bluuebunny/crossref_metadata_2025 huggingface dataset:
Enforcedbcl-xLGene Expression Restored Splenic B Lymphocyte Development in BAFF-R Mutant Mice
Authors: Amanna Ian J. , Dingwall Jennifer P. , Hayes Colleen E.
Published: April 2014

The TNFR family member BAFF-R facilitates peripheral B cell development, although it is unclear whether it promotes survival of B cells, or also initiates a differentiation program. We show that disruption of the BAFF-R encoding gene Tnfrsf13c in strain A/WySnJ mice causes a progressive decline in peripheral B cell numbers, beginning at the transitional 1 developmental stage and continuing through the mature peripheral B cell stage. Bcl-xL overexpression in A/WySnJ B cells decreased the turnover of transitional B cells, as determined by 5-bromo-2′-deoxyuridine labeling, and restored follicular B cell development. We conclude that the mutant A/WySnJ allele of Tnfrsf13c can be complemented through the survival signal provided by Bcl-xL.
Influenza A Virus Infection Inhibits the Efficient Recruitment of Th2 Cells into the Airways and the Development of Airway Eosinophilia
Authors: Wohlleben Gisela , Müller Justus , Tatsch Ursula , Hambrecht Christine , Herz Udo , Renz Harald , Schmitt Edgar , Moll Heidrun , Erb Klaus J.
Published: April 2014

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8+ T cells secreting IFN-γ, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-γ- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-γ, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.
TGF-β1 Regulates Lymphocyte Homeostasis by Preventing Activation and Subsequent Apoptosis of Peripheral Lymphocytes
Authors: Bommireddy Ramireddy , Saxena Vijay , Ormsby Ilona , Yin Moying , Boivin Gregory P. , Babcock George F. , Singh Ram R. , Doetschman Thomas
Published: April 2014

TGF-β1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-β1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-β1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1−/− mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-γ and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1−/− T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1−/− T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4+ T cells have elevated intracellular Ca2+ levels. However, upon subsequent stimulation in vitro, increases in Ca2+ levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1−/− splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca2+ levels.
CD5 Inhibits Signaling at the Immunological Synapse Without Impairing Its Formation
Authors: Brossard Cédric , Semichon Monique , Trautmann Alain , Bismuth Georges
Published: April 2014

Physiologically, Ag detection by T cells occurs at the immunological synapse (IS) formed at the interface with an APC. CD5 is considered as an inhibitory molecule for Ag receptor-mediated signals in T cells. However, the influence of CD5 at the IS on synapse formation and functioning has not yet been reported. We demonstrate here that CD5 is recruited and tightly colocalized with CD3 in different human and murine IS. Following transfection in a CD5-negative T cell line of CD5 fused to the green fluorescent protein, we show that CD5 recruitment includes a fast Ag-independent and a slower Ag-dependent component. In video-imaging recordings of doubly transfected cells, the movements of CD3 and CD5 show similar kinetics, and the amount of CD3 recruited to the synapse is unaffected by CD5 expression. Moreover, APC-T cell adhesion is unchanged in CD5-expressing cells. Despite this, the extent of tyrosine phosphorylation at the synapse and the amplitude of calcium responses induced by Ag recognition are both decreased by CD5. These inhibitions increase with CD5 membrane levels. They also requires the pseudo-immunoreceptor tyrosine-based activation motif expressed in the cytoplasmic domain of the molecule. Thus, CD5 is rapidly recruited at the IS and lowers the T cell response elicited by Ag presentation by targeting downstream signaling events without affecting IS formation.
IKKβ Is Required for Peripheral B Cell Survival and Proliferation
Authors: Li Zhi-Wei , Omori Sidne A. , Labuda Tord , Karin Michael , Rickert Robert C.
Published: April 2014

NF-κB activity in mammalian cells is regulated through the IκB kinase (IKK) complex, consisting of two catalytic subunits (IKKα and IKKβ) and a regulatory subunit (IKKγ). Targeted deletion of Ikkβ results in early embryonic lethality, thus complicating the examination of IKKβ function in adult tissues. Here we describe the role of IKKβ in B lymphocytes made possible by generation of a mouse strain that expresses a conditional Ikkβ allele. We find that the loss of IKKβ results in a dramatic reduction in all peripheral B cell subsets due to associated defects in cell survival. IKKβ-deficient B cells are also impaired in mitogenic responses to LPS, anti-CD40, and anti-IgM, indicating a general defect in the ability to activate the canonical NF-κB signaling pathway. These findings are consistent with a failure to mount effective Ab responses to T cell-dependent and independent Ags. Thus, IKKβ provides a requisite role in B cell activation and maintenance and thus is a key determinant of humoral immunity.
Naive T Cell Recruitment to Nonlymphoid Tissues: A Role for Endothelium-Expressed CC Chemokine Ligand 21 in Autoimmune Disease and Lymphoid Neogenesis
Authors: Weninger Wolfgang , Carlsen Hege S. , Goodarzi Mahmoud , Moazed Farzad , Crowley Maura A. , Baekkevold Espen S. , Cavanagh Lois L. , von Andrian Ulrich H.
Published: April 2014

