bluuebunny/crossref_metadata_2025 huggingface dataset:
Premature TCRαβ Expression and Signaling in Early Thymocytes Impair Thymocyte Expansion and Partially Block Their Development
Authors: Lacorazza H. Daniel , Tuček-Szabo Carolyn , Vasović Ljiljana V. , Remus Kristin , Nikolich-Žugich Janko
Published: April 2014

In thymocyte ontogeny, Tcr-a genes rearrange after Tcr-b genes. TCRαβ transgenic (Tg) mice have no such delay, consequently expressing rearranged TCRαβ proteins early in the ontogeny. Such mice exhibit reduced thymic cellularity and accumulate mature, nonprecursor TCR+CD8−4− thymocytes, believed to be caused by premature Tg TCRαβ expression via unknown mechanism(s). Here, we show that premature expression of TCRαβ on early thymocytes curtails thymocyte expansion and impairs the CD8−4− → CD8+4+ transition. This effect is accomplished by two distinct mechanisms. First, the early formation of TCRαβ appears to impair the formation and function of pre-TCR, consistent with recently published results. Second, the premature TCRαβ contact with intrathymic MHC molecules further pronounces the block in proliferation and differentiation. These results suggest that the benefit of asynchronous Tcr-a and Tcr-b rearrangement is not only to minimize waste during thymopoiesis, but also to simultaneously allow proper expression/function of the pre-TCR and to shield CD8−4− thymocytes from TCRαβ signals that impair thymocyte proliferation and CD8−4− → CD8+4+ transition.
Anergy and not Clonal Ignorance Determines the Fate of B Cells that Recognize a Physiological Autoantigen
Authors: Rojas Mauricio , Hulbert Chrys , Thomas James W.
Published: April 2014

Autoantibodies to insulin arise spontaneously in the insulin autoimmune syndrome and in type I diabetes. In addition, administration of insulin to individuals without autoimmune disease routinely results in Abs that bind autologous hormone. These observations and findings in transgenic models of tolerance led to an inference that physiological levels of hormones and growth factors, such as insulin, are not sufficient to induce tolerance in B cells, a state termed clonal ignorance. In contrast, we have discovered that virtually all conventional B cells expressing a low affinity anti-insulin transgene interact with endogenous insulin and are effectively silenced for Ig production and for T cell-dependent immune responses. A fraction of transgenic B cells escapes silencing and functions autonomously to produce insulin Abs that may lower fasting blood sugars similar to an insulin autoimmune syndrome. These B cells have characteristics of a B1-like subset and are depleted by hypotonic peritoneal lysis. These findings question the concept of clonal ignorance and show that physiological concentrations of Ag may effectively silence conventional B cells even when the affinity for autoantigen is low. Self-reactivity may arise in the repertoire because of compartmental differences that govern the fate of B cells and not as a result of true clonal ignorance.
Oncostatin M-Induced Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase-3 Genes Expression in Chondrocytes Requires Janus Kinase/STAT Signaling Pathway
Authors: Li Wen Qing , Dehnade Faramaze , Zafarullah Muhammad
Published: April 2014

Oncostatin M (OSM), a member of the IL-6 superfamily of cytokines, is elevated in patients with rheumatoid arthritis and, in synergy with IL-1, promotes cartilage degeneration by matrix metalloproteinases (MMPs). We have previously shown that OSM induces MMP and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression in chondrocytes by protein tyrosine kinase-dependent mechanisms. In the present study, we investigated signaling pathways regulating the induction of MMP and TIMP-3 genes by OSM. We demonstrate that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, JAK2, JAK3, and STAT1 as well as extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase 1/2 mitogen-activated protein kinases in primary bovine and human chondrocytes. A JAK3-specific inhibitor blocked OSM-stimulated STAT1 tyrosine phosphorylation, DNA-binding activity of STAT1 as well as collagenase-1 (MMP-1), stromelysin-1 (MMP-3), collagenase-3 (MMP-13), and TIMP-3 RNA expression. In contrast, a JAK2-specific inhibitor, AG490, had no impact on these events. OSM-induced ERK1/2 activation was also not affected by these inhibitors. Similarly, curcumin (diferuloylmethane), an anti-inflammatory agent, suppressed OSM-stimulated STAT1 phosphorylation, DNA-binding activity of STAT1, and c-Jun N-terminal kinase activation without affecting JAK1, JAK2, JAK3, ERK1/2, and p38 phosphorylation. Curcumin also inhibited OSM-induced MMP-1, MMP-3, MMP-13, and TIMP-3 gene expression. Thus, OSM induces MMP and TIMP-3 genes in chondrocytes by activating JAK/STAT and mitogen-activated protein kinase signaling cascades, and interference with these pathways may be a useful approach to block the catabolic actions of OSM.
Eukaryotic Translation Initiation Factor-6 Enhances Histamine and IL-2 Production in Mast Cells
Authors: Oh Chad K. , Filler Scott G. , Cho Seong H.
Published: April 2014

