal expression of fibrinogen leading to the pathogenic conditions. Transient transfection and EMSA were performed to investigate the molecular mechanism of IL-6-induced γ-fibrinogen gene expression in hepatic cells. Using progressively deleted 5′ fragments of the γ-fibrinogen promoter coupled to chloramphenicol acetyltransferase gene, an IL-6 responsive element located between positions −273 and −259 was identified. Mutation of this element abrogates IL-6 responsiveness of the γ-fibrinogen promoter. Interaction of this promoter with a zinc finger transcription factor, serum amyloid A activating factor (SAF)-1, was demonstrated by EMSA. Furthermore, overexpression of wild-type SAF-1 in transfected liver cells can increase transcription of the γ-fibrinogen promoter. These data show that transcription factor SAF-1 is involved in the regulation of IL-6-mediated induction of the human γ-fibrinogen gene in liver cells.
Local Blockade of Allergic Airway Hyperreactivity and Inflammation by the Poxvirus-Derived Pan-CC-Chemokine Inhibitor vCCI
Authors: Dabbagh Karim , Xiao Yun , Smith Craig , Stepick-Biek Pamela , Kim Sung G. , Lamm Wayne J. E. , Liggitt Denny H. , Lewis David B.
Published: April 2014

Allergen-induced asthma is characterized by chronic pulmonary inflammation, reversible bronchoconstriction, and airway hyperreactivity to provocative stimuli. Multiple CC-chemokines, which are produced by pulmonary tissue in response to local allergen challenge of asthmatic patients or experimentally sensitized rodents, chemoattract leukocytes from the circulation into the lung parenchyma and airway, and may also modify nonchemotactic function. To determine the therapeutic potential of local intrapulmonary CC-chemokine blockade to modify asthma, a recombinant poxvirus-derived viral CC-chemokine inhibitor protein (vCCI), which binds with high affinity to rodent and human CC-chemokines in vitro and neutralizes their biological activity, was administered by the intranasal route. Administration of vCCI to the respiratory tract resulted in dramatically improved pulmonary physiological function and decreased inflammation of the airway and the lung parenchyma. In contrast, vCCI had no significant effect on the circulating levels of total or allergen-specific IgE, allergen-specific cytokine production by peripheral lymph node T cells, or peritoneal inflammation after local allergen challenge, indicating that vCCI did not alter systemic Ag-specific immunity or chemoattraction at extrapulmonary sites. Together, these findings emphasize the importance of intrapulmonary CC-chemokines in the pathogenesis of asthma, and the therapeutic potential of generic and local CC-chemokine blockade for this and other chronic diseases in which CC-chemokines are locally produced.
Deficiency in Inducible Nitric Oxide Synthase Results in Reduced Atherosclerosis in Apolipoprotein E-Deficient Mice
Authors: Detmers Patricia A. , Hernandez Melba , Mudgett John , Hassing Heide , Burton Charlotte , Mundt Steven , Chun Sam , Fletcher Dan , Card Deborah J. , Lisnock JeanMarie , Weikel Reneé , Bergstrom James D. , Shevell Diane E. , Hermanowski-Vosatka Anne , Sparrow Carl P. , Chao Yu-Sheng , Rader Daniel J. , Wright Samuel D. , Puré Ellen
Published: April 2014

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)−/− mice that were also deficient in iNOS. ApoE−/− and iNOS−/− mice were cross-bred to produce apoE−/−/iNOS−/− mice and apoE−/−/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30–50% reduction in apoE−/−/iNOS−/− mice compared with apoE−/−/iNOS+/+ mice. Although the size of the lesions in apoE−/−/iNOS−/− mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45–70% reductions in both male and female apoE−/−/iNOS−/− mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.
Inducing Tolerance by Intranasal Administration of Long Peptides in Naive and Primed CBA/J Mice
Authors: Astori Mireille , von Garnier Christophe , Kettner Alexander , Dufour Nathalie , Corradin Giampietro , Spertini François
Published: April 2014