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP+ cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP+ cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 ± 10%) and ulcerative colitis (66 ± 1%) expressed CCL21, and many perivascular CD45RA+ naive T cells were found in these tissues, but not in psoriasis, where CCL21+ vessels were rare (17 ± 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.
A Role of CXC Chemokine Ligand 12/Stromal Cell-Derived Factor-1/Pre-B Cell Growth Stimulating Factor and Its Receptor CXCR4 in Fetal and Adult T Cell Development in Vivo
Authors: Ara Toshiaki , Itoi Manami , Kawabata Kenji , Egawa Takeshi , Tokoyoda Koji , Sugiyama Tatsuki , Fujii Nobutaka , Amagai Takashi , Nagasawa Takashi
Published: April 2014

The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12−/− or CXCR4−/− embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3−CD4−CD8− triple-negative T cell precursors at the CD44−CD25+ and CD44−CD25− stages, and CD4+CD8+ double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4−/− fetal liver cells, the reduction in donor-derived thymocytes compared with wild-type chimeras was much more severe than the reduction in donor-derived myeloid lineage cells in bone marrow. Triple negative CD44+CD25+ T cell precursors exhibited survival response to CXCL12 in the presence of stem cell factor as well as migratory response to CXCL12. Thus, it may be that CXCL12 and CXCR4 are involved in the expansion of T cell precursors in both fetal and adult thymus in vivo. Finally, enforced expression of bcl-2 did not rescue impaired T cell development in CXCR4−/− embryos or impaired reconstitution of CXCR4−/− thymocytes in competitively repopulated mice, suggesting that defects in T cell development caused by CXCR4 mutation are not caused by reduced expression of bcl-2.
Maternal Autoantibody Triggers De Novo T Cell-Mediated Neonatal Autoimmune Disease
Authors: Setiady Yulius Y. , Samy Eileen T. , Tung Kenneth S. K.
Published: April 2014

Although human maternal autoantibodies may transfer transient manifestation of autoimmune disease to their progeny, some neonatal autoimmune diseases can progress, leading to the loss of tissue structure and function. In this study we document that murine maternal autoantibody transmitted to progeny can trigger de novo neonatal pathogenic autoreactive T cell response and T cell-mediated organ-specific autoimmune disease. Autoantibody to a zona pellucida 3 (ZP3) epitope was found to induce autoimmune ovarian disease (AOD) and premature ovarian failure in neonatal, but not adult, mice. Neonatal AOD did not occur in T cell-deficient pups, and the ovarian pathology was transferable by CD4+ T cells from diseased donors. Interestingly, neonatal AOD occurred only in pups exposed to ZP3 autoantibody from neonatal days 1–5, but not from day 7 or day 9. The disease susceptibility neonatal time window was not related to a propensity of neonatal ovaries to autoimmune inflammation, and it was not affected by infusion of functional adult CD4+CD25+ T cells. However, resistance to neonatal AOD in 9-day-old mice was abrogated by CD4+CD25+ T cell depletion. Finally, neonatal AOD was blocked by Ab to IgG-FcR, and interestingly, the disease was not elicited by autoantibody to a second, independent native ZP3 B cell epitope. Therefore, a new mechanism of neonatal autoimmunity is presented in which epitope-specific autoantibody stimulates de novo autoimmune pathogenic CD4+ T cell response.
Endogenous IL-17 as a Mediator of Neutrophil Recruitment Caused by Endotoxin Exposure in Mouse Airways
Authors: Miyamoto Masahide , Prause Olof , Sjöstrand Margareta , Laan Martti , Lötvall Jan , Lindén Anders
Published: April 2014