Eukaryotic translation initiation factor (eIF)-6 is known to be important in ribosome biogenesis. Previously, we have discovered that eIF-6 mRNA is induced in lung in a murine model of asthma. We also found that there was enhanced eIF-6 expression in mast cells stimulated with PMA plus calcium ionophore. Therefore, we hypothesized that the induction of eIF-6 enhances the production of bioactive mediators by mast cells upon allergic stimulation. In the current study, we found that eIF-6 mRNA was rapidly induced in murine mast cells stimulated by FcεRI cross-linking, which is a major physiologic stimulant for mast cells. eIF-6 was also induced in human mast cells upon stimulation. The increase in eIF-6 gene expression in murine mast cells was blocked by therapeutic agents such as dexamethasone and cyclosporin A. To determine the location and function of eIF-6, murine mast cells were transfected with a construct that overexpressed enhanced green fluorescent protein-tagged eIF-6. These experiments demonstrated that eIF-6 was localized predominantly in the nucleolus of the mast cells. Also, overexpression of enhanced green fluorescent protein/eIF-6 enhanced the production of histamine and IL-2, but not IL-4 by stimulated murine mast cells. These results suggest that eIF-6 regulates the production of selected bioactive mediators in allergic diseases. This is the first demonstration of a biologic function of eIF-6 in mammalian cells.
Multiple Antigen-Specific Processing Pathways for Activating Naive CD8+ T Cells In Vivo
Authors: Norbury Christopher C. , Princiotta Michael F. , Bacik Igor , Brutkiewicz Randy R. , Wood Philip , Elliott Tim , Bennink Jack R. , Yewdell Jonathan W.
Published: April 2014

Current knowledge of the processing of viral Ags into MHC class I-associated ligands is based almost completely on in vitro studies using nonprofessional APCs (pAPCs). This is two steps removed from real immune responses to pathogens and vaccines, in which pAPCs activate naive CD8+ T cells in vivo. Rational vaccine design requires answers to numerous questions surrounding the function of pAPCs in vivo, including their abilities to process and present peptides derived from endogenous and exogenous viral Ags. In the present study, we characterize the in vivo dependence of Ag presentation on the expression of TAP by testing the immunogenicity of model Ags synthesized by recombinant vaccinia viruses in TAP1−/− mice. We show that the efficiency of TAP-independent presentation in vitro correlates with TAP-independent activation of naive T cells in vivo and provide the first in vivo evidence for proteolytic processing of antigenic peptides in the secretory pathway. There was, however, a clear exception to this correlation; although the presentation of the minimal SIINFEKL determinant from chicken egg OVA in vitro was strictly TAP dependent, it was presented in a TAP-independent manner in vivo. In vivo presentation of the same peptide from a fusion protein retained its TAP dependence. These results show that determinant-specific processing pathways exist in vivo for the generation of antiviral T cell responses. We present additional findings that point to cross-priming as the likely mechanism for these protein-specific differences.
p62dokNegatively Regulates CD2 Signaling in Jurkat Cells
Authors: Némorin Jean-Guy , Laporte Pierre , Bérubé Geneviève , Duplay Pascale
Published: April 2014

p62dok belongs to a newly identified family of adaptor proteins. In T cells, the two members that are predominantly expressed, p56dok and p62dok, are tyrosine phosphorylated upon CD2 or CD28 stimulation, but not upon CD3 ligation. Little is known about the biological role of Dok proteins in T cells. In this study, to evaluate the importance of p62dok in T cell function, we generated Jurkat clones overexpressing p62dok. Our results demonstrate that overexpression of p62dok in Jurkat cells has a dramatic negative effect on CD2-mediated signaling. The p62dok-mediated inhibition affects several biochemical events initiated by CD2 ligation, such as the increase of intracellular Ca2+, phospholipase Cγ1 activation, and extracellular signal-regulated kinase 1/2 activation. Importantly, these cellular events are not affected in the signaling cascade induced by engagement of the CD3/TCR complex. However, both CD3- and CD2-induced NF-AT activation and IL-2 secretion are impaired in p62dok-overexpressing cells. In addition, we show that CD2 but not CD3 stimulation induces p62dok and Ras GTPase-activating protein recruitment to the plasma membrane. These results suggest that p62dok plays a negative role at multiple steps in the CD2 signaling pathway. We propose that p62dok may represent an important negative regulator in the modulation of the response mediated by the TCR.
The NK Cell MHC Class I Receptor Ly49A Detects Mutations on H-2Dd Inside and Outside of the Peptide Binding Groove
Authors: Matsumoto Naoki , Yokoyama Wayne M. , Kojima Somei , Yamamoto Kazuo
Published: April 2014

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2Dd and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in α1/α2 domain of H-2Dd. Here we have substituted polymorphic as well as conserved residues of H-2Dd α1/α2 domain (when compared with H-2Kd, which does not interact with Ly49A). We then tested the ability of the H-2Dd mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2Dd residue into the corresponding H-2Kd residue (N30D, D77S, or A99F) in H-2Dd partially abrogated the interaction between Ly49A and H-2Dd. Introduction of the three mutations into H-2Dd completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2Dd that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2Dd reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: α1/α2, α3, and β2-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.
B Cell-Deficient (μMT) Mice Have Alterations in the Cytokine Microenvironment of the Gut-Associated Lymphoid Tissue (GALT) and a Defect in the Low Dose Mechanism of Oral Tolerance
Authors: Gonnella Patricia A. , Waldner Hans Peter , Weiner Howard L.
Published: April 2014

