bluuebunny/crossref_metadata_2025 huggingface dataset:
Tid1 Is Required for T Cell Transition from Double-Negative 3 to Double-Positive Stages
Authors: Lo Jeng-Fan , Zhou He , Fearns Colleen , Reisfeld Ralph A. , Yang Young , Lee Jiing-Dwan
Published: April 2014

Tid1, a DnaJ cochaperone protein, is the mammalian homologue of the Drosophila tumor suppressor Tid56 whose antitumor function is most likely mediated through its capacity to regulate cell differentiation in imaginal discs. We suspected that the mammalian counterpart, tid1, may also be involved in regulating cell differentiation. To investigate this, we exploited the system of T cell development to examine whether tid1 plays a role in this well-defined process. Mice with tid1 specifically deleted in T cells developed thymic atrophy, with dramatic reduction of double-positive and single-positive thymocytes in the tid1−/− thymus. Although the subpopulations of tid1−/− double-negative (DN) 1–3 thymocytes were normal, the subpopulation of DN4 thymocytes was measurably smaller because of reduced proliferation and significant cell death. Immature tid1−/− thymocytes show normal VDJ β-chain rearrangement and pre-TCR and CD3 expression in both DN3 and DN4 thymocytes, but in DN4 thymocytes, there was significantly reduced expression of the antiapoptotic bcl-2 gene. Restoring the expression level of Bcl-2 protein in tid1−/− thymus by introduction of a transgenic human bcl-2 gene resulted in reversal of the developmental defects in tid1−/− thymus. Together, these results demonstrate that tid1 is critical in early thymocyte development, especially during transition from the DN3 to double-positive stages, possibly through its regulation of bcl-2 expression, which provides survival signals.
CpG-Independent Synergistic Induction of β-Chemokines and a Dendritic Cell Phenotype by Orthophosphorothioate Oligodeoxynucleotides and Granulocyte-Macrophage Colony-Stimulating Factor in Elutriated Human Primary Monocytes
Authors: Wang Jinhai , Alvarez Raymond , Roderiquez Gregory , Guan Ennan , Caldwell Quincy , Wang Jiun , Phelan Michael , Norcross Michael A.
Published: April 2014

Chemokines attract leukocytes bearing the relevant chemokine receptors and regulate innate immune responses. CpG oligodeoxynucleotides (ODN) and GM-CSF are potent vaccine adjuvants and in combination induce enhanced Th1 responses by mechanisms yet to be determined. We have examined combinations of CpG- or non-CpG-ODN and GM-CSF for effects on the production of chemokines and the differentiation of monocytes to dendritic cells. High levels of the Th1-attracting, HIV-1-inhibitory chemokines, CCL3/MIP-1α and CCL4/MIP-1β, were induced in human primary monocytes when CpG- or non-CpG-ODN was combined with GM-CSF, but not with IL-4 or IFN-γ. The synergistic induction of β-chemokines by non-CpG-ODN was phosphorothioate (PS) chemistry dependent and inhibited by blocking endosome maturation/acidification and ERK1/2 activation. Chemokine and TLR9 mRNAs were induced by PS-ODN. Cells treated with non-CpG PS-ODN and GM-CSF expressed dendritic cell marker CD83 and high levels of HLA-DR and costimulatory molecules, and were CD14− or CD14dim, consistent with monocyte differentiation into a dendritic cell phenotype. The induction of CD83 and β-chemokines was tyrosine phosphorylation dependent. Secreted CCL3 and CCL4 were detected as a heterodimer. Our results indicate the CpG-independent synergy between PS-ODN and GM-CSF mediated through chemokine and dendritic cell induction. In addition, our observations suggest that PS-ODN plus GM-CSF may be useful as potent ex vivo dendritic cell differentiation/maturation agents for dendritic cell therapy and as vaccine adjuvants for tumor and infectious microorganisms, including HIV-1.
Impaired TGF-β Responses in Peripheral T Cells of Gαi2−/− Mice
Authors: Wu Jim Y. , Jin YongZhu , Edwards Robert A. , Zhang Yujin , Finegold Milton J. , Wu Mei X.
Published: April 2014

