bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammatory Response and Glutathione Peroxidase in a Model of Stroke
Authors: Ishibashi Nobuya , Prokopenko Olga , Reuhl Kenneth R. , Mirochnitchenko Oleg
Published: April 2014

Stroke is one of the leading causes of death in major industrial countries. Many factors contribute to the cellular damage resulting from ischemia/reperfusion (I/R). Experimental data indicate an important role for oxidative stress and the inflammatory cascade during I/R. We are testing the hypothesis that the mechanism of protection against I/R damage observed in transgenic mice overexpressing human antioxidant enzymes (particularly intracellular glutathione peroxidase) involves the modulation of inflammatory response as well as reduced sensitivity of neurons to cytotoxic cytokines. Transgenic animals show significant reduction of expression of chemokines, IL-6, and cell death-inducing ligands as well as corresponding receptors in a focal cerebral I/R model. Reduction of DNA binding activity of consensus and potential AP-1 binding sites in mouse Fas ligand promoter sequence was observed in nuclear extracts from transgenic mice overexpressing intracellular glutathione peroxidase compared with normal animals following I/R. This effect was accompanied by modulation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway. Cultured primary neurons from the transgenic mice demonstrated protection against hypoxia/reoxygenation injury as well as cytotoxicity after TNF-α and Fas ligand treatment. These results indicate that glutathione peroxidase-sensitive reactive oxygen species play an important role in regulation of cell death during cerebral I/R by modulating intrinsic neuronal sensitivity as well as brain inflammatory reactions.
The Role of E-Selectin, P-Selectin, and Very Late Activation Antigen-4 in T Lymphocyte Migration to Dermal Inflammation
Authors: Issekutz Andrew C. , Issekutz Thomas B.
Published: April 2014

T lymphocyte infiltration into inflamed tissues is thought to involve lymphocyte rolling on vascular endothelial cells. Because both selectin and α4 integrin adhesion molecules can mediate leukocyte rolling, the contribution of these receptors to lymphocyte migration to inflammation was examined. The recruitment of 111In-labeled spleen T cells to intradermal sites injected with IFN-γ, TNF-α, LPS, poly inosine-cytosine, and Con A was measured in the rat, and the effect of blocking mAbs to E-selectin, P-selectin, very late activation Ag-4 (VLA-4), and LFA-1 was determined on this T cell migration in vivo. Anti-E-selectin and anti-P-selectin mAbs each inhibited 10–40 and 20–48%, respectively, of the T lymphocyte migration to the inflammatory sites, depending on the stimulus. Blocking VLA-4 inhibited 50% of the migration to all of the lesions except Con A. Treatment with both anti-VLA-4 and anti-E-selectin mAbs inhibited up to 85% of the lymphocyte accumulation, while P-selectin and VLA-4 blockade in combination was not more effective than VLA-4 blockade alone in TNF-α, IFN-γ, LPS, and poly inosine-cytosine lesions. Inhibiting E-selectin, P-selectin, and VLA-4 together nearly abolished lymphocyte migration to all inflammatory sites. Anti-LFA-1 mAb strongly inhibited lymphocyte accumulation by itself, and this inhibition was not significantly further reduced by E- or P-selectin blockade. Thus, T cell migration to dermal inflammation is dependent on E-selectin, P-selectin, and VLA-4, likely because these three receptors are required for rolling of memory T lymphocytes, but VLA-4 and E-selectin are especially important for lymphocyte infiltration in these tissues.
Lyn Tyrosine Kinase Is Important for IL-5-Stimulated Eosinophil Differentiation
Authors: Stafford Susan , Lowell Clifford , Sur Sanjiv , Alam Rafeul
Published: April 2014

IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5Rα is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5Rα in eosinophils. To identify IL-5Rα-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5Rα under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5Rα. Lyn kinase phosphorylates both IL-5Rα and βcR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn−/− mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5Rα signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.
Autocrine Induction of the Human Pro-IL-1β Gene Promoter by IL-1β in Monocytes
Authors: Toda Yoko , Tsukada Junichi , Misago Masahiro , Kominato Yoshihiko , Auron Philip E. , Tanaka Yoshiya
Published: April 2014

IL-1β is produced primarily by activated monocytes/macrophages. We report in this study that IL-1β induces the human pro-IL-1β (IL1B) gene promoter in human THP-1 monocytic cells. The −131 to +12 minimal IL1B promoter was induced by IL-1β in a dose-dependent manner. The promoter possesses two important transcription factor binding motifs, one for an ETS family transcription factor Spi-1 (PU.1), and the other a binding site for NF-IL6 (CCAAT/enhancer binding protein β). Autocrine promoter activity was completely inhibited by mutation of the Spi-1 site. Mutation of the NF-IL6 binding motif caused partial loss of activity. EMSAs using THP-1 cell nuclear extracts indicated that IL-1β significantly induced Spi-1 binding to its target site within the IL1B promoter that was maximal at 1 h after stimulation, correlating with the kinetics of IL-1β induction. The importance of Spi-1 was supported by our observation that Spi-1-deficient EL4 thymocytes exhibited IL-1β-induced activity only after transfection with a Spi-1 expression vector. Moreover, TNFR-associated factor 6 also required Spi-1 to activate the promoter. Transfection studies using Spi-1 mutant constructs showed that the TATA-binding protein binding and glutamine-rich domains of Spi-1 were important for IL-1β induction, whereas LPS induction required the proline, glutamic acid, serine, and threonine-rich domain containing serine 148 as well as the TATA-binding protein and glutamine-rich domains. We conclude that the IL1B promoter is an IL-1β-responsive sequence as a result of its ability to bind Spi-1 in response to IL-1β.
Functional Genomic Analysis of Type II IL-1β Decoy Receptor: Potential for Gene Therapy in Human Arthritis and Inflammation
Authors: Attur Mukundan G. , Dave Mandar N. , Leung Mary Y. , Cipolletta Christine , Meseck Marcia , Woo Savio L. C. , Amin Ashok R.
Published: April 2014