We have previously demonstrated that administration of the recently described cytokine IL-17 in rat airways in vivo recruits and activates neutrophils locally. In the current study, we examined whether endogenous IL-17 is involved in mediating neutrophil recruitment caused by endotoxin exposure in mouse airways. Our in vivo data show that local endotoxin exposure causes the release of free, soluble IL-17 protein 6 h later. Systemic pretreatment with a neutralizing anti-IL-17 Ab almost completely inhibits neutrophil recruitment 24 h, but not 6 h, after endotoxin exposure in the airways. Pretreatment with neutralizing anti-IL-6 and anti-macrophage inflammatory protein (MIP)-2 Abs inhibits neutrophil recruitment caused by local endotoxin exposure and IL-17, respectively. Our in vitro data show that endotoxin exposure stimulates the release of soluble IL-17 protein in T lymphocytes harvested from lung and spleen, respectively, and that this cytokine release requires coculture with airway macrophages. Intracellular IL-17 protein is detected in T lymphocytes from spleen but not in airway macrophages after coculture and stimulation of these two cell types. Finally, anti-IL-17 does not alter endotoxin-induced release of IL-6 and MIP-2 from T lymphocytes and airway macrophages in coculture. In conclusion, our results indicate that endotoxin exposure causes the release of IL-17 from T lymphocytes and that this cytokine release requires the presence of macrophages. Once released, endogenous IL-17 acts in part by inducing local release of neutrophil-mobilizing cytokines such as IL-6 and MIP-2, from nonlymphocyte, nonmacrophage cells, and this contributes to recruitment of neutrophils in the airways. These IL-17-related mechanisms constitute potential targets for pharmacotherapy against exaggerated neutrophil recruitment in airway disease.
The Paradox of Immune Molecular Recognition of α-Galactosylceramide: Low Affinity, Low Specificity for CD1d, High Affinity for αβ TCRs
Authors: Cantu Carlos , Benlagha Kamel , Savage Paul B. , Bendelac Albert , Teyton Luc
Published: April 2014

CD1 resembles both class I and class II MHC but differs by the important aspect of presenting lipid/glycolipids, instead of peptides, to T cells. Biophysical studies of lipid/CD1 interactions have been limited, and kinetics of binding are in contradiction with functional studies. We have revisited this issue by designing new assays to examine the loading of CD1 with lipids. As expected for hydrophobic interactions, binding affinity was not high and had limited specificity. Lipid critical micelle concentration set the limitation to these studies. Once loaded onto CD1d, the recognition of glycolipids by αβ T cell receptor was studied by surface plasmon resonance using soluble Vα14-Vβ8.2 T cell receptors. The Vα14 Jα18 chain could be paired with NK1.1 cell-derived Vβ chain, or any Vβ8 chain, to achieve high affinity recognition of α-galactosylceramide. Biophysical analysis indicated little effect of temperature or ionic strength on the binding interaction, in contrast to what has been seen in peptide/MHC-TCR studies. This suggests that there is less accommodation made by this TCR in recognizing α-galactosylceramide, and it can be assumed that the most rigid part of the Ag, the sugar moiety, is critical in the interaction.
Point Mutations in or Near the Antigen-Binding Groove of HLA-DR3 Implicate Class II-Associated Invariant Chain Peptide Affinity as a Constraint on MHC Class II Polymorphism
Authors: Doebele Robert C. , Pashine Achal , Liu Wendy , Zaller Dennis M. , Belmares Michael , Busch Robert , Mellins Elizabeth D.
Published: April 2014

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II αβ dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations βS11F, βS13Y, βQ70R, βK71E, βK71N, and βR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as αG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.
Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to ExpelTrichuris murisInfection
Authors: deSchoolmeester Matthew L. , Little Matthew C. , Rollins Barrett J. , Else Kathryn J.
Published: April 2014

Despite a growing understanding of the role of cytokines in immunity to intestinal helminth infections, the importance of chemokines has been neglected. As a chemokine with both chemoattractive properties and an ability to shape the quality of the adaptive immune response, CC chemokine ligand 2 (CCL2) was investigated as an attractive candidate for controlling resistance to these types of infection, which require highly polarized Th cell responses. We show here for the first time that CCL2 plays an important role in the development of resistance to infection by the gastrointestinal nematode Trichuris muris. Thus, in the absence of CCL2, worm expulsion does not occur, and the lymph node draining the site of infection becomes a Th1-promoting environment. Elevated levels of IL-12 are produced by polarizing APCs, and the composition of the APC environment itself is perturbed, with reduced numbers of macrophages.
Origin of Mannose-Binding Lectin-Associated Serine Protease (MASP)-1 and MASP-3 Involved in the Lectin Complement Pathway Traced Back to the Invertebrate, Amphioxus
Authors: Endo Yuichi , Nonaka Masaru , Saiga Hidetoshi , Kakinuma Yuji , Matsushita Akiko , Takahashi Minoru , Matsushita Misao , Fujita Teizo
Published: April 2014