To assess the capacity of a peptide-based immunotherapy to induce systemic tolerance via the nasal route, we designed three long overlapping peptides of 44–60 aa covering the entire sequence of phospholipase A2 (PLA2), a major bee venom allergen. Both prophylactic and therapeutic intranasal administrations of long peptides to PLA2-hypersensitive CBA/J mice induced specific T cell tolerance to the native allergen. In prophylactic conditions, this tolerance was marked by a suppression of subsequent specific IgE response, whereas the therapeutic approach in presensitized mice induced a more than 60% decrease in PLA2-specific IgE. This decline was associated with a shift in the cytokine response toward a Th1 profile, as demonstrated by decreased PLA2-specific IgG1 and enhanced IgG2a levels, and by a decline in the specific IL-4/IFN-γ ratios. T cell transfer from long peptide-tolerized mice to naive animals abrogated the expected anti-PLA2 IgE and IgG1 Ab response, as well as specific T cell proliferation, but enhanced specific IgG2a response upon sensitization with PLA2. These events were strongly suggestive of a clonal anergy affecting more profoundly Th2 than the Th1 subsets. In conclusion, these results demonstrate that allergen-derived long peptides delivered via the nasal mucosa may offer an alternative to immunotherapy with native allergens without the inherent risk of systemic anaphylactic reactions. Moreover, long peptides, in contrast to immunotherapy strategies based on short peptides, have the advantage of covering all potential T cell epitopes, and may represent novel and safe tools for the therapy of allergic diseases.
The Combination of Apoptotic U937 Cells and Lupus IgG Is a Potent IFN-α Inducer
Authors: Båve Ullvi , Alm Gunnar V. , Rönnblom Lars
Published: April 2014

Patients with active systemic lupus erythematosus (SLE) have signs of an ongoing IFN-α production, that may be of pathogenic significance in the disease. We previously showed that SLE patients have an IFN-α-inducing factor in blood, probably consisting of complexes containing anti-DNA Abs and immunostimulatory DNA. The DNA component could be derived from apoptotic cells, because SLE patients have been reported to have both increased apoptosis and reduced clearance of apoptotic cell material. In the present study, we therefore investigated whether apoptotic cells, together with IgG from SLE patients, could act as an IFN-α inducer in normal PBMC in vitro. We found that apoptotic cells of the myeloid leukemia cell line U937 as well as four other cell lines (MonoMac6, H9, Jurkat, U266) could induce IFN-α production in PBMC when combined with IgG from SLE patients. The IFN-α production by PBMC was much enhanced when PBMC were costimulated by IFN-α2b. The ability of IgG from different SLE patients to promote IFN-α induction by apoptotic U937 cells was associated with the presence of anti-ribonucleoprotein Abs, but not clearly with occurrence of anti-DNA Abs. These results suggest that apoptotic cells in the presence of autoantibodies can cause production of a clearly immunostimulatory cytokine, which is IFN-α. This mechanism for induction of IFN-α production could well be operative also in vivo, explain the IFN-α production seen in SLE patients, and be important in the pathogenesis of SLE.
NK-Mediated Elimination of Mutant Lymphocytes that Have Lost Expression of MHC Class I Molecules
Authors: Kusunoki Yoichiro , Kyoizumi Seishi , Honma Masamitsu , Kubo Yoshiko , Ohnishi Hisashi , Hayashi Tomonori , Seyama Toshio
Published: April 2014