Gene expression arrays show that human epithelial cells and human arthritis-affected cartilage lack detectable amounts of mRNA for IL-1 antagonizing molecules: IL-1Ra and IL-1RII, but constitutively express IL-1. Functional genomic analysis was performed by reconstituting human IL-1RII expression in various IL-1RII-deficient cell types to examine its antagonist role using gene therapy approaches. Adenovirus-expressing IL-1RII when transduced into human and bovine chondrocytes, human and rabbit synovial cells, human epithelial cells, and rodent fibroblasts expressed membrane IL-1RII and spontaneously released functional soluble IL-1RII. The IL-1RII+ (but not IL-1RII−) cells were resistant to IL-1β-induced, NO, PGE2, IL-6, and IL-8 production or decreased proteoglycan synthesis. IL-1RII inhibited the function of IL-1 in chondrocytes and IL-1- and TNF-α-induced inflammatory mediators in human synovial and epithelial cells. IL-1RII+ chondrocytes were more resistant to induction of NO and PGE2 by IL-1β compared with IL-1RII− cells incubated with a 10-fold (weight) excess of soluble type II IL-1R (sIL-1RII) protein. In cocultures, IL-1RII+ synovial cells released sIL-1RII, which in a paracrine fashion protected chondrocytes from the effects of IL-1β. Furthermore, IL-1RII+ (but not IL-1RII−) chondrocytes when transplanted onto human osteoarthritis-affected cartilage in vitro, which showed spontaneous release of sIL-1RII for 20 days, inhibited the spontaneous production of NO and PGE2 in cartilage in ex vivo. In summary, reconstitution of IL-1RII in IL-1RII− cells using gene therapy approaches significantly protects cells against the autocrine and paracrine effects of IL-1 at the signaling and transcriptional levels.
G-CSF Therapy of Ongoing Experimental Allergic Encephalomyelitis Via Chemokine- and Cytokine-Based Immune Deviation
Authors: Zavala Flora , Abad Sébastien , Ezine Sophie , Taupin Véronique , Masson Annie , Bach Jean-François
Published: April 2014

Converging evidence that G-CSF, the hemopoietic growth factor of the myeloid lineage, also exerts anti-inflammatory and pro-Th2 effects, prompted us to evaluate its direct therapeutic potential in autoimmune diseases. Here we report a novel activity of G-CSF in experimental allergic encephalomyelitis, a murine model for multiple sclerosis, driven by Th1-oriented autoaggressive cells. A short 7-day treatment with G-CSF, initiated at the onset of clinical signs, provided durable protection from experimental autoimmune encephalomyelitis. G-CSF-treated mice displayed limited demyelination, reduced recruitment of T cells to the CNS, and very discrete autoimmune inflammation, as well as barely detectable CNS mRNA levels of cytokines and chemokines. In the periphery, G-CSF treatment triggered an imbalance in the production by macrophages as well as autoreactive splenocytes of macrophage inflammatory protein-1α and monocyte chemoattractant protein-1, the prototypical pro-Th1 and pro-Th2 CC chemokines, respectively. This chemokine imbalance was associated with an immune deviation of the autoreactive response, with reduced IFN-γ and increased IL-4 and TGF-β1 levels. Moreover, G-CSF limited the production of TNF-α, a cytokine also associated with early CNS infiltration and neurological deficit. These findings support the potential application of G-CSF in the treatment of human autoimmune diseases such as multiple sclerosis, taking advantage of the wide clinical favorable experience with this molecule.
Mechanism of Action of Combined Short-Term CTLA4Ig and Anti-CD40 Ligand in Murine Systemic Lupus Erythematosus
Authors: Wang Xiaobo , Huang Weiqing , Mihara Masahiko , Sinha Jayashree , Davidson Anne
Published: April 2014