Null mutation of heterotrimeric G protein α2 inhibitory subunit (Gαi2) induces Th1-skewed hyperimmune responses in the colon, leading to chronic colitis and the development of colonic adenocarcinoma. However, the underlying molecular mechanisms and cellular basis, in particular, for the role of Gαi2 in regulating immune responses, are poorly understood. We show here that peripheral T cells from Gαi2-deficient mice do not respond normally to the inhibitory effects of TGF-β on proliferation and cytokine production, revealing a previously unappreciated cross-talk between these two signaling pathways. Lack of Gαi2 resulted in decreased phosphorylation of Smad2 and Smad3 in T cells at the basal levels as well as at the late but not early phase of TGF-β stimulation, which appears to be ascribed to differential expression of neither cell surface TGF-β receptors nor Smad7. The altered phosphorylation of Smad proteins involves phospholipase C-mediated signaling, a downstream signaling molecule of Gαi2, because phospholipase C inhibitors could restore Smad2 and Smad3 phosphorylation in Gαi2−/− T cells at levels comparable to that in wild-type T cells. Moreover, adoptive transfer of Gαi2-deficient T cells into immunocompromised mice rendered an otherwise resistant mouse strain susceptible to trinitrobenzesulfonic acid-induced colitis, suggesting that an impaired response of Gαi2-deficient T cells to TGF-β may be one of the primary defects accounting for the observed colonic Th1-skewed hyperimmune responses. These findings shed new lights on the molecular and cellular basis of how Gαi2 down-regulates immune responses, contributing to the maintenance of mucosal tolerance.
Endosomal Translocation of Vertebrate DNA Activates Dendritic Cells via TLR9-Dependent and -Independent Pathways
Authors: Yasuda Kei , Yu Philipp , Kirschning Carsten J. , Schlatter Beatrix , Schmitz Frank , Heit Antje , Bauer Stefan , Hochrein Hubertus , Wagner Hermann
Published: April 2014

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.
TGF-α Regulates TLR Expression and Function on Epidermal Keratinocytes
Authors: Miller Lloyd S. , Sørensen Ole E. , Liu Philip T. , Jalian H. Ray , Eshtiaghpour Deborah , Behmanesh Behnaz E. , Chung Woosin , Starner Timothy D. , Kim Jenny , Sieling Peter A. , Ganz Tomas , Modlin Robert L.
Published: April 2014

The expression of TLRs on epithelial cells provides a first line of defense against invading pathogens. We investigated the regulated expression and function of TLR5 and TLR9 on human keratinocytes, because we found by immunohistochemistry that these TLRs are expressed in distinct layers of the epidermis. We found that TGF-α, a growth and differentiation factor that is present during wound healing and in psoriasis, increased the expression of both TLR5 and TLR9 on keratinocytes. In addition, TGF-α regulated the function of TLR5 and TLR9, because activation with their respective ligands enhanced the production of IL-8 and human β-defensins. These findings provide evidence that TGF-α up-regulates TLR expression and function, augmenting host defense mechanisms at epithelial surfaces.
Transcriptional Profiling of γδ T Cells Identifies a Role for Vitamin D in the Immunoregulation of the Vγ9Vδ2 Response to Phosphate-Containing Ligands
Authors: Chen Lanfen , Cencioni Maria Teresa , Angelini Daniela F. , Borsellino Giovanna , Battistini Luca , Brosnan Celia F.
Published: April 2014

Vitamin D is a steroid hormone that, in addition to its well-characterized role in calcium/phosphate metabolism, has been found to have regulatory properties for immune system function. The nuclear vitamin D receptor is widely expressed in tissues, but has also been shown to be regulated by hormones, growth factors, and cytokines. In this study we show that activation of human Vδ2Vγ9 T cells by nonpeptidic monoalkyl phosphates such as isopentenyl pyrophosphate leads to the up-regulation of the vitamin D receptor via a pathway that involves the classical isoforms of protein kinase C. We further show that this receptor is active by demonstrating that the ligand 1α,25-dihydroxyvitamin D3 (vitD3) significantly inhibits in a dose-dependent fashion phospholigand-induced γδ T cell expansion, IFN-γ production, and CD25 expression. We also show that vitD3 negatively regulates signaling via Akt and ERK and, at high concentrations, potentiates Ag-induced cell death. As such, these data provide further support for the immunoregulatory properties of vitamin D, and suggest that the ability of vitD3 to negatively regulate the proinflammatory activity of γδ T cells may contribute to the protection this vitamin affords against inflammatory and autoimmune disorders dependent upon Th1-type responses.
An Ancient Role for a Prokineticin Domain in Invertebrate Hematopoiesis
Authors: Söderhäll Irene , Kim Young-A , Jiravanichpaisal Pikul , Lee So-Young , Söderhäll Kenneth
Published: April 2014