Peripheral immune tolerance following i.v. administration of Ag has been shown to occur in the absence of B cells. Because different mechanisms have been identified for i.v. vs low dose oral tolerance and B cells are a predominant component of the gut-associated lymphoid tissue (GALT) they may play a role in tolerance induction following oral Ag. To examine the role of B cells in oral tolerance we fed low doses of OVA or myelin oligodendrocyte glycoprotein to B cell-deficient (μMT) and wild-type C57BL/6 mice. Results showed that the GALT of naive wild-type and μMT mice was characterized by major differences in the cytokine microenvironment. Feeding low doses of 0.5 mg OVA or 250 μg myelin oligodendrocyte glycoprotein resulted in up-regulation of IL-4, IL-10, and TGF-β in the GALT of wild-type but not μMT mice. Upon stimulation of popliteal node cells, in vitro induction of regulatory cytokines TGF-β and IL-10 was observed in wild-type but not μMT mice. Greater protection against experimental autoimmune encephalomyelitis was found in wild-type mice. Oral tolerance in μMT and wild-type mice was found to proceed by different mechanisms. Anergy was observed from 0.5 mg to 250 ng in μMT mice but not in wild-type mice. Increased Ag was detected in the lymph of μMT mice. No cytokine-mediated suppression was found following lower doses from 100 ng to 500 pg in either group. These results demonstrate the importance of the B cell for the induction of cytokine-mediated suppression associated with low doses of Ag.
Spontaneous and Continuous Cyclooxygenase-2-Dependent Prostaglandin E2 Production by Stromal Cells in the Murine Small Intestine Lamina Propria: Directing the Tone of the Intestinal Immune Response
Authors: Newberry Rodney D. , McDonough Jacquelyn S. , Stenson William F. , Lorenz Robin G.
Published: April 2014

The mechanisms allowing the gastrointestinal immune system to avoid an inappropriate inflammatory response to nonpathogenic luminal Ags are poorly understood. We have previously described a role for cyclooxygenase (COX)-2-dependent arachidonic acid metabolites produced by the murine small intestine lamina propria in controlling the immune response to a dietary Ag. To better understand the role of COX-2-dependent arachidonic acid metabolites produced by the lamina propria, we examined the pattern of expression and the cellular source of COX-2 and COX-2-dependent PGE2. We now demonstrate that non-bone marrow-derived lamina propria stromal cells have basal COX-2 expression and that COX-2-dependent PGE2 production by these cells is spontaneous and continuous. The other mucosal and nonmucosal lymphoid compartments examined do not share this phenotype. In contrast to the majority of descriptions of COX-2 expression, COX-2 expression by lamina propria stromal cells is not dependent upon exogenous stimuli, including adhesion, LPS signaling via Toll-like receptor 4, or the proinflammatory cytokines TNF-α, IFN-γ, and IL-1β. These findings, in conjunction with the known immunomodulatory capacities of PGs, suggest that COX-2 expression by the small intestine lamina propria is a basal state contributing to the hyporesponsiveness of the intestinal immune response.
GIF Inhibits Th Effector Generation by Acting on Antigen-Presenting B Cells
Authors: Sugie Katsuji , Huang Jianyong
Published: April 2014

Glycosylation-inhibiting factor (GIF) is a 13-kDa cytokine secreted from T cells. Administration of bioactive recombinant GIF inhibits IgG1 and IgE Ab responses in vivo. Treatment of B cells with the cytokine reduces the secretion of IgG1 and IgE induced by LPS and IL-4. To examine the effect on cognate T-B interaction, GIF was added to low-density B cells from MD4 transgenic (Tg) mice, which express B cell receptor specific for hen egg lysozyme (HEL). The B cells were subsequently pulsed with HEL-OVA conjugate and cultured with OVA-specific naive CD4 T cells from DO11.10 Tg mice. Treatment of Ag-presenting B cells with GIF reduced expansion and IL-2 secretion of naive T cells and rendered them hyporesponsive to antigenic restimulation, resulting in 50–95% reduction of IL-4 and IFN-γ secretion upon restimulation with Ag. GIF dramatically inhibited Th effector generation when it was added to B cells before pulsing with HEL-OVA, whereas it showed little to no effect when added after B cells were pulsed with Ag. GIF was more effective when B cells from MD4 Tg mice were pulsed with HEL-OVA than when they were pulsed with OVA. This cytokine did not affect Th effector generation when B cells or irradiated splenocytes pulsed with OVA323–339 peptide stimulated naive DO11.10 T cells. Confocal microscopy revealed that GIF inhibited internalization of HEL by B cells from MD4 Tg mice. Therefore, the cytokine may regulate early steps of Ag presentation involving B cell receptors to diminish Th effector generation from naive CD4 T cells.
IFN-γ/TNF-α Synergism as the Final Effector in Autoimmune Diabetes: A Key Role for STAT1/IFN Regulatory Factor-1 Pathway in Pancreatic β Cell Death
Authors: Suk Kyoungho , Kim Sunshin , Kim Yun-Hee , Kim Kyoung-Ah , Chang Inik , Yagita Hideo , Shong Minho , Lee Myung-Shik
Published: April 2014