Mutant cells generated in vivo can be eliminated when mutated gene products are presented as altered MHC/peptide complexes and recognized by T cells. Diminished expression of MHC/peptide complexes enables mutant cells to escape recognition by T cells. In the present study, we tested the hypothesis that mutant lymphocytes lacking expression of MHC class I molecules are eliminated by autologous NK cells. In H-2b/k F1 mice, the frequency of H-2Kb-negative T cells was higher than that of H-2Kk-negative T cells. The frequency of H-2K-deficient T cells increased transiently after total body irradiation. During recovery from irradiation, H-2Kk-negative T cells disappeared more rapidly than H-2Kb-negative T cells. The disappearance of H-2K-deficient T cells was inhibited by administration of Ab against asialo-GM1. H-2Kk-negative T cells showed higher sensitivity to autologous NK cells in vitro than H-2Kb/k heterozygous or H-2Kb-negative T cells. Adding syngeneic NK cells to in vitro cultures prevented emergence of mutant cells lacking H-2Kk expression but had little effect on the emergence of mutant cells lacking H-2Kb expression. Results in the H-2b/k F1 strain correspond with the sensitivity of parental H-2-homozygous cells in models of marrow graft rejection. In H-2b/d F1 mice, there was no significant difference between the frequencies of H-2Kb-negative and H-2Kd-negative T cells, although the frequencies of mutant cells were different after radiation exposure among the strains examined. H-2b/d F1 mice also showed rapid disappearance of the mutant T cells after irradiation, and administration of Ab against asialo-GM1 inhibited the disappearance of H-2K-deficient T cells in H-2b/d F1 mice. Our results provide direct evidence that autologous NK cells eliminate mutant cell populations that have lost expression of self-MHC class I molecules.
Ligation of the WC1 Receptor Induces γδ T Cell Growth Arrest Through Fumonisin B1-Sensitive Increases in Cellular Ceramide
Authors: Kirkham Paul A. , Takamatsu Haru-Hisa , Lam Eric W.-F. , Parkhouse R. M. E.
Published: April 2014

Ceramide is a powerful regulator of cell fate, inducing either apoptosis or growth arrest. We have previously shown that an Ab to the γδ T cell-specific orphan receptor, WC1, is able to induce growth arrest in proliferating IL-2-dependent γδ T cells. We now show that this WC1-mediated growth arrest is associated with an increase in cellular ceramide, in the absence of any measurable changes in acidic/neutral sphingomyelinase activity. Moreover, cell-permeable analogues of ceramide also mimicked WC1-induced growth arrest along with an associated decrease in pocket protein expression and phosphorylation status. An important role for ceramide in WC1-induced growth arrest was confirmed by demonstrating that the specific ceramide synthase inhibitor fumonisin B1 blocked WC1-induced growth arrest and the associated molecular effects on the pocket proteins. Finally, we observed constitutive expression of both antiapoptotic factors bcl-2 and bcl-X, the former having increased expression upon WC1 stimulation. It is therefore proposed that ligation of WC1 leads to an accumulation in cellular ceramide through activation of ceramide synthase. This in turn results in a decreased overall expression of the pocket proteins pRb and p107, their hypophosphorylation, and an eventual growth arrest of the γδ T cell. To our knowledge, these results demonstrate for the first time that cell surface receptor-mediated ceramide synthase activation can affect cell fate through increases in cellular ceramide and provide further evidence that the orphan receptor WC1 regulates γδ T cell biology through a novel signaling pathway.
Distinct Requirements for IFNs and STAT1 in NK Cell Function
Authors: Lee Chien-Kuo , Rao Dharma T. , Gertner Rachel , Gimeno Ramon , Frey Alan B. , Levy David E.
Published: April 2014

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNα receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1−/− mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1−/− bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-γ were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1−/− mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-α and IFN-γ are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.
Monocyte-Derived CD1a+ and CD1a− Dendritic Cell Subsets Differ in Their Cytokine Production Profiles, Susceptibilities to Transfection, and Capacities to Direct Th Cell Differentiation
Authors: Chang Chia-Chun J. , Wright Anne , Punnonen Juha
Published: April 2014

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-γ, but they remained CD1a− and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.
Mutant Mouse Lysozyme Carrying a Minimal T Cell Epitope of Hen Egg Lysozyme Evokes High Autoantibody Response
Authors: Tsujihata Yoshiyuki , So Takanori , Chijiiwa Yuki , Hashimoto Yoshio , Hirata Masato , Ueda Tadashi , Imoto Taiji
Published: April 2014