Short-term combination therapy with the costimulatory antagonists CTLA4Ig and anti-CD40 ligand induces prolonged suppression of disease onset in New Zealand Black/New Zealand White F1 systemic lupus erythematosus-prone mice. To determine the mechanism for this effect, 20- to 22-wk-old New Zealand Black/New Zealand White F1 mice were treated with six doses each of CTLA4Ig and anti-CD40 ligand Ab over 2 wk. Combination-treated mice, but not mice treated with either agent alone, had prolonged survival and the production of pathogenic IgG anti-dsDNA Ab was suppressed. Twenty weeks after completion of treatment the frequency of activated B cells producing anti-dsDNA Ab was decreased, and the abnormal transition of T cells from the naive to the memory compartment was blocked. Combination treatment partially suppressed class switching and decreased the frequency of somatic mutations in the VHBW-16 gene, which is expressed by pathogenic anti-DNA Abs. Treated mice were still able to respond to the hapten oxazolone when it was given 8 wk after treatment initiation, and they mounted a somatically mutated IgG anti-oxazolone response that was noncross-reactive with dsDNA. Fifty to 60% of previously treated mice, but only 14% of previously untreated mice, responded within 2–3 wk to a second course of therapy given at the onset of fixed proteinuria and remained well for a further 3–4 mo. Although this treatment had no immediate effect on serum anti-dsDNA Abs or on the abnormal T cell activation observed in sick mice, 25% of treated mice lived for >18 mo compared with 5% of untreated controls. These results suggest that the effect of costimulatory blockade in remission induction must be mediated by a different mechanism than is demonstrated in the disease prevention studies.
Cutting Edge: Identification of c-Rel-Dependent and -Independent Pathways of IL-12 Production During Infectious and Inflammatory Stimuli
Authors: Mason Nicola , Aliberti Julio , Caamano Jorge C. , Liou Hsiou-Chi , Hunter Christopher A.
Published: April 2014

The production of IL-12 is required for immunity to many intracellular pathogens. Recent studies have shown that c-Rel, a member of the NF-κB family of transcription factors, is essential for LPS-induced IL-12p40 production by macrophages. In this study, we demonstrate that c-Rel is also required for IL-12p40 production by macrophages in response to Corynebacterium parvum, CpG oligodeoxynucleotides, anti-CD40 and low molecular weight hyaluronic acid. However, c-Rel−/− mice infected with Toxoplasma gondii produce comparable amounts of IL-12p40 to infected wild-type mice and have an IL-12-dependent mechanism of resistance to this infection. Furthermore, c-Rel was not required for IL-12p40 production by macrophages or dendritic cells in response to soluble Toxoplasma Ag, and neutrophils from c-Rel−/− mice contain normal amounts of preformed IL-12p40. Together these studies reveal the presence of c-Rel-dependent pathways critical for IL-12p40 production in response to inflammatory stimuli and demonstrate a novel c-Rel-independent pathway of IL-12p40 production during toxoplasmosis.
A Role for Cathepsin L and Cathepsin S in Peptide Generation for MHC Class II Presentation
Authors: Hsieh Chyi-Song , deRoos Paul , Honey Karen , Beers Courtney , Rudensky Alexander Y.
Published: April 2014

The enzymes that degrade proteins to peptides for presentation on MHC class II molecules are poorly understood. The cysteinal lysosomal proteases, cathepsin L (CL) and cathepsin S (CS), have been shown to process invariant chain, thereby facilitating MHC class II maturation. However, their role in Ag processing is not established. To examine this issue, we generated embryonic fibroblast lines that express CL, CS, or neither. Expression of CL or CS mediates efficient degradation of invariant chain as expected. Ag presentation was evaluated using T cell hybridoma assays as well as mass spectroscopic analysis of peptides eluted from MHC class II molecules. Interestingly, we found that the majority of peptides are presented regardless of CL or CS expression, although these proteases often alter the relative levels of the peptides. However, for a subset of Ags, epitope generation is critically regulated by CL or CS. This result suggests that these cysteinal proteases participate in Ag processing and generate qualitative and quantitative differences in the peptide repertoires displayed by MHC class II molecules.
L-Selectin Is Not Required for T Cell-Mediated Autoimmune Diabetes
Authors: Friedline Randall H. , Wong Carmen P. , Steeber Douglas A. , Tedder Thomas F. , Tisch Roland
Published: April 2014

Administration of anti-L-selectin (CD62L) mAb to neonatal nonobese diabetic (NOD) mice mediates long term protection against the development of insulitis and overt diabetes. These results suggested that CD62L has a key role in the general function of β cell-specific T cells. To further examine the role of CD62L in the development of type 1 diabetes, NOD mice lacking CD62L were established. The onset and frequency of overt diabetes were equivalent among CD62L+/+, CD62L+/−, and CD62L−/− NOD littermates. Furthermore, patterns of T cell activation, migration, and β cell-specific reactivity were similar in NOD mice of all three genotypes. Adoptive transfer experiments with CD62L−/− CD4+ T cells prepared from BDC2.5 TCR transgenic mice revealed no apparent defects in migration to pancreatic lymph nodes, proliferation in response to β cell Ag, or induction of diabetes in NOD.scid recipients. In conclusion, CD62L expression is not essential for the development of type 1 diabetes in NOD mice.
Differential In Vivo Persistence of Two Subsets of Memory Phenotype CD8 T Cells Defined by CD44 and CD122 Expression Levels
Authors: Walzer Thierry , Arpin Christophe , Beloeil Laurent , Marvel Jacqueline
Published: April 2014

The existence of distinct subsets of memory CD8 T cells with different characteristics is now well established. In this work, we describe two subsets of mouse CD8 T cells with memory characteristics that coexist in primed thymectomized TCR-transgenic F5 mice and that share some properties with the human central and effector memory cells. The first subset corresponds to CD8 T cells generated following nucleoprotein 68 peptide priming which are CD44intCD122−nucleoprotein 68/H-2Db tetramer+ and express high levels of CCR7 mRNA. In contrast, CD8 T cells in the second subset are CD44highCD122+, are heterogeneous in terms of Ag specificity, and express low levels of CCR7 mRNA. We have studied the functional characteristics and the persistence of these two subsets in thymectomized mice. CD44int CD8 T cells persist like naive cells; i.e., they are slowly lost with time. However, surviving cells maintain their phenotype and memory characteristics for the entire life span of the animal. In contrast, CD44high CD8 T cells are persistent and accumulate in thymectomized but not euthymic mice. This is correlated with an increased in vivo proliferative and survival potential of these cells. These results show that acquisition of enhanced functional characteristics and long-term persistence by memory T cells are independent. This may have important consequences for the design of specific vaccine.
T Cell Immunity to Lymphoma Following Treatment with Anti-CD40 Monoclonal Antibody
Authors: Tutt Alison L. , O’Brien Lyn , Hussain Akmal , Crowther Graham R. , French Ruth R. , Glennie Martin J.
Published: April 2014