Fas ligand (FasL), perforin, TNF-α, IL-1, and NO have been considered as effector molecule(s) leading to β cell death in autoimmune diabetes. However, the real culprit(s) in β cell destruction have long been elusive, despite intense investigation. We and others have demonstrated that FasL is not a major effector molecule in autoimmune diabetes, and previous inability to transfer diabetes to Fas-deficient nonobese diabetic (NOD)-lpr mice was due to constitutive FasL expression on lymphocytes from these mice. Here, we identified IFN-γ/TNF-α synergism as the final effector molecules in autoimmune diabetes of NOD mice. A combination of IFN-γ and TNF-α, but neither cytokine alone, induced classical caspase-dependent apoptosis in insulinoma and pancreatic islet cells. IFN-γ treatment conferred susceptibility to TNF-α-induced apoptosis on otherwise resistant insulinoma cells by STAT1 activation followed by IFN regulatory factor (IRF)-1 induction. IRF-1 played a central role in IFN-γ/TNF-α-induced cytotoxicity because inhibition of IRF-1 induction by antisense oligonucleotides blocked IFN-γ/TNF-α-induced cytotoxicity, and transfection of IRF-1 rendered insulinoma cells susceptible to TNF-α-induced cytotoxicity. STAT1 and IRF-1 were expressed in pancreatic islets of diabetic NOD mice and colocalized with apoptotic cells. Moreover, anti-TNF-α Ab inhibited the development of diabetes after adoptive transfer. Taken together, our results indicate that IFN-γ/TNF-α synergism is responsible for autoimmune diabetes in vivo as well as β cell apoptosis in vitro and suggest a novel signal transduction in IFN-γ/TNF-α synergism that may have relevance in other autoimmune diseases and synergistic anti-tumor effects of the two cytokines.
Nonobese Diabetic Mice Display Elevated Levels of Class II-Associated Invariant Chain Peptide Associated with I-Ag7 on the Cell Surface
Authors: Bhatnagar Aparna , Milburn Peter J. , Lobigs Mario , Blanden Robert V. , Gautam Anand M.
Published: April 2014

Peptide presentation by MHC class II molecules plays a pivotal role in determining the peripheral T cell repertoire as a result of both positive and negative selection in the thymus. Homozygous I-Ag7 expression imparts susceptibility to autoimmune diabetes in the nonobese diabetic mouse, and recently, it has been proposed that this arises from ineffectual peptide binding. Following biosynthesis, class II molecules are complexed with class II-associated invariant chain peptides (CLIP), which remain associated until displaced by Ag-derived peptides. If I-Ag7 is a poor peptide binder, then this may result in continued occupation by CLIP to the point of translocation to the cell surface. To test this hypothesis we generated affinity-purified polyclonal antisera that recognized murine CLIP bound to class II molecules in an allele-independent fashion. We have found abnormally high natural levels of cell surface class II occupancy by CLIP on nonobese diabetic splenic B cells. Experiments using I-A-transfected M12.C3 cells showed that I-Ag7 alone was associated with elevated levels of CLIP, suggesting that this was determined solely by the amino acid sequence of the class II molecule. These results indicated that an intrinsic property of I-Ag7 would affect both the quantity and the repertoire of self-peptides presented during thymic selection.
STAT6 Mediates Eotaxin-1 Expression in IL-4 or TNF-α-Induced Fibroblasts
Authors: Hoeck Jutta , Woisetschläger Maximilian
Published: April 2014

Eosinophils are attracted to sites of allergic inflammation by a number of chemoattractants including eotaxin-1. This chemokine can be secreted from epithelial cells and fibroblasts after IL-4 and TNF-α stimulation in a synergistic fashion. TNF-α activated gene expression at the transcriptional level in a STAT6-dependent manner, because: 1) eotaxin-1 promoter luciferase constructs were TNF-α inducible in STAT6-defective HEK293 cells only on cotransfection of STAT6 expression vector, an effect that was partially mediated by activation-induced binding of NF-κB proteins to a composite STAT6/NF-κB element; 2) reporter constructs defective in STAT6 DNA binding did not respond to TNF-α stimulation; 3) eotaxin-1 protein secretion was detected only in STAT6-transfected HEK293 cell supernatants on TNF-α treatment; and 4) a trans-dominant negative STAT6 protein inhibited TNF-α-induced eotaxin-1 secretion in primary fibroblasts. TNF-α inducibility of the IL-8 and monocyte chemoattractant protein-1 genes was not dependent on STAT6 expression in the same experimental systems. The inducing effect of IL-4 and IL-13 was also mediated by STAT6. The synergistic effect of IL-4 and TNF-α observed at the RNA and the protein level was not seen at the promoter level. The data demonstrate that both IL-4 and TNF-α induce eotaxin-1 expression at the level of transcription via a STAT6-mediated pathway.
Cysteinylation of MHC Class II Ligands: Peptide Endocytosis and Reduction Within APC Influences T Cell Recognition
Authors: Haque M. Azizul , Hawes John W. , Blum Janice S.
Published: April 2014