Self proteins including foreign T cell epitope induce autoantibodies. We evaluated the relationship between the size of foreign Ag introduced into self protein and the magnitude of autoantibody production. Mouse lysozyme (ML) was used as a model self protein, and we prepared three different ML derivatives carrying T cell epitope of hen egg white lysozyme (HEL) 107–116, i.e., heterodimer of ML and HEL (ML-HEL), chimeric lysozyme that has residue 1–82 of ML and residue 83–130 of HEL in its sequence (chiMH), and mutant ML that has triple mutations rendering the most potent T cell epitope of HEL (sequence 107–116). Immunization of BALB/c mice with these three ML derivatives induced anti-ML autoantibody responses, whereas native ML induced no detectable response. In particular, mutML generated a 104 times higher autoantibody titer than did ML-HEL. Anti-HEL107–116 T cell-priming activities were almost similar among the ML derivatives. The heterodimerization of mutant ML and HEL led to significant reduction of the autoantibody response, whereas the mixture did not. These results show that size of the nonself region in modified self Ag has an important role in determining the magnitude of the autoantibody response, and that decrease in the foreign region in a modified self protein may cause high-titered autoantibody response.
Bacterial Lipopolysaccharide, TNF-α, and Calcium Ionophore Under Serum-Free Conditions Promote Rapid Dendritic Cell-Like Differentiation in CD14+ Monocytes Through Distinct Pathways That Activate NF-κB
Authors: Lyakh Lyudmila A. , Koski Gary K. , Telford William , Gress Ronald E. , Cohen Peter A. , Rice Nancy R.
Published: April 2014

To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-α, or calcium ionophore (CI). Within 48–96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC. However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-α production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS. Nevertheless, all three inducing agents activated members of the NF-κB family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-κB. In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway.
Cytotoxic T Cells Specifically Induce Fas on Target Cells, Thereby Facilitating Exocytosis-Independent Induction of Apoptosis
Authors: Simon Markus M. , Waring Paul , Lobigs Mario , Nil Ahmed , Tran Thao , Hla Ron Tha , Chin Seow , Müllbacher Arno
Published: April 2014

Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.
CD8+ T Cells Rapidly Acquire NK1.1 and NK Cell-Associated Molecules Upon Stimulation In Vitro and In Vivo
Authors: Assarsson Erika , Kambayashi Taku , Sandberg Johan K. , Hong Seokmann , Taniguchi Masaru , Van Kaer Luc , Ljunggren Hans-Gustaf , Chambers Benedict J.
Published: April 2014

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRαβ+ cells have been described as being either CD4+CD8− or CD4−CD8−. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Vα14-Jα281 TCR chain in conjunction with either a Vβ2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRαβ+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1−/−, and Jα281−/− mice that lack classical NKT cells. Unlike classical NKT cells, 50–60% of these NK1.1+TCRαβ+ cells express CD8 and have a diverse TCR Vβ repertoire. Purified NK1.1−CD8α+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rβ+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.
A Profound Deficiency in Thymic Progenitor Cells in Mice Lacking Jak3
Authors: Baird Allison M. , Lucas Julie A. , Berg Leslie J.
Published: April 2014

Humans and mice with genetic deficiencies that lead to loss of signaling through common γ-chain (γc)-containing cytokine receptors have severe defects in B and T lymphocytes. In humans, these deficiencies lead to a complete absence of T cells, whereas in mice, small thymuses give rise to normal numbers of peripheral T cells. We have examined the first wave of developing T cells in Jak3−/−, IL-7−/−, and IL-7Rα−/− fetal mice, and have found a near absence of thymic progenitor cells. This deficiency is highlighted by the complete inability of Jak3−/− progenitor cells to reconstitute T cell development in the presence of competing wild-type cells. These data clearly demonstrate a strong common basis for the T cell deficiencies in mice and humans lacking γc/Jak3 signaling pathways.
Structure-Guided Identification of C3d Residues Essential for Its Binding to Complement Receptor 2 (CD21)
Authors: Clemenza Liliana , Isenman David E.
Published: April 2014

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.
A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain
Authors: Flicker Sabine , Vrtala Susanne , Steinberger Peter , Vangelista Luca , Bufe Albrecht , Petersen Arnd , Ghannadan Minoo , Sperr Wolfgang R. , Valent Peter , Norderhaug Lars , Bohle Barbara , Stockinger Hannes , Suphioglu Cenk , Ong Eng Kok , Kraft Dietrich , Valenta Rudolf
Published: April 2014

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an α helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.
Alanine Scanning Mutants of Rat Proinsulin I Show Functional Diversity of Anti-Insulin Monoclonal Antibodies
Authors: Tikhomirov O. Yu , Thomas James W.
Published: April 2014