Hemopoietic development requires firm control of cell proliferation and differentiation. Although recent research has revealed conserved function of transcription factors and signaling pathways regulating lineage commitment in hemopoietic development in Drosophila melanogaster and vertebrates, little is known about hemopoietic cytokines among the invertebrate phyla. In the present study, we show that differentiation and growth of hemopoietic stem cells in vitro from an invertebrate, Pacifastacus leniusculus, require an endogenous cytokine-like factor, astakine, containing a prokineticin (PK) domain. Astakine induces a strong hematopoiesis response in live animals. An astakine homologue was also found in the shrimp, Penaeus monodon. So far, PK domains are only identified in vertebrates, in which they, for example, direct angiogenic growth. Our finding of the first PK-like cytokine characterized from any invertebrate provides novel information concerning the evolution of growth factors and blood cell development.
Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells
Authors: Nasr Isam W. , Wang Yinong , Gao Ge , Deng Songyan , Diggs Lonnette , Rothstein David M. , Tellides George , Lakkis Fadi G. , Dai Zhenhua
Published: April 2014

Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8+ T cells but induces more Ag-specific CD4+CD25+ regulatory T cells than in a conventional site. These CD4+CD25+ cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8+ T cells or deletion of CD25+ T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.
Enforced Expression of Spi-B Reverses T Lineage Commitment and Blocks β-Selection
Authors: Lefebvre Juliette M. , Haks Mariëlle C. , Carleton Michael O. , Rhodes Michele , Sinnathamby Gomathinayagam , Simon M. Celeste , Eisenlohr Laurence C. , Garrett-Sinha Lee Ann , Wiest David L.
Published: April 2014

The molecular changes that restrict multipotent murine thymocytes to the T cell lineage and render them responsive to Ag receptor signals remain poorly understood. In this study, we report our analysis of the role of the Ets transcription factor, Spi-B, in this process. Spi-B expression is acutely induced coincident with T cell lineage commitment at the CD4−CD8−CD44−CD25+ (DN3) stage of thymocyte development and is then down-regulated as thymocytes respond to pre-TCR signals and develop beyond the β-selection checkpoint to the CD4−CD8−CD44−CD25− (DN4) stage. We found that dysregulation of Spi-B expression in DN3 thymocytes resulted in a dose-dependent perturbation of thymocyte development. Indeed, DN3 thymocytes expressing approximately five times the endogenous level of Spi-B were arrested at the β-selection checkpoint, due to impaired induction of Egr proteins, which are important molecular effectors of the β-selection checkpoint. T lineage-committed DN3 thymocytes expressing even higher levels of Spi-B were diverted to the dendritic cell lineage. Thus, we demonstrate that the prescribed modulation of Spi-B expression is important for T lineage commitment and differentiation beyond the β-selection checkpoint; and we provide insight into the mechanism underlying perturbation of development when that expression pattern is disrupted.
Mechanisms of Dimethyl Sulfoxide Augmentation of IL-1β Production
Authors: Xing Liyu , Remick Daniel G.
Published: April 2014

Expression of the inflammatory cytokine IL-1β occurs in various inflammatory diseases, and IL-1β production is regulated at multiple levels. There are conflicting reports about the effects of antioxidants on IL-1β production. In this study, we investigated the regulatory role of the antioxidant DMSO on LPS-stimulated IL-1β gene expression in human PBMC and in vivo. This study demonstrated that 1% DMSO increased LPS-stimulated (50 ng/ml) IL-1β secretion in a dose- and time-dependent manner without altering TNF or IL-6. DMSO also elevated IL-1β secretion by PBMC in response to exogenous superoxide anions. Despite the increase in IL-1β, there was no augmentation of NF-κB with the addition of DMSO. The steady state mRNA coding for IL-1β following LPS stimulation was also increased. Cycloheximide studies demonstrated that the DMSO augmentation of IL-1β mRNA did not require de novo protein synthesis, and studies with actinomycin D showed that DMSO did not alter the half-life of IL-1β mRNA, suggesting that DMSO did not change the stability of IL-1β mRNA. Experiments using a reporter vector containing the 5′-flanking region of the human IL-1β gene revealed that DMSO augmented LPS-induced IL-1β reporter activity. In vivo, treatment of mice with DMSO significantly increased plasma levels of IL-1β after endotoxin challenge. These data indicate that DMSO directly increases LPS-stimulated IL-1β protein production through the mechanisms of augmenting promoter activity and increasing mRNA levels.
IFN-γ-Stimulated Transcriptional Activation by IFN-γ-Activated Transcriptional Element-Binding Factor 1 Occurs via an Inducible Interaction with CAAAT/Enhancer-Binding Protein-β
Authors: Meng Qingjun , Raha Abhijit , Roy Sanjit , Hu Junbo , Kalvakolanu Dhananjaya V.
Published: April 2014