In this study we demonstrate that treatment with anti-CD40 mAb eradicates a range of mouse lymphomas (BCL1, A31, A20, and EL4), but only when used against i.v. tumor doses in excess of 107 cells. Only partial protection was seen against smaller tumor loads. We saw no evidence that anti-CD40 mAb changed the phenotype of the lymphomas or inhibited their growth in the initial period following treatment, but it did result in a rapid expansion of cytotoxic CD8+ cells that was able to clear the neoplastic disease and provide long-term protection against tumor rechallenge. The CTL responses were blocked by mAb against a range of coreceptors and cytokines, including CD8, B7-1, B7-2, LFA-1, and IFN-γ, but not CD4 or CTLA-4, indicating the presence of a conventional cellular Th1 response. Furthermore, we found evidence of cross-recognition between lymphomas (BCL1 and A20) as measured by cytotoxicity and IFN-γ responses in vitro and using tumor rechallenge experiments, suggesting common target Ags. Finally, although anti-CD40 was shown to stimulate NK cell killing, we could find no role for these cells in controlling tumor growth. These data underline the ability of anti-CD40 mAb to potentiate CTL responses and the potency of cellular immunity in eradicating large quantities of syngeneic tumor.
CD40 Ligand Functions Non-Cell Autonomously to Promote Deletion of Self-Reactive Thymocytes
Authors: Williams Joy A. , Sharrow Susan O. , Adams Anthony J. , Hodes Richard J.
Published: April 2014

CD40 ligand (CD40L)-deficient mice have been shown to have a defect in negative selection of self-reactive T cells during thymic development. However, the mechanism by which CD40L promotes deletion of autoreactive thymocytes has not yet been elucidated. We have studied negative selection in response to endogenous superantigens in CD40L-deficient mice and, consistent with previous reports, have found a defect in negative selection in these mice. To test the requirement for expression of CD40L on T cells undergoing negative selection, we have generated chimeric mice in which CD40L wild-type and CD40L-deficient thymocytes coexist. We find that both CD40L wild-type and CD40L-deficient thymocytes undergo equivalent and efficient negative selection when these populations coexist in chimeric mice. These results indicate that CD40L can function in a non-cell-autonomous manner during negative selection. Deletion of superantigen-reactive thymocytes was normal in B7-1/B7-2 double-knockout mice, indicating that CD40-CD40L-dependent negative selection is not solely mediated by B7 up-regulation and facilitation of B7-dependent T cell signaling. Finally, although the absence of CD40-CD40L interactions impairs negative selection of autoreactive CD4+ and CD8+ cells during thymic development, we find that self-reactive T cells are deleted in the mature CD4+ population through a CD40L-independent pathway.
Impact of Antigen Presentation on TCR Modulation and Cytokine Release: Implications for Detection and Sorting of Antigen-Specific CD8+ T Cells Using HLA-A2 Wild-Type or HLA-A2 Mutant Tetrameric Complexes
Authors: Jäger Elke , Salter Russell , Castelli Chiara , Höhn Hanni , Freitag Kirsten , Karbach Julia , Neukirch Claudia , Necker Antje , Knuth Alexander , Maeurer Markus J.
Published: April 2014

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/ζ-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8+ T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanine→valine aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR ζ-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/ζ-chain regulation and cytokine release, including IL-2, IFN-γ, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.
A Role for CCR9 in T Lymphocyte Development and Migration
Authors: Uehara Shoji , Grinberg Alexander , Farber Joshua M. , Love Paul E.
Published: April 2014

CCR9 mediates chemotaxis in response to CCL25/thymus-expressed chemokine and is selectively expressed on T cells in the thymus and small intestine. To investigate the role of CCR9 in T cell development, the CCR9 gene was disrupted by homologous recombination. B cell development, thymic αβ-T cell development, and thymocyte selection appeared unimpaired in adult CCR9-deficient (CCR9−/−) mice. However, competitive transplantation experiments revealed that bone marrow from CCR9−/− mice was less efficient at repopulating the thymus of lethally irradiated Rag-1−/− mice than bone marrow from littermate CCR9+/+ mice. CCR9−/− mice had increased numbers of peripheral γδ-T cells but reduced numbers of γδTCR+ and CD8αβ+αβTCR+ intraepithelial lymphocytes in the small intestine. Thus, CCR9 plays an important, although not indispensable, role in regulating the development and/or migration of both αβ− and γδ− T lymphocytes.
ThePmed1Gene Promoter of Human FcγRIIIA Can Function as a NK/T Cell-Specific Restriction Element, Which Involves Binding of Sp1 Transcription Factor
Authors: Heusohn Frank , Wirries Gundula , Schmidt Reinhold E. , Gessner J. Engelbert
Published: April 2014