In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a “molten globule” may contribute to the diversity and autoreactivity of the anti-insulin repertoire.
Signal Joint Formation Is Also Impaired in DNA-Dependent Protein Kinase Catalytic Subunit Knockout Cells
Authors: Fukumura Ryutaro , Araki Ryoko , Fujimori Akira , Tsutsumi Yoko , Kurimasa Akihiro , Li Gloria C. , Chen David J. , Tatsumi Kouichi , Abe Masumi
Published: April 2014

The effort to elucidate the mechanism of V(D)J recombination has given rise to a dispute as to whether DNA-dependent protein kinase catalytic subunit (DNA-PKcs) contributes to signal joint formation (sjf). Observations reported to date are confusing. Analyses using DNA-PKcs-deficient cells could not conclude the requirement of DNA-PKcs for sjf, because sjf can be formed by end-joining activities which are diverse among cells other than those participating in V(D)J recombination. Here, we observed V(D)J recombination in DNA-PKcs knockout cells and showed that both signal and coding joint formation were clearly impaired in the cells. Subsequently, to directly demonstrate the requirement of DNA-PKcs for sjf, we introduced full-length cDNA of DNA-PKcs into the knockout cells. Furthermore, several mutant DNA-PKcs cDNA constructs designed from mutant cell lines (irs-20, V3, murine scid, and SX9) were also introduced into the cells to obtain further evidence indicating the involvement of DNA-PKcs in sjf. We found as a result that the full-length cDNA complemented the aberrant sjf and that the mutant cDNAs constructs also partially complemented it. Lastly, we looked at whether the kinase activity of DNA-PKcs is necessary for sjf and, as a result, demonstrated a close relationship between them. Our observations clearly indicate that the DNA-PKcs controls not only coding joint formation but also the sjf in V(D)J recombination through its kinase activity.
Allergy-Associated Polymorphisms of the FcεRIβ Subunit Do Not Impact Its Two Amplification Functions
Authors: Donnadieu Emmanuel , Cookson William O. , Jouvin Marie-Hélène , Kinet Jean-Pierre
Published: April 2014

Two variants of the β-chain of the high affinity IgE receptor FcεRI, I181L-V183L and E237G, have been found associated with allergy. We have previously shown that the β-chain plays at least two distinct amplifier functions. It amplifies FcεRI surface expression and signaling, resulting in an estimated 12- to 30-fold amplification of downstream events. To test the hypothesis that the I181L-V183L and E237G β variants may be functionally relevant and could directly contribute to an allergic phenotype, we have evaluated the functional impact of the β variants on the two amplifier functions of β. We found that these variants have no direct effect on the β amplifier functions. However, the possibility remains that these variants are in linkage disequilibrium with other more relevant polymorphisms or are affecting unknown β-chain functions.
Group BStreptococcusInduces Apoptosis in Macrophages
Authors: Fettucciari Katia , Rosati Emanuela , Scaringi Lucia , Cornacchione Paola , Migliorati Graziella , Sabatini Rita , Fetriconi Ilaria , Rossi Ruggero , Marconi Pierfrancesco
Published: April 2014

Group B Streptococcus (GBS) is a pathogen that has developed some strategies to resist host immune defenses. Because phagocytic killing is an important pathogenetic mechanism for bacteria, we investigated whether GBS induces apoptosis in murine macrophages. GBS type III strain COH31 r/s (GBS-III) first causes a defect in cell membrane permeability, then at 24 h, apoptosis. Apoptosis was confirmed by several techniques based on morphological changes and DNA fragmentation. Cytochalasin D does not affect apoptosis, suggesting that GBS-III needs not be within the macrophage cytoplasm to promote apoptosis. Inhibition of host protein synthesis prevents apoptosis, whereas inhibition of caspase-1 or -3, does not. Therefore, GBS can trigger an apoptotic pathway independent of caspase-1 and -3, but dependent on protein synthesis. Inhibition of apoptosis by EGTA and PMA, and enhancement of apoptosis by calphostin C and GF109203X suggests that an increase in the cytosolic calcium level and protein kinase C activity status are important in GBS-induced apoptosis. Neither alteration of plasma membrane permeability nor apoptosis were induced by GBS grown in conditions impeding hemolysin expression or when we used dipalmitoylphosphatidylcholine, which inhibited GBS β-hemolytic activity, suggesting that GBS β-hemolysin could be involved in apoptosis. β-Hemolysin, by causing membrane permeability defects, could allow calcium influx, which initiates macrophage apoptosis. GBS also induces apoptosis in human monocytes but not in tumor lines demonstrating the specificity of its activity. This study suggests that induction of macrophage apoptosis by GBS is a novel strategy to overcome host immune defenses.
Exaggerated Human Monocyte IL-10 Concomitant to Minimal TNF-α Induction by Heat-Shock Protein 27 (Hsp27) Suggests Hsp27 Is Primarily an Antiinflammatory Stimulus
Authors: De Asit K. , Kodys Karen M. , Yeh Berhan S. , Miller-Graziano Carol
Published: April 2014