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4+ T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.
Identification of a Novel Lipopolysaccharide-Inducible Gene with Key Features of Both a Kinase Anchor Proteins and chs1/beige Proteins
Authors: Wang Jia-Wang , Howson Julie , Haller Edward , Kerr William G.
Published: April 2014

Mutations in chs1/beige result in a deficiency in intracellular transport of vesicles that leads to a generalized immunodeficiency in mice and humans. The function of NK cells, CTL, and granulocytes is impaired by these mutations, indicating that polarized trafficking of vesicles is controlled by CHS1/beige proteins. However, a molecular explanation for this defect has not been identified. Here we describe a novel gene with orthologues in mice, humans, and flies that contains key features of both chs1/beige and A kinase anchor genes. We designate this novel gene lba for LPS-responsive, beige-like anchor gene. Expression of lba is induced after LPS stimulation of B cells and macrophages. In addition, lba is expressed in many other tissues in the body and has three distinct mRNA isoforms that are differentially expressed in various tissues. Strikingly, LBA-green-fluorescent protein (GFP) fusion proteins are localized to vesicles after LPS stimulation. Confocal microscopy indicates this protein is colocalized with the trans-Golgi complex and some lysosomes. Further analysis by immunoelectron microscopy demonstrates that LBA-GFP fusion protein can localize to endoplasmic reticulum, plasma membrane, and endocytosis vesicles in addition to the trans-Golgi complex and lysosomes. We hypothesize that LBA/CHS1/BG proteins function in polarized vesicle trafficking by guiding intracellular vesicles to activated receptor complexes and thus facilitate polarized secretion and/or membrane deposition of immune effector molecules.
Diversity of Epitope and Cytokine Profiles for Primary and Secondary Influenza A Virus-Specific CD8+ T Cell Responses
Authors: Belz Gabrielle T. , Xie Weidong , Doherty Peter C.
Published: April 2014

Screening with the flow cytometric IFN-γ assay has led to the identification of a new immunogenic peptide (SSYRRVPGI) from the influenza PB1 polymerase (PB1703–711) and a mimotope (ISPLMVAYM) from the PB2 polymerase (PB2198–206). CD8+ T cells specific for KbPB1703 make both IFN-γ and TNF-α following stimulation with both peptides. The CD8+ KbPB1703+ population kills PB2198-pulsed targets, but cell lines stimulated with PB2198 neither bind the KbPB1703 tetramer nor become CTL. This CD8+KbPB1703+ population is prominent in the primary response to an H3N2 virus, although it is much less obvious following secondary challenge of H1N1-primed mice. Even so, we can now account for >40% of the CD8+ T cells in a primary influenza pneumonia and >85% of those present after H3N2 → H1N1 challenge. Profiles of IFN-γ and TNF-α staining following in vitro stimulation have been traced for the four most prominent influenza peptides through primary and secondary responses into long-term memory. The DbNP366 epitope that is immunodominant after the H3N2 → H1N1 challenge shows the lowest frequencies of CD8+ IFN-γ+TNF-α+ cells for >6 wk, and the intensity of IFN-γ staining is also low for the first 3 wk. By 11 wk, however, the IFN-γ/TNF-α profiles look to be similar for all four epitopes. At least by the criterion of cytokine production, there is considerable epitope-related functional diversity in the influenza virus-specific CD8+ T cell response. The results for the KbPB1703 epitope and the PB2198 mimotope also provide a cautionary tale for those using the cytokine staining approach to identity antigenic peptides.
An Increase in Circulating Mast Cell Colony-Forming Cells in Asthma
Authors: Mwamtemi Hadija Hemed , Koike Kenichi , Kinoshita Tatsuya , Ito Susumu , Ishida Shuichi , Nakazawa Yozo , Kurokawa Yumi , Shinozaki Koji , Sakashita Kazuo , Takeuchi Kouichi , Shiohara Masaaki , Kamijo Takehiko , Yasui Yozo , Ishiguro Akira , Kawano Yoshifumi , Kitano Kiyoshi , Miyazaki Hiroshi , Kato Takashi , Sakuma Shozo , Komiyama Atsushi
Published: April 2014

We compared a potential to generate mast cells among various sources of CD34+ peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34+ PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34+ PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34+ PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34+ PB cells between the two groups. In the presence of TPO, CD34+ PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34+ PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.
Macrophages Transfected with Adenovirus to Express IL-4 Reduce Inflammation in Experimental Glomerulonephritis
Authors: Kluth David C. , Ainslie Clare V. , Pearce Wayne P. , Finlay Sian , Clarke Daniel , Anegon Ignacio , Rees Andrew J.
Published: April 2014