The low-affinity receptor for IgG (human FcγRIIIA) is selectively expressed by a subset of T lymphocytes, NK cells, and macrophages. To understand the mechanisms underlying this pattern of cell type-specific expression, we initially identified alternative promoters, Pmed1/2 and Pprox, in the 5′ end of the FcγRIIIA gene. In this study, we focused on the Pmed1 promoter and demonstrated this 93-bp region to be highly specific in governing restriction to NK/T cell lines. This property of Pmed1 is context independent and can extend to a disparate promoter. Deletion analysis defined a contribution of two separate elements located to the 5′ 21-bp (−942/−922) and 3′ 72-bp (−921/−850) regions of Pmed1 in conferring NK/T cell specificity. The 5′ part of Pmed1 contains binding sites for Sp1 and NK element-recognizing factors and substitution mapping studies revealed a critical requirement of the Sp1-I site. The importance of Sp1 protein to regulate maximal Pmed1 promoter activity was further established by EMSAs and cotransfection experiments in Sp1-null Drosophila SL2 cells. Our data suggest that Sp1 can contribute, in part, to NK/T cell restriction and further indicate that the FcγRIIIA Pmed1 sequence might be useful to direct the NK/T cell-specific expression of heterologous genes.
Eotaxin/CCL11 Suppresses IL-8/CXCL8 Secretion from Human Dermal Microvascular Endothelial Cells
Authors: Cheng Sara S. , Lukacs Nicholas W. , Kunkel Steven L.
Published: April 2014

The CC chemokine eotaxin/CCL11 is known to bind to the receptor CCR3 on eosinophils and Th2-type lymphocytes. In this study, we demonstrate that CCR3 is expressed on a subpopulation of primary human dermal microvascular endothelial cells and is up-regulated by TNF-α. We found that incubation of human dermal microvascular endothelial cells with recombinant eotaxin/CCL11 suppresses TNF-α-induced production of the neutrophil-specific chemokine IL-8/CXCL8. The eotaxin/CCL11-suppressive effect on endothelial cells was not seen on IL-1β-induced IL-8/CXCL8 release. Eotaxin/CCL11 showed no effect on TNF-α-induced up-regulation of growth-related oncogene-α or IFN-γ-inducible protein-10, two other CXC chemokines tested, and did not affect production of the CC chemokines monocyte chemoattractant protein-1/CCL2 and RANTES/CCL5, or the adhesion molecules ICAM-1 and E-selectin. These results suggest that eotaxin/CXCL11 is not effecting a general suppression of TNF-αR levels or signal transduction. Suppression of IL-8/CXCL8 was abrogated in the presence of anti-CCR3 mAb, pertussis toxin, and wortmannin, indicating it was mediated by the CCR3 receptor, Gi proteins, and phosphatidylinositol 3-kinase signaling. Eotaxin/CCL11 decreased steady state levels of IL-8/CXCL8 mRNA in TNF-α-stimulated cells, an effect mediated in part by an acceleration of IL-8 mRNA decay. Eotaxin/CCL11 may down-regulate production of the neutrophil chemoattractant IL-8/CXCL8 by endothelial cells in vivo, acting as a negative regulator of neutrophil recruitment. This may play an important biological role in the prevention of overzealous inflammatory responses, aiding in the resolution of acute inflammation or transition from neutrophilic to mononuclear/eosinophilic inflammation.
Dendritic Cells Pulsed with Fungal RNA Induce Protective Immunity toCandida albicansin Hematopoietic Transplantation
Authors: Bacci Angela , Montagnoli Claudia , Perruccio Katia , Bozza Silvia , Gaziano Roberta , Pitzurra Lucia , Velardi Andrea , d’Ostiani Cristiana Fe’ , Cutler Jim E. , Romani Luigina
Published: April 2014

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-γ-producing CD4+ donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.
Protection Against Influenza Virus Infection in Polymeric Ig Receptor Knockout Mice Immunized Intranasally with Adjuvant-Combined Vaccines
Authors: Asahi Yasuko , Yoshikawa Tomoki , Watanabe Izumi , Iwasaki Takuya , Hasegawa Hideki , Sato Yuko , Shimada Shin-ichiro , Nanno Masanobu , Matsuoka Yoshiaki , Ohwaki Makoto , Iwakura Yoichiro , Suzuki Yujiro , Aizawa Chikara , Sata Tetutaro , Kurata Takeshi , Tamura Shin-ichi
Published: April 2014

The role of secretory IgA in conferring cross-protective immunity was examined in polymeric (p)IgR knockout (KO) mice immunized intranasally with different inactivated vaccines prepared from A/PR/8/34 (H1N1), A/Yamagata/120/86 (H1N1), A/Beijing/262/95 (H1N1), and B/Ibaraki/2/85 viruses and infected with the A/PR/8/34 virus in the upper respiratory tract (RT)-restricting volume. In wild-type mice, immunization with A/PR/8/34 or its variant (A/Yamagata/120/86 and A/Beijing/262/95) vaccines conferred complete protection or partial cross-protection against infection, while the B-type virus vaccine failed to provide protection. The protection or cross-protection was accompanied by an increase in the nasal A/PR/8/34 hemagglutinin-reactive IgA concentration, which was estimated to be >30 times the serum IgA concentration and much higher than the nasal IgG concentration. In contrast, the blockade of transepithelial transport of dimeric IgA in pIgR-KO mice reduced the degree of protection or cross-protection, in parallel with the marked increase in serum IgA concentration and the decrease in nasal IgA concentration (∼20 and 0.3 times those in wild-type mice, respectively). The degree of the reduction of protection or cross-protection was moderately reversed by the low but non-negligible level of nasal IgA, transudates from the accumulated serum IgA. These results, together with the absence of the IgA-dependent cross-protection in the lower RT and the unaltered level of nasal or serum IgG in wild-type and pIgR-KO mice, confirm that the actively secreted IgA plays an important role in cross-protection against variant virus infection in the upper RT, which cannot be substituted by serum IgG.
Heat Shock Proteins gp96 and hsp70 Activate the Release of Nitric Oxide by APCs
Authors: Panjwani Naveed N. , Popova Lana , Srivastava Pramod K.
Published: April 2014