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.
Retrocyclin, an Antiretroviral θ-Defensin, Is a Lectin
Authors: Wang Wei , Cole Alexander M. , Hong Teresa , Waring Alan J. , Lehrer Robert I.
Published: April 2014

θ-Defensins are circular octadecapeptides that contain an internal tridisulfide ladder. Because retrocyclin-1, an ancestral hominid θ-defensin, can protect human cells in vitro from infection by T- and M-tropic strains of HIV-1, we used surface plasmon resonance techniques to study its binding to glycoproteins and glycolipids implicated in HIV-1 entry. Retrocyclin-1 bound with high affinity to gp120 (Kd, 35.4 nM), CD4 (Kd, 31 nM), and galactosylceramide (Kd, 24.1 nM). Neither a circular form of retrocyclin without its tridisulfide ladder nor its β-hairpin precursor with these disulfides intact bound gp120 or CD4 effectively. Retrocyclin also bound fetuin, an extensively glycosylated protein, with high affinity, but it did not bind nonglycosylated gp120 or BSA. However, retrocyclin did bind to a neoglycoprotein, BSA, with covalently attached sugar residues. Experiments with glycosidase-treated fetuin, gp120, and CD4 revealed that both O-linked and N-linked sugars were used as binding sites. In a panel of retrocyclin variants, binding to immobilized gp120 and CD4 were highly correlated to each other and to the peptide’s ability to protect human PBMCs from infection by HIV-1. Although small, cysteine-rich antimicrobial peptides with lectin-like properties exist in plants, θ-defensins are the first such molecules to be identified in vertebrates. Retrocyclin’s ability to recognize and bind carbohydrate-containing surface molecules is integrally related to its ability to protect cells from HIV-1 infection.
CpG Oligodeoxynucleotides Protect Normal and SIV-Infected Macaques fromLeishmaniaInfection
Authors: Verthelyi Daniela , Gursel Mayda , Kenney Richard T. , Lifson Jeffrey D. , Liu Shuying , Mican Joan , Klinman Dennis M.
Published: April 2014

Oligodeoxynucleotides containing CpG motifs (CpG ODNs) mimic microbial DNA and activate effectors of the innate immune response, which limits the spread of pathogens and promotes an adaptive immune response. CpG ODNs have been shown to protect mice from infection with intracellular pathogens. Unfortunately, CpG motifs that optimally stimulate humans are only weakly active in mice, mandating the use of nonhuman primates to monitor the activity and safety of “human” CpG ODNs in vivo. This study demonstrates that CpG ODN treatment of rhesus macaques significantly reduces the severity of the lesions caused by a challenge with Leishmania. Leishmania superinfection is common in immunocompromised hosts, particularly those infected with HIV. This study shows that PBMCs from HIV-infected subjects respond to stimulation with CpG ODNs. To determine whether CpG ODNs can protect retrovirus-infected primates, SIV-infected macaques were treated with CpG ODNs and then challenged with Leishmania. Both lesion size and parasite load were significantly reduced in the CpG-treated animals. These findings support the clinical development of CpG ODNs as immunoprotective agents in normal and HIV-infected patients.
IL-1 Receptor Type 1 Gene-Deficient Mice Demonstrate an Impaired Host Defense Against Pneumococcal Meningitis
Authors: Zwijnenburg Petra J. G. , van der Poll Tom , Florquin Sandrine , Roord John J. , van Furth A. Marceline
Published: April 2014

The fatality rate associated with Streptococcus pneumoniae meningitis remains high despite adequate antibiotic treatment. IL-1 is an important proinflammatory cytokine, which is up-regulated in brain tissue after the induction of meningitis. To determine the role of IL-1 in pneumococcal meningitis we induced meningitis by intranasal inoculation with 8 × 104 CFU of S. pneumoniae and 180 U of hyaluronidase in IL-1R type I gene-deficient (IL-1R−/−) mice and wild-type mice. Meningitis resulted in elevated IL-1α and IL-1β mRNA and protein levels in the brain. The absence of an intact IL-1 signal was associated with a higher susceptibility to develop meningitis. Furthermore, the lack of IL-1 impaired bacterial clearance, as reflected by an increased number of CFU in cerebrospinal fluid of IL-1R−/− mice. The characteristic pleocytosis of meningitis was not significantly altered in IL-1R−/− mice, but meningitis was associated with lower brain levels of cytokines. The mortality was significantly higher and earlier in the course of the disease in IL-1R−/− mice. These results demonstrate that endogenous IL-1 is required for an adequate host defense in pneumococcal meningitis.
IL-12 Receptor β1 and Toll-Like Receptor 4 Increase IL-1β- and IL-18-Associated Myocarditis and Coxsackievirus Replication
Authors: Fairweather DeLisa , Yusung Susan , Frisancho Sylvia , Barrett Masheka , Gatewood Shannon , Steele Ronelle , Rose Noel R.
Published: April 2014