Nephrotoxic nephritis (NTN) is characterized by acute macrophage-dependent inflammation and serves as a model of human glomerulonephritis. In this study we have transfected rat macrophages with recombinant adenovirus expressing IL-4 (Ad-IL4) and demonstrated that these transfected macrophages develop fixed properties as a result of transfection, as shown by reduced NO production in response to IFN-γ and TNF. Ad-IL4-transfected macrophages localized with enhanced efficiency to inflamed glomeruli after renal artery injection in rats with NTN compared with adenovirus expressing β-galactosidase (Ad-βgal)-transfected macrophages and produced elevated levels of the cytokine in glomeruli in vivo for up to 4 days. The delivery of IL-4-expressing macrophages produced a marked reduction in the severity of albuminuria (day 2 albuminuria, 61 ± 15 mg/24 h) compared with unmodified NTN (day 2 albuminuria, 286 ± 40 mg/24 h; p < 0.01), and this was matched by a reduction in the number of ED1-positive macrophages infiltrating the glomeruli. Interestingly, the injection of IL-4-expressing macrophages into single kidney produced a marked reduction in the numbers of ED1-positive macrophages in the contralateral noninjected kidney, an effect that could not be mimicked by systemic delivery of IL-4-expressing macrophages. This implies that the presence of IL-4-expressing macrophages in a single kidney can alter the systemic development of the inflammatory response. Macrophage transfection and delivery provide a valuable system to study and modulate inflammatory disease and highlight the feasibility of macrophage-based gene therapy.
Nonphlogistic Clearance of Late Apoptotic Neutrophils by Macrophages: Efficient Phagocytosis Independent of β2 Integrins
Authors: Ren Yi , Stuart Lynda , Lindberg Frederik P. , Rosenkranz Alexander R. , Chen Yongmei , Mayadas Tanya N. , Savill John
Published: April 2014

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-α. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the αvβ3 vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from αm−/− or β2−/− mice were used. We conclude that specific nonphlogistic, β2 integrin-independent mechanisms involving thrombospondin-1 and αvβ3 allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.
Specific Immunotherapy by Genetically Engineered APCs: The “Guided Missile” Strategy
Authors: Wu Bo , Wu Jian-Ming , Miagkov Alexei , Adams Robert N. , Levitsky Hyam I. , Drachman Daniel B.
Published: April 2014

We tested the hypothesis that APCs genetically engineered to present an Ag and to express Fas ligand (FasL) simultaneously can target and eliminate Ag-specific T cells. Transgenic T cells specific for influenza hemagglutinin (HA) were used as targets. We prepared recombinant vaccinia virus vectors (VVV) to transfer the gene constructs individually or simultaneously into APCs. We prevented unwanted viral replication by attenuating the VVVs with psoralen-UV light treatment. For presentation of the HA Ag, APCs were transduced with cDNA for HA flanked by sequences of the lysosome-associated membrane protein that direct efficient processing and presentation of the Ag by APCs. As a “warhead” for the APCs, we transduced them with the gene for FasL, which induces apoptosis of Fas-expressing activated T cells. To protect the transduced APCs from self-destruction by FasL, we transferred cDNA for a truncated form of Fas-associated death domain, which inhibits Fas-mediated cell death. Our results show that the engineered APCs effectively expressed the genes of interest. APCs transduced with VVV carrying all three gene constructs specifically killed HA-transgenic T cells in culture. Coculture with T cells specific for an unrelated Ag (OVA) had no significant effect. Our in vitro findings show that APCs can be genetically engineered to target and kill Ag-specific T cells and represent a promising novel strategy for the specific treatment of autoimmune diseases.
Reactivity of Anti-Proliferating Cell Nuclear Antigen (PCNA) Murine Monoclonal Antibodies and Human Autoantibodies to the PCNA Multiprotein Complexes Involved in Cell Proliferation
Authors: Takasaki Yoshinari , Kogure Toshiaki , Takeuchi Ken , Kaneda Kazuhiko , Yano Tetsuro , Hirokawa Kaoru , Hirose Sachiko , Shirai Toshikazu , Hashimoto Hiroshi
Published: April 2014

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the “protein machinery” for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.
Cutting Edge: Evidence for a Signaling Partnership Between Urokinase Receptors (CD87) and L-Selectin (CD62L) in Human Polymorphonuclear Neutrophils
Authors: Sitrin Robert G. , Pan Pauline M. , Blackwood R. Alexander , Huang Jibiao , Petty Howard R.
Published: April 2014

Leukocyte urokinase plasminogen activator receptors (uPARs) cluster at adhesion interfaces and at migratory fronts where they participate in adhesion, chemotaxis, and proteolysis. uPAR aggregation triggers activation signaling even though this glycolipid-anchored protein must associate with membrane-spanning proteins to access the cell interior. This study demonstrates a novel partnership between uPAR and L-selectin in human polymorphonuclear neutrophils. Fluorescence resonance energy transfer demonstrated a direct physical association between uPAR and L-selectin. To examine the role of L-selectin in uPAR-mediated signaling, uPAR was cross-linked and intracellular Ca2+ concentrations were measured by spectrofluorometry. A mAb reactive against the carbohydrate binding domain (CBD) of L-selectin substantially inhibited uPAR-mediated Ca2+ mobilization, whereas mAbs against the β2 integrin complement receptor 3 (CR3), another uPAR-binding adhesion protein, had no effect. Similarly, fucoidan, a sulfated polysaccharide that binds to L-selectin CBD, inhibited the Ca2+ signal. We conclude that uPAR associates with the CBD region of L-selectin to form a functional signaling complex.
Suppression of IgE Responses in CD23-Transgenic Animals Is Due to Expression of CD23 on Nonlymphoid Cells
Authors: Payet-Jamroz Margaret , Helm Shirley L. T. , Wu Jiuhua , Kilmon Michelle , Fakher Mohamed , Basalp Aynur , Tew John G. , Szakal Andras K. , Noben-Trauth Nancy , Conrad Daniel H.
Published: April 2014