NO is a cytotoxic and immunomodulatory cytokine produced by macrophages and dendritic cells. We show that stimulation of murine and human macrophages with the heat shock proteins gp96 and hsp70 results in induction of inducible NO synthase and the production of NO. The release of NO by monocytes exposed to hsp60 has been documented previously. Immature, but not mature, dendritic cells respond in the same manner. The activity of heat shock proteins is relatively unaffected by an antagonist of LPS, and is abrogated by heat denaturation. Macrophages have been shown previously to produce NO in response to stimulation with IFN-γ; stimulation of macrophages with mixtures of IFN-γ and gp96 or hsp70 leads to a synergistic production of NO. The present observations extend the roles of these heat shock proteins in innate immune responses to another potent and highly conserved function of APC.
Interaction of Antibodies to Proteinase 3 (Classic Anti-Neutrophil Cytoplasmic Antibody) with Human Renal Tubular Epithelial Cells: Impact on Signaling Events and Inflammatory Mediator Generation
Authors: Hattar Katja , Grandel Ulrich , Bickenbach Annette , Schwarting Andreas , Mayet Werner-Johannes , Bux Jürgen , Jessen Sönke , Fischer Claus , Seeger Werner , Grimminger Friedrich , Sibelius Ulf
Published: April 2014

Among the anti-neutrophil cytoplasmic Abs (ANCA), those targeting proteinase 3 (PR3) have a high sensitivity and specificity for Wegener’s granulomatosis (WG). A pathogenetic role for these autoantibodies has been proposed due to their capacity of activating neutrophils in vitro. Recently, PR3 was also detected in human renal tubular epithelial cells (TEC). In the present study, the effect of murine monoclonal anti-PR3 Abs (anti-PR3) and purified c-ANCA targeting PR3 from WG serum on isolated human renal tubular cell signaling and inflammatory mediator release was characterized. Priming of TEC with TNF-α resulted in surface expression of PR3, as quantified in immunofluorescence studies and by flow cytometry. Moreover, PR3 was immunoprecipitated on surface-labeled TEC. Primed TEC responded to anti-PR3 with a dose- and time-dependent activation of phosphoinositide hydrolysis, resulting in a remarkable accumulation of inositolphosphates. Control IgG was entirely ineffective, whereas PR3-ANCA reproduced the phosphoinositide response. The signaling response was accompanied by a pronounced release of superoxidanion into the cell supernatant. Moreover, large amounts of PGE2 and, to a lesser extent, of thromboxane B2, the stable metabolite of TxA2, were secreted from anti-PR3-stimulated TEC. In parallel, a rise in intracellular cAMP levels was observed, which was blocked by the cyclooxygenase inhibitor indomethacin. We conclude that anti-PR3 Abs directly target renal TECs, thereby provoking pronounced activation of the phosphoinositide-related signal transduction pathway. Associated metabolic events such as the release of reactive oxygen species and lipid mediators may directly contribute to the development of renal lesions and loss of kidney function in WG.
Murine Sclerodermatous Graft-Versus-Host Disease, a Model for Human Scleroderma: Cutaneous Cytokines, Chemokines, and Immune Cell Activation
Authors: Zhang Yan , McCormick Laura L. , Desai Snehal R. , Wu Caiyun , Gilliam Anita C.
Published: April 2014

Murine sclerodermatous graft-vs-host disease (Scl GVHD) models human scleroderma, with prominent skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA. Fibrosis in Scl GVHD may be driven by infiltrating TGF-β1-producing mononuclear cells. Here we characterize the origin and types of those cutaneous effector cells, the cytokine and chemokine environments, and the effects of anti-TGF-β Ab on skin fibrosis, immune cell activation markers, and collagen and cytokine synthesis. Donor cells infiltrating skin in Scl GVHD increase significantly at early time points post-transplantation and are detectable by PCR analysis of Y-chromosome sequences when female mice are transplanted with male cells. Cutaneous monocyte/macrophages and T cells are the most numerous cells in Scl GVHD compared with syngeneic controls. These immune cells up-regulate activation markers (MHC class II I-Ad molecules and class A scavenger receptors), suggesting Ag presentation by cutaneous macrophages in early fibrosing disease. Early elevated cutaneous mRNA expression of TGF-β1, but not TGF-β2 or TGF-β3, and elevated C-C chemokines macrophage chemoattractant protein-1, macrophage inflammatory protein-1α, and RANTES precede subsequent skin and lung fibrosis. Therefore, TGF-β1-producing donor mononuclear cells may be critical effector cells, and C-C chemokines may play important roles in the initiation of Scl GVHD. Abs to TGF-β prevent Scl GVHD by effectively blocking the influx of monocyte/macrophages and T cells into skin and by abrogating up-regulation of TGF-β1, thereby preventing new collagen synthesis. The Scl GVHD model is valuable for testing new interventions in early fibrosing diseases, and chemokines may be new potential targets in scleroderma.
Increased Severity of Experimental Allergic Encephalomyelitis in lyn−/− Mice in the Absence of Elevated Proinflammatory Cytokine Response in the Central Nervous System
Authors: Du Caigan , Sriram Subramaniam
Published: April 2014