Th1-type immune responses, mediated by IL-12-induced IFN-γ, protect the host from most viral infections. To investigate the role of IL-12 and IFN-γ on the development of Coxsackievirus B3 (CB3)-induced myocarditis, we examined the level of inflammation, viral replication, and cytokine production in IL-12Rβ1- and IFN-γ-deficient mice following CB3 infection. We report that IL-12Rβ1 deficiency results in decreased viral replication and inflammation in the heart, while IFN-γ deficiency exacerbates CB3 replication. Importantly, decreased IL-1β and IL-18 levels in IL-12Rβ1-deficient hearts correlated directly with decreased myocardial inflammation. Because IL-1β and IL-18 were associated with myocardial inflammation, we examined the effect of TLR4 deficiency on CB3 infection and myocarditis. We found that TLR4-deficient mice also had significantly reduced levels of myocarditis, viral replication, and IL-1β/IL-18, just as we had observed in IL-12Rβ1-deficient mice. This is the first report that TLR4 influences CB3 replication. These results show that IL-12Rβ1 and TLR4 exacerbate CB3 infection and myocarditis while IFN-γ protects against viral replication. The remarkable similarities between the effects of IL-12Rβ1 and TLR4 suggest that these receptors share common downstream pathways that directly influence IL-1β and IL-18 production, and confirm that IL-1β and IL-18 play a significant role in the pathogenesis of CB3-induced myocarditis. These findings have important implications not only for the pathogenesis of myocarditis, but for other autoimmune diseases triggered by viral infections.
Microsomal Prostaglandin E Synthase-1 Is a Major Terminal Synthase That Is Selectively Up-Regulated During Cyclooxygenase-2-Dependent Prostaglandin E2 Production in the Rat Adjuvant-Induced Arthritis Model
Authors: Claveau David , Sirinyan Mirna , Guay Jocelyne , Gordon Robert , Chan Chi-Chung , Bureau Yves , Riendeau Denis , Mancini Joseph A.
Published: April 2014

To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to ≥90% with a potency similar to that of recombinant rat mPGES-1 (IC50 = 2.4 μM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE2 production. Prostacyclin (measured as 6-keto-PGF1α) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE2 in this model.
Mitochondrial Potential and Reactive Oxygen Intermediates in Antigen-Specific CD8+ T Cells During Viral Infection
Authors: Grayson Jason M. , Laniewski Nathan G. , Lanier J. Gibson , Ahmed Rafi
Published: April 2014

Following many viral infections, there are large expansions of Ag-specific CD8+ T cells. After viral clearance, mechanisms exist to ensure that the vast majority of effector cells undergo apoptosis. In studies of thymocyte apoptosis, loss of mitochondrial potential (ΔΨm) and excess production of reactive oxygen intermediates have been implicated as key events in cellular apoptosis. The purpose of the experiments presented in this work was to determine these parameters in Ag-specific CD8+ T cells during a physiological response such as viral infection. Using lymphocytic choriomeningitis virus infection of mice, we found that Ag-specific CD8+ effector T cells that had undergone recent TCR stimulation had an increased ΔΨm. These cells also had increased levels of superoxide. As these cells progressed through the contraction of the immune response, their potential decreased, but superoxide levels remained similar to naive cells. One of the consequences of reduced mitochondrial potential is membrane permeability and subsequent caspase activation. We examined both the enzymatic activity and levels of cleaved caspase 3, an effector caspase, and could only detect increased levels in Ag-specific CD8+ T cells on day 5 postinfection, a time point in which virus was still present. This contrasts with Ag-specific effector cells examined during the contraction phase that had no detectable caspase activity directly ex vivo. These data suggest that the apoptotic program begins earlier than previously expected on day 5, during the expansion phase.
Δ12-Prostaglandin J2, a Plasma Metabolite of Prostaglandin D2, Causes Eosinophil Mobilization from the Bone Marrow and Primes Eosinophils for Chemotaxis
Authors: Heinemann Akos , Schuligoi Rufina , Sabroe Ian , Hartnell Adele , Peskar Bernhard A.
Published: April 2014