Serum IgE is suppressed in CD23-transgenic (Tg) mice where B cells and some T cells express high levels of CD23, suggesting that CD23 on B and T cells may cause this suppression. However, when Tg B lymphocytes were compared with controls in B cell proliferation and IgE synthesis assays, the two were indistinguishable. Similarly, studies of lymphokine production suggested that T cell function in the Tg animals was normal. However, adoptive transfer studies indicated that suppression was seen when normal lymphocytes were used to reconstitute Tg mice, whereas reconstitution of controls with Tg lymphocytes resulted in normal IgE responses, suggesting that critical CD23-bearing cells are irradiation-resistant, nonlymphoid cells. Follicular dendritic cells (FDC) are irradiation resistant, express surface CD23, and deliver iccosomal Ag to B cells, prompting us to reason that Tg FDC may be a critical cell. High levels of transgene expression were observed in germinal centers rich in FDC and B cells, and IgE production was inhibited when Tg FDCs were cultured with normal B cells. In short, suppressed IgE production in CD23-Tg mice appears to be associated with a population of radioresistant nonlymphoid cells. FDCs that interface with B cells in the germinal center are a candidate for explaining this CD23-mediated IgE suppression.
Coregulation of CXC Chemokine Receptor and CD4 Expression on T Lymphocytes During Allogeneic Activation
Authors: Ebert Lisa M. , McColl Shaun R.
Published: April 2014

Upon activation, naive T cells alter their migratory patterns, acquiring the ability to move through peripheral tissues as well as the general lymphoid circulation. Although the mechanisms responsible for these alterations are not well understood, changes in chemokine receptor expression may play a critical role. To investigate these changes, the expression patterns of two chemokine receptors, CXCR3 and CXCR4, were compared on CD4+ T cells following activation in the MLR. By day 9 of activation, expression of the inflammatory chemokine receptor CXCR3 was up-regulated, while expression of the homeostatic chemokine receptor CXCR4 was down-regulated. Alterations in receptor expression occurred almost exclusively on a subpopulation of T cells that expressed higher levels of CD4. These CD4high T cells demonstrated many characteristics of activated T cells and had undergone division in the MLR. By day 9 of culture, the majority of CXCR3+ and CXCR4− cells had divided and had acquired an activated/memory phenotype (CD45RA− CD45RO+ CD69+ CD25+). The levels of transcripts for both CXCR3 and CXCR4 were increased upon allo-activation. The discrepancy between levels of CXCR4 mRNA and surface protein was not due to sequestration of the receptor in intracellular compartments, as CXCR4 was not detectable intracellularly. However, intracellular CXCR3 was readily detectable. Finally, cells from allogeneic cultures demonstrated enhanced migration toward IFN-inducible T cell α chemoattractant and reduced migration toward stromal cell-derived factor-1 compared with syngeneic controls, thus suggesting that the observed switch in receptor expression may at least partly contribute to the differential patterns of migration displayed by naive and memory T cells.
CD80 Costimulation Is Required for Th2 Cell Cytokine Production But Not for Antigen-Specific Accumulation and Migration into the Lung
Authors: Harris Nicola L. , Prout Melanie , Peach Robert J. , Fazekas de St. Groth Barbara , Ronchese Franca
Published: April 2014

The CD28 ligands CD80 and CD86 are expressed on APC, and both provide costimulatory function. However, the reason for the expression of two separate CD28 ligands remains unclear. We have previously shown that blockade of CD80 costimulation by Y100F-Ig, a CTL-associated Ag-4 (CTLA4)-Ig mutant that does not bind CD86, inhibits the development of lung inflammatory immune responses, but does not affect blood eosinophilia or Ab production. Each of those responses was inhibited by treatment with CTLA4-Ig, which binds both CD80 and CD86. To clarify the mechanism underlying these observations we have developed a model of lung inflammation using adoptively transferred CD4+ T cells expressing a Vα11+Vβ3+ transgenic TCR specific for I-Ek and moth cytochrome c. Treatment with Y100F-Ig inhibited the induction of lung eosinophilia in adoptively transferred mice. However, Y100F-Ig did not detectably affect the accumulation of Ag-specific T cells at the site of peptide deposit or in the draining lymphoid tissues. Acquisition of an activated phenotype and expression of adhesion molecules required for migration into the lung were modestly affected. Importantly, treatment with Y100F-Ig diminished the ability of T cells to produce the cytokines IL-4 and IL-5 following intranasal challenge with Ag. All the responses examined were severely inhibited by treatment with CTLA4-Ig. We conclude that T cells require CD80 costimulation for the optimal production of IL-5 following intranasal administration of Ag. Decreased IL-5 production is the most likely explanation for the diminished airway eosinophilia observed.
Modulation of Inhaled Antigen-Induced IgE Tolerance by Ongoing Th2 Responses in the Lung
Authors: Hurst Stephen D. , Seymour Brian W. P. , Muchamuel Tony , Kurup Viswanath P. , Coffman Robert L.
Published: April 2014