lyn, a member of the src kinase family, is an important signaling molecule in B cells. lyn−/− mice display hyperactive B-1 cells and IgM hyperglobulinemia. The role of lyn on T cell function and development of Th1-mediated inflammatory disease is not known. Therefore, we examined the effect of disruption of the lyn gene on the development of experimental allergic encephalomyelitis (EAE), a well-established Th1-mediated autoimmune disease. Following immunization with myelin oligodendrocyte protein (MOG) p35-55, lyn−/− mice had higher clinical and pathological severity scores of EAE when compared with wild type (WT). The increase in the severity of EAE in lyn−/− mice was not associated with a commensurate increase in the production of proinflammatory cytokines in the CNS. lyn−/− mice with EAE showed elevation in serum anti-IgM MOG Ab levels over that seen in WT mice, along with a modest increase in the mRNA levels of complement C5 and its receptor, C5aR, in the spinal cord. Transfer of serum from MOG-immunized lyn−/− mice worsened EAE in WT mice, suggesting a pathogenic role for anti-MOG IgM Abs in EAE. These observations underscore the potential role of lyn in regulation of Th1-mediated disease and the role of autoantibodies and complement in the development of EAE.
Cutting Edge: Polarized Th Cell Response Induction by Transferred Antigen-Pulsed Dendritic Cells Is Dependent on IL-4 or IL-12 Production by Recipient Cells
Authors: MacDonald Andrew S. , Pearce Edward J.
Published: April 2014

To assess the influence of dendritic cell (DC) production of polarizing cytokines on Th2 and Th1 development we transferred Ag-pulsed DC generated from wild-type, IL-4−/−, or IL-12−/− mice into wild-type, IL-4−/−, or IL-12−/− recipients. We found that DC IL-4 was not necessary for Th2 induction and that, surprisingly, DC IL-12 was not an absolute requirement for Th1 development. However, DC IL-12 production facilitated optimal Th1 response development. Critically, recipient ability to produce IL-4 or IL-12 was essential for either Th2 or Th1 development. These data help delineate the source and importance of IL-4 and IL-12 in the process of induction of polarized T cell responses by DC.
Cutting Edge: Analysis of Human Vα24+CD8+ NK T Cells Activated by α-Galactosylceramide-Pulsed Monocyte-Derived Dendritic Cells
Authors: Takahashi Tsuyoshi , Chiba Shigeru , Nieda Mie , Azuma Takeshi , Ishihara Soichiro , Shibata Yoichi , Juji Takeo , Hirai Hisamaru
Published: April 2014

Human Vα24+ NKT cells constitute a counterpart of mouse Vα14+ NKT cells, both of which use an invariant TCR-α chain. The human Vα24+ NKT cells as well as mouse Vα14+ NKT cells are activated by glycolipids in a CD1d-restricted manner and produce many immunomodulatory cytokines, possibly affecting the immune balance. In mice, it has been considered from extensive investigations that Vα14+CD8+ NKT cells that express invariant TCR do not exist. Here we introduce human Vα24+CD8+ NKT cells. These cells share important features of Vα24+ NKT cells in common, but in contrast to CD4−CD8− (double-negative) or CD4+ Vα24+ NKT cells, they do not produce IL-4. Our discovery may extend and deepen the research field of Vα24+ NKT cells as well as help to understand the mechanism of the immune balance-related diseases.
Cutting Edge: Single-Chain Trimers of MHC Class I Molecules Form Stable Structures That Potently Stimulate Antigen-Specific T Cells and B Cells
Authors: Yu Yik Y. L. , Netuschil Nikolai , Lybarger Lonnie , Connolly Janet M. , Hansen Ted H.
Published: April 2014

We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-β2-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.
Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development In Vivo
Authors: Johansson-Lindbom Bengt , Borrebaeck Carl A. K.
Published: April 2014

Protective immunity depends upon the capability of the immune system to properly adapt the response to the nature of an infectious agent. CD4+ Th cells are implicated in this orchestration by secreting a polarized pattern of cytokines. Although Th2 development in animal models and in human cells in vitro to a large extent depends on IL-4, the nature of the cells that provide the initial IL-4 in vivo is still elusive. In this report, we describe the anatomical localization as well as the identity of IL-4-producing cells in human tonsil, a representative secondary lymphoid organ. We demonstrate that IL-4 production is a normal and intrinsic feature of germinal center (GC) B cells. We also show that expression of IL-4 is highly confined to the GCs, in which the B cells constitute the prevalent cellular source. Furthermore, immunofluorescence analysis of colon mucosa reveals a strikingly similar pattern of IL-4-expressing cells compared with tonsils, demonstrating that IL-4 production from GC B cells is not a unique feature of the upper respiratory tract. Our results show that GCs provide the most appropriate microenvironment for IL-4-dependent Th2 polarization in vivo and imply a critical role for GC B cells in this differentiation process.
Transgenic Expression of Numb Inhibits Notch Signaling in Immature Thymocytes But Does Not Alter T Cell Fate Specification
Authors: French Michelle B. , Koch Ute , Shaye Rachel E. , McGill Melanie A. , Dho Sascha E. , Guidos Cynthia J. , McGlade C. Jane
Published: April 2014