Unlike more well-studied large heat shock proteins (hsp) that induce both T cell antiinflammatory (IL-10, IL-4) and macrophage proinflammatory (TNF-α, IL-15, IL-12) cytokines, hsp27, a small hsp, has been primarily identified as a substrate of mitogen-activated protein kinase-activated protein kinase-2 involved in the p38 signaling pathway and activated during monocyte IL-10 production. Hsp27 can also act as an endogenous protein circulating in the serum of breast cancer patients and a protein whose induction correlates to protection from LPS shock. However, the cytokine-stimulating properties of hsp27 have been unexplored. In this study, exogenous hsp27 is demonstrated for the first time as a potent activator of human monocyte IL-10 production, but only a modest inducer of TNF-α. Although exogenous hsp27 stimulation activated all three monocyte mitogen-activated protein kinase pathways (extracellular signal-related kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38), only p38 activation was sustained and required for hsp27 induction of monocyte IL-10, while both ERK 1/2 and p38 activation were required for induction of TNF-α when using the p38 inhibitor SB203580 or the ERK inhibitor PD98059. Hsp27’s transient activation of the c-Jun N-terminal kinase pathway, which can down-regulate IL-10, may contribute to its potent IL-10 induction. Hsp27’s ERK 1/2 activation was also less sustained than activation by stimuli like LPS, possibly contributing to its modest TNF-α induction. The failure of either PD98059 or anti-TNF-α Ab to substantially inhibit IL-10 induction implied that hsp27 induces IL-10 via activation of p38 signaling independently of TNF-α activation and may be predominantly an antiinflammatory monokine stimulus.
Capsaicin Inhibits Platelet-Activating Factor-Induced Cytosolic Ca2+ Rise and Superoxide Production
Authors: Choi Se-Young , Ha Hyunjung , Kim Kyong-Tai
Published: April 2014

Platelet-activating factor (PAF) is an important participant in the inflammatory process. We studied the regulation of PAF activity by capsaicin in human promyelocytic leukemia HL-60 cells. Capsaicin inhibited PAF-induced superoxide production in a concentration-dependent manner. In addition to PAF, the fMLP- and extracellular ATP-induced superoxide productions were inhibited by capsaicin, whereas PMA-induced superoxide production was not affected. In the PAF-stimulated cytosolic Ca2+ increase, capsaicin inhibited in particular the sustained portion of the raised Ca2+ level without attenuation of the peak height. In the absence of extracellular Ca2+, the PAF-induced Ca2+ elevation was not inhibited by capsaicin because capsaicin only inhibited the Ca2+ influx from the extracellular space. In addition, capsaicin did not affect PAF-induced inositol 1,4,5-trisphosphate production, suggesting that phospholipase C activation by PAF is not affected by capsaicin. Store-operated Ca2+ entry (SOCE) induced by thapsigargin was inhibited by capsaicin in a concentration-dependent manner. This capsaicin effect was also observed on thapsigargin-induced Ba2+ and Mn2+ influx. Furthermore, capsaicin’s inhibitory effect on the thapsigargin-induced Ca2+ rise overlapped with that of SK&F96365, an inhibitor of SOCE. Both capsaicin and SK&F96365 also inhibited PAF-induced cytosolic superoxide generation in HL-60 cells differentiated by all-trans-retinoic acid. Our data suggest that capsaicin exerts its anti-inflammatory effect by inhibiting SOCE elicited via PLC activation, which occurs upon PAF activation and results in the subsequent superoxide production.