The normal response to inhaled Ag is the absence of Ag-specific IgE and cytokine production to later Ag challenges. Although the mechanism of this aerosol-induced IgE tolerance is not completely understood, it may prevent sensitization to inhaled Ags, which could otherwise lead to allergy and asthma. We examined the consequences of ongoing Th1 and Th2 responses in the lungs of mice during OVA inhalation to mimic conditions that may subvert tolerance and lead to sensitization. We found that concurrent, secondary Th2 lung responses to keyhole limpet hemocyanin or primary responses to Nippostrongylus larvae or Asperigillus fumagatus extract prevented establishment of IgE tolerance to aerosolized OVA. Intranasal rIL-4 given before OVA aerosolization also prevented establishment of tolerance, whereas concurrent Th1 responses to influenza virus or Mycobacterium bovis bacillus Calmette-Guérin had no effect. However, once established, aerosol tolerance to OVA could not be completely broken by OVA rechallenge concurrent with a secondary Th2 response to keyhole limpet hemocyanin or A. fumagatus extract, or by intranasal rIL-4. These data suggest that the immune status of the lung of an individual may profoundly influence the initial response to inhaled Ag, and that aerosol-induced IgE tolerance may not be appropriately established in individuals undergoing concurrent, Th2-mediated responses to Ags or pathogens.
Migration and Maturation of Human Colonic Dendritic Cells
Authors: Bell Sally J. , Rigby Rachael , English Nicholas , Mann Steven D. , Knight Stella C. , Kamm Michael A. , Stagg Andrew J.
Published: April 2014

Dendritic cells (DC) in the colon may regulate intestinal immunity but remain poorly characterized. In this study a CD11c+HLA-DR+lin− (CD3−CD14−CD16−CD19−CD34−) population has been identified by flow cytometry in cells obtained by rapid collagenase digestion of human colonic and rectal biopsies. These day 0 (d0) CD11c+HLA-DR+lin− cells comprised ∼0.6% of the mononuclear cells obtained from the lamina propria, were endocytically active, and had the phenotype of immature DC; they were CD40+ and expressed low levels of CD83 and CD86, but little or no CD80 or CD25. Similar d0 DC populations were isolated from the colonic mucosa of healthy controls and from both inflamed and noninflamed tissue from patients with Crohn’s disease. The lamina propria also contained a population of cells capable of migrating out of biopsies during an overnight culture and differentiating into mature DC with lower levels of endocytic activity and high cell surface expression of CD40, CD80, CD86, CD83, and CD25. This mature DC population was a potent stimulator of an allogeneic mixed leukocyte (MLR). Overnight culture of cells isolated by enzymatic digestion on d0 yielded DC with a phenotype intermediate between that of the d0 cells and that of the cells migrating out overnight. Overnight culture of colonic cells in which DC and HLA-DR+lin+ cells were differentially labeled with FITC-dextran suggested that some of the maturing DC might differentiate from HLA-DR+lin+ progenitors. This study presents the first analysis of the phenotype, maturational status, and migratory activity of human gut DC.
Impaired Fas Signaling Pathway Is Involved in Defective T Cell Apoptosis in Autoimmune Murine Arthritis
Authors: Zhang Jian , Bárdos Tamás , Mikecz Katalin , Finnegan Alison , Glant Tibor T.
Published: April 2014

Proteoglycan (PG)-induced arthritis (PGIA) is a novel autoimmune murine model for rheumatoid arthritis induced by immunization with cartilage PG in susceptible BALB/c mice. In this model, hyperproliferation of peripheral CD4+ T cells has been observed in vitro with Ag stimulation, suggesting the breakdown of peripheral tolerance. Activation-induced cell death (AICD) is a major mechanism for peripheral T cell tolerance. A defect in AICD may result in autoimmunity. We report in this study that although CD4+ T cells from both BALB/c and B6 mice, identically immunized with human cartilage PG or OVA, express equally high levels of Fas at the cell surface, CD4+ T cells from human cartilage PG-immunized BALB/c mice, which develop arthritis, fail to undergo AICD. This defect in AICD in PGIA may lead to the accumulation of autoreactive Th1 cells in the periphery. The impaired AICD in PGIA might be ascribed to an aberrant expression of Fas-like IL-1β-converting enzyme-inhibitory protein, which precludes caspase-8 activation at the death-inducing signaling complex, and subsequently suppresses the caspase cascade initiated by Fas-Fas ligand interaction. Moreover, this aberrant expression of Fas-like IL-1β-converting enzyme-inhibitory protein may also mediate TCR-induced hyperproliferation of CD4+ T cells from arthritic BALB/c mice. Our data provide the first insight into the molecular mechanism(s) of defective AICD in autoimmune arthritis.