The conserved adaptor protein Numb is an intrinsic cell fate determinant that functions by antagonizing Notch-mediated signal transduction. The Notch family of membrane receptors controls cell survival and cell fate determination in a variety of organ systems and species. Recent studies have identified a role for mammalian Notch-1 signals at multiple stages of T lymphocyte development. We have examined the role of mammalian Numb (mNumb) as a Notch regulator and cell fate determinant during T cell development. Transgenic overexpression of mNumb under the control of the Lck proximal promoter reduced expression of several Notch-1 target genes, indicating that mNumb antagonizes Notch-1 signaling in vivo. However, thymocyte development, cell cycle, and survival were unperturbed by mNumb overexpression, even though transgenic Numb was expressed at an early stage in thymocyte development (CD4−CD8−CD3− cells that were CD44+CD25+ or CD44−CD25+; double-negative 2/3). Moreover, bone marrow from mNumb transgenic mice showed no defects in thymopoiesis in competitive repopulation experiments. Our results suggest that mNumb functions as a Notch-1 antagonist in immature thymocytes, but that suppression of Notch-1 signaling at this stage does not alter γδ/αβ or CD4/CD8 T cell fate specification.
Donor T Cell Activation Initiates Small Bowel Allograft Rejection Through an IFN-γ-Inducible Protein-10-Dependent Mechanism
Authors: Zhang Zheng , Kaptanoglu Levent , Haddad Wael , Ivancic David , Alnadjim Ziad , Hurst Stephen , Tishler Darren , Luster Andrew D. , Barrett Terrence A. , Fryer Jonathan
Published: April 2014

The poor success in controlling small bowel (SB) allograft rejection is partially attributed to the unique immune environment in the donor intestine. We hypothesized that Ag-induced activation of donor-derived T cells contributes to the initiation of SB allograft rejection. To address the role of donor T cell activation in SB transplantation, SB grafts from DO11.10 TCR transgenic mice (BALB/c, H-2Ld+) were transplanted into BALB/c (isografts), or single class I MHC-mismatched (Ld-deficient) BALB/c H-2dm2 (dm2, H-2Ld−) mutant mice (allografts). Graft survival was followed after injection of control or antigenic OVA323–339 peptide. Eighty percent of SB allografts developed severe rejection in mice treated with antigenic peptide, whereas <20% of allografts were rejected in mice treated with control peptide (p < 0.05). Isografts survived >30 days regardless of OVA323–339 administration. Activation of donor T cells increased intragraft expression of proinflammatory cytokine (IFN-γ) and CXC chemokine IFN-γ-inducible protein-10 mRNA and enhanced activation and accumulation of host NK and T cells in SB allografts. Treatment of mice with neutralizing anti-IFN-γ-inducible protein-10 mAb increased SB allograft survival in Ag-treated mice (67%; p < 0.05) and reduced accumulation of host T cells and NK cells in the lamina propria but not mesenteric lymph nodes. These results suggest that activation of donor T cells after SB allotransplantation induces production of a Th1-like profile of cytokines and CXC chemokines that enhance infiltration of host T cells and NK cells in SB allografts. Blocking this pathway may be of therapeutic value in controlling SB allograft rejection.
Naive CD4+ T Cells Exhibit Distinct Expression Patterns of Cytokines and Cell Surface Molecules on Their Primary Responses to Varying Doses of Antigen
Authors: Ise Wataru , Totsuka Mamoru , Sogawa Yoshitaka , Ametani Akio , Hachimura Satoshi , Sato Takehito , Kumagai Yoshihiro , Habu Sonoko , Kaminogawa Shuichi
Published: April 2014

The amount of an Ag used for stimulation affects the type and magnitude of T cell responses. In this study we have investigated the primary response of naive CD4+ T cells derived from OVA-specific TCR-transgenic mice (OVA23-3) upon stimulation with varying doses of the antigenic peptide, OVA323–339. IL-4 expression was maximal with 50 nM Ag and decreased significantly with increasing doses. In contrast, IFN-γ expression, which was also detected at 50 nM Ag, increased with increasing doses. The expression patterns of mRNA for the Th2-specific transcription factors GATA-3 and c-Maf were parallel to that of IL-4. These expression profiles were not altered by the addition of anti-IL-4 plus anti-IL-12 mAbs, suggesting that cytokine receptor signaling is not essential. Naive CD4+ T cells stimulated with 5 nM Ag elicited IgM secretion from cocultured B cells, whereas those stimulated with 50 nM Ag or more elicited apoptosis of B cells. This may be because at lower doses of Ag (5 nM), naive CD4+ T cells express CD40 ligand and OX40, whereas at higher doses (50 nM), they express Fas ligand. Clearly, the expression of each type of molecule depends on the Ag dose, and different molecules had different expression patterns. Thus, in the primary response, naive CD4+ T cells can exhibit different functions depending on the dose of Ag.