PGD2, a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD2 is rapidly transformed into its major metabolite Δ12-PGJ2, the effect of which on eosinophil migration has not yet been characterized. In this study we found that Δ12-PGJ2 was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD2, PGJ2, or 15-deoxy-Δ12,14-PGJ2. Moreover, pretreatment of eosinophils with Δ12-PGJ2 markedly enhanced the chemotactic response to eotaxin, and in this respect Δ12-PGJ2 was more effective than PGD2. Δ12-PGJ2-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that Δ12-PGJ2 signaled through the same receptor, CRTH2, as PGD2. Finally, Δ12-PGJ2 was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that Δ12-PGJ2 is present in the systemic circulation at relevant levels, a role for this PGD2 metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.
IL-1-Driven Endogenous IL-10 Production Protects Against the Systemic and Local Acute Inflammatory Response Following Intestinal Reperfusion Injury
Authors: Souza Danielle G. , Guabiraba Rodrigo , Pinho Vanessa , Bristow Adrian , Poole Stephen , Teixeira Mauro M.
Published: April 2014

TNF-α release and action are central in the pathogenesis of the local and systemic inflammatory responses that occur after intestinal reperfusion. In this study we examined whether IL-1 participated in the cascade of events leading to TNF-α production and TNF-α-mediated injury following reperfusion of the ischemic superior mesenteric artery in rats. Blockade of the action of IL-1 by the use of anti-IL-1 antiserum or administration of IL-1R antagonist (IL-1ra), a natural antagonist of IL-1Rs, resulted in marked enhancement of reperfusion-associated tissue injury, TNF-α expression, and lethality. In contrast, there was marked decrease in IL-10 production. Facilitation of IL-1 action by administration of anti-IL-1ra, which antagonizes endogenous IL-1ra, or exogenous administration of rIL-1β suppressed reperfusion-induced tissue pathology, TNF-α production, and lethality, but increased IL-10 production. Exogenous administration of IL-10 was effective in preventing the increase in tissue or plasma levels of TNF-α, the exacerbated tissue injury, and lethality. An opposite effect was observed after treatment with anti-IL-10, demonstrating a role for endogenous production of IL-10 in modulating exacerbated reperfusion-associated tissue pathology and lethality. Finally, pretreatment with anti-IL-10 reversed the protective effect of IL-1β on reperfusion-associated lethality. Thus, IL-1 plays a major role in driving endogenous IL-10 production and protects against the TNF-α-dependent systemic and local acute inflammatory response following intestinal reperfusion injury.
Tyrosine Phosphorylation of I-κB Kinase α/β by Protein Kinase C-Dependent c-Src Activation Is Involved in TNF-α-Induced Cyclooxygenase-2 Expression
Authors: Huang Wei-Chien , Chen Jun-Jie , Inoue Hiroyasu , Chen Ching-Chow
Published: April 2014

The signaling pathway involved in TNF-α-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-α- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-α- or TPA-induced I-κB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-κBα degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-α or TPA. Overexpression of the constitutively active PKCα (PKCα A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-κB-inducing kinase (NIK) (KA), or IKKβ (KM) mutant. The dominant-negative PKCα (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKα/β was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-α or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKβ, were identified to be crucial for NF-κB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKβ induced by TNF-α or TPA. These data suggest that, in addition to activating NIK, TNF-α also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKα/β, and go on to activate NF-κB, via serine phosphorylation and degradation of IκB-α, and, finally, to initiate COX-2 expression.
IFN-β Gene Deletion Leads to Augmented and Chronic Demyelinating Experimental Autoimmune Encephalomyelitis
Authors: Teige Ingrid , Treschow Alexandra , Teige Anna , Mattsson Ragnar , Navikas Vaidrius , Leanderson Tomas , Holmdahl Rikard , Issazadeh-Navikas Shohreh
Published: April 2014

Since the basic mechanisms behind the beneficial effects of IFN-β in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-β gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN-β knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells to proliferate or produce IFN-γ in response to recall Ag. Consequently, we addressed the effect of IFN-β on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN-β KO mice acquired a higher incidence and augmented EAE regardless of the source of T cells. This shows that the anti-inflammatory effect of endogenous IFN-β is predominantly exerted on the effector phase of the disease. Histopathological investigations of CNS in the effector phase revealed an extensive microglia activation and TNF-α production in IFN-β KO mice; this was virtually absent in wt littermates. This coincided with an increase in effector functions of T cells in IFN-β KO mice, as measured by IFN-γ and IL-4 production. We suggest that lack of endogenous IFN-β in CNS leads to augmented microglia activation, resulting in a sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits.
Necroinflammatory Liver Disease in BALB/c Background, TGF-β1-Deficient Mice Requires CD4+ T Cells
Authors: Rudner Lynnie A. , Lin Jack T. , Park Il-Kyoo , Cates Justin M. M. , Dyer Darci A. , Franz Douglas M. , French Margaret A. , Duncan Elizabeth M. , White Hillary D. , Gorham James D.
Published: April 2014

The etiology of autoimmune liver disease is poorly understood. BALB/c mice deficient in the immunoregulatory cytokine TGF-β1 spontaneously develop necroinflammatory liver disease, but the immune basis for the development of this pathology has not been demonstrated. Here, we show that BALB/c-TGF-β1−/− mice exhibit abnormal expansion in hepatic mononuclear cells (MNCs) compared with wild-type littermate control mice, particularly in the T cell and macrophage lineages. To test whether lymphocytes of the adaptive immune system are required for the spontaneous development of necroinflammatory liver disease, BALB/c-TGF-β1−/− mice were rendered deficient in B and T cells by crossing them with BALB/c-recombinase-activating gene 1−/− mice. BALB/c-TGF-β1−/−/recombinase-activating gene 1−/− double-knockout mice showed extended survival and did not develop necroinflammatory liver disease. The cytolytic activity of BALB/c-TGF-β1−/− hepatic lymphocytes was assessed using an in vitro CTL assay. CTL activity was much higher in BALB/c-TGF-β1−/− hepatic MNCs compared with littermate control hepatic MNCs and was particularly pronounced in the CD4+ T cell subset. Experimental depletion of CD4+ T cells in young BALB/c-TGF-β1−/− mice prevented the subsequent development of necroinflammatory liver disease, indicating that CD4+ T cells are essential for disease pathogenesis in vivo. These data definitively establish an immune-mediated etiology for necroinflammatory liver disease in BALB/c-TGF-β1−/− mice and demonstrate the importance of CD4+ T cells in disease pathogenesis in vivo. Furthermore, TGF-β1 has a critical role in homeostatic regulation of the hepatic immune system, inhibiting the development or expansion of hepatic cytolytic CD4+ T cells.
Albumin-Based Drug Delivery as Novel Therapeutic Approach for Rheumatoid Arthritis
Authors: Wunder Andreas , Müller-Ladner Ulf , Stelzer Ernst H. K. , Funk Jürgen , Neumann Elena , Stehle Gerd , Pap Thomas , Sinn Hannsjörg , Gay Steffen , Fiehn Christoph
Published: April 2014

We reported recently that albumin is a suitable drug carrier for targeted delivery of methotrexate (MTX) to tumors. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumors and are metabolized by malignant cells. MTX, covalently coupled to human serum albumin (MTX-HSA) for cancer treatment, is currently being evaluated in phase II clinical trials. Because synovium of patients with rheumatoid arthritis (RA) shares various features observed also in tumors, albumin-based drug targeting of inflamed joints might be an attractive therapeutic approach. Therefore, the pharmacokinetics of albumin and MTX in a mouse model of arthritis was examined. Additionally, uptake of albumin by synovial fibroblasts of RA patients and the efficacy of MTX and MTX-HSA in arthritic mice were studied. The results show that when compared with MTX, significantly higher amounts of albumin accumulate in inflamed paws, and significantly lower amounts of albumin are found in the liver and the kidneys. The protein is metabolized by human synovial fibroblasts in vitro and in vivo. MTX-HSA was significantly more effective in suppression of the onset of arthritis in mice than was MTX. In conclusion, albumin appears to be a suitable drug carrier in RA, most likely due to effects on synovial fibroblasts, which might increase therapeutic efficacy and reduce side effects of MTX.
Transfer of Severe Experimental Autoimmune Encephalomyelitis by IL-12- and IL-18-Potentiated T Cells Is Estrogen Sensitive
Authors: Ito Atsushi , Matejuk Agata , Hopke Corwyn , Drought Heather , Dwyer Jami , Zamora Alex , Subramanian Sandhya , Vandenbark Arthur A. , Offner Halina
Published: April 2014

The aim of this study was to evaluate the roles of IL-18 and IL-12 in potentiating the encephalitogenic activity of T cell lines specific for myelin oligodendrocyte glycoprotein (MOG35–55). MOG-specific T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-12 or IL-18 alone transferred only mild experimental autoimmune encephalomyelitis (EAE) into a low percentage of recipients. However, T cells cocultured with both cytokines transferred aggressive clinical and histological EAE into all recipients. Coculture of T cells with IL-12 enhanced the secretion of IFN-γ, but not TNF-α, whereas coculture with IL-18 enhanced the secretion of TNF-α, but not INF-γ. However, coculture with both IL-18 and IL-12 induced high levels of both TNF-α and IFN-γ. Additionally, IL-12 selectively enhanced mRNA expression of CCR5, whereas IL-18 selectively enhanced the expression of CCR4 and CCR7, and CCR4 and CCR5 were coexpressed on the surface of T cells cocultured with IL-12 and IL-18. Finally, estrogen treatment, previously found to inhibit both TNF-α and IFN-γ production, completely abrogated all signs of passive EAE. These data demonstrate that optimal potentiation of encephalitogenic activity can be achieved by conditioning MOG-specific T cells with the combination of IL-12 and IL-18, which, respectively, induce the secretion of IFN-γ/CCR5 and TNF-α/CCR4/CCR7, and that estrogen treatment, which is known to inhibit both proinflammatory cytokines, can completely ablate this aggressive form of passive EAE.
CC Chemokine Ligand 1 Promotes Recruitment of Eosinophils But Not Th2 Cells During the Development of Allergic Airways Disease
Authors: Bishop Benjamin , Lloyd Clare M.
Published: April 2014

One of the characteristic features of allergic asthma is recruitment of large numbers of inflammatory cells including eosinophils and Th2 lymphocytes to the lung. This influx of inflammatory cells is thought to be a controlled and coordinated process mediated by chemokines and their receptors. It is thought that distinct, differential expression of chemokine receptors allows selective migration of T cell subtypes in response to the chemokines that bind these receptors. Th2 cells preferentially express CCR8 and migrate selectively to its ligand, CC chemokine ligand (CCL)1. We studied the role of the CCR8 ligand, CCL1, in the specific recruitment of Th2 cells and eosinophils to the lung in a murine model of allergic airway disease. We have demonstrated for the first time that CCL1 is up-regulated in the lung following allergen challenge. Moreover, a neutralizing Ab to CCL1 reduced eosinophil migration to the lung, but had no effect on recruitment of Th2 cells following allergen challenge. In addition, there was no change in airway hyperresponsiveness or levels of Th2 cytokines. In a Th2 cell transfer system of pulmonary inflammation, anti-CCL1 also failed to affect recruitment of Th2 cells to the lung following allergen challenge. Significantly, intratracheal instillation of rCCL1 increased recruitment of eosinophils but not Th2 cells to the lung in allergen-sensitized and -challenged mice. In summary, our results indicate that CCL1 is important for the pulmonary recruitment of eosinophils, rather than allergen-specific Th2 cells, following allergen challenge.
Differential Contribution of IL-4 and STAT6 vs STAT4 to the Development of Lupus Nephritis
Authors: Singh Ram Raj , Saxena Vijay , Zang Song , Li Lily , Finkelman Fred D. , Witte David P. , Jacob Chaim O.
Published: April 2014

Mechanisms that initiate lupus nephritis and cause progression to end-stage renal disease remain poorly understood. In this study, we show that lupus-prone New Zealand Mixed 2410 mice that develop a severe glomerulosclerosis and rapidly progressive renal disease overexpress IL-4 in vivo. In these mice, STAT6 deficiency or anti-IL-4 Ab treatment decreases type 2 cytokine responses and ameliorates kidney disease, particularly glomerulosclerosis, despite the presence of high levels of IgG anti-dsDNA Abs. STAT4 deficiency, however, decreases type 1 and increases type 2 cytokine responses, and accelerates nephritis, in the absence of high levels of IgG anti-dsDNA Abs. Thus, STAT6 and IL-4 may selectively contribute to the development of glomerulosclerosis, whereas STAT4 may play a role in autoantibody production.
The Naive B Cell Repertoire Predisposes to Antigen-Induced Systemic Lupus Erythematosus
Authors: Wang Chuansheng , Khalil Magi , Ravetch Jeffrey , Diamond Betty
Published: April 2014

It is clear that the development of an autoimmune disease usually depends on both a genetic predisposition and an environmental trigger. In this study, we demonstrate that BALB/c mice develop a lupus-like serology following immunization with a peptide mimetope of DNA, while DBA/2 mice do not. We further demonstrate that the critical difference resides within the B cell compartment and that the naive B cell repertoire of DBA/2 mice has fewer B cells specific for the DNA mimetope. Differences in the strength of B cell receptor signaling exist between these two strains and may be responsible for the difference in disease susceptibility. BALB/c mice possess more autoreactive cells in the native repertoire; they display a weaker response to Ag and exhibit less Ag-induced apoptosis of B cells. DBA/2 mice, in contrast, display a stronger B cell receptor signal and more stringent central tolerance. This correlates with resistance to lupus induction. Thus, the degree to which autoreactive B cells have been eliminated from the naive B cell repertoire is genetically regulated and may determine whether a nonspontaneously autoimmune host will develop autoimmunity following exposure to Ag.