IFN-γ-activated transcriptional element (GATE)-binding factor 1 (GBF1) was identified as a transactivator that induces gene expression through GATE, a novel IFN-inducible element. Although it can induce gene expression, it is an extremely weak DNA-binding protein on its own. GATE also binds another transcription factor, C/EBP-β. Therefore, we explored whether GBF1 physically interacts with C/EBP-β to induce IFN-γ-regulated transcription. In response to IFN-γ, C/EBP-β undergoes phosphorylation at a critical ERK1/2 phosphorylation motif. Mutational inactivation of this motif and/or interference with the ERK1/2 activation prevented the IFN-γ-induced interactions between GBF1 and C/EBP-β. A 37-aa long peptide derived from the GBF1 protein can associate with C/EBP-β in an IFN-inducible manner. These results identify a converging point for two transactivators that exert their effects through a single response element. Together, our studies identify a novel regulatory mechanism that controls IFN-induced transcription.
Electroporation as a “Prime/Boost” Strategy for Naked DNA Vaccination against a Tumor Antigen
Authors: Buchan Sarah , Grønevik Eirik , Mathiesen Iacob , King Catherine A. , Stevenson Freda K. , Rice Jason
Published: April 2014

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetnus toxin, amplification of anti-tumor Ab, CD4+, and CD8+ T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8+ T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8+ T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.
Binding of the Complement Inhibitor C4bp to Serogroup BNeisseria meningitidis
Authors: Jarva Hanna , Ram Sanjay , Vogel Ulrich , Blom Anna M. , Meri Seppo
Published: April 2014

Neisseria meningitidis (meningococcus) is an important cause of meningitis and sepsis. Currently, there is no effective vaccine against serogroup B meningococcal infection. Host defense against neisseriae requires the complement system (C) as indicated by the fact that individuals deficient in properdin or late C components (C6-9) have an increased susceptibility to recurrent neisserial infections. Because the classical pathway (CP) is required to initiate efficient complement activation on neisseriae, meningococci should be able to evade it to cause disease. To test this hypothesis, we studied the interactions of meningococci with the major CP inhibitor C4b-binding protein (C4bp). We tested C4bp binding to wild-type group B meningococcus strain (H44/76) and to 11 isogenic mutants thereof that differed in capsule expression, lipo-oligosaccharide sialylation, and/or expression of either porin (Por) A or PorB3. All strains expressing PorA bound radiolabeled C4bp, whereas the strains lacking PorA bound significantly less C4bp. Increased binding was observed under hypotonic conditions. Deleting PorB3 did not influence C4bp binding, but the presence of polysialic acid capsule reduced C4bp binding by 50%. Bound C4bp remained functionally active in that it promoted the inactivation of C4b by factor I. PorA-expressing strains were also more resistant to C lysis than PorA-negative strains in a serum bactericidal assay. Binding of C4bp thus helps Neisseria meningitidis to escape CP complement activation.
Exchanging ESAT6 with TB10.4 in an Ag85B Fusion Molecule-Based Tuberculosis Subunit Vaccine: Efficient Protection and ESAT6-Based Sensitive Monitoring of Vaccine Efficacy
Authors: Dietrich Jes , Aagaard Claus , Leah Robert , Olsen Anja W. , Stryhn Anette , Doherty T. Mark , Andersen Peter
Published: April 2014

Previously we have shown that Ag85B-ESAT-6 is a highly efficient vaccine against tuberculosis. However, because the ESAT-6 Ag is also an extremely valuable diagnostic reagent, finding a vaccine as effective as Ag85B-ESAT-6 that does not contain ESAT-6 is a high priority. Recently, we identified a novel protein expressed by Mycobacterium tuberculosis designated TB10.4. In most infected humans, TB10.4 is strongly recognized, raising interest in TB10.4 as a potential vaccine candidate and substitute for ESAT-6. We have now examined the vaccine potential of this protein and found that vaccination with TB10.4 induced a significant protection against tuberculosis. Fusing Ag85B to TB10.4 produced an even more effective vaccine, which induced protection against tuberculosis comparable to bacillus Calmette-Guérin vaccination and superior to the individual Ag components. Thus, Ag85B-TB10 represents a new promising vaccine candidate against tuberculosis. Furthermore, having now exchanged ESAT-6 for TB10.4, we show that ESAT-6, apart from being an excellent diagnostic reagent, can also be used as a reagent for monitoring vaccine efficacy. This may open a new way for monitoring vaccine efficacy in clinical trials.
Cyclic AMP Response Element-Binding Protein Positively Regulates Production of IFN-γ by T Cells in Response to a Microbial Pathogen
Authors: Samten Buka , Howard Susan T. , Weis Steven E. , Wu Shiping , Shams Homayoun , Townsend James C. , Safi Hassan , Barnes Peter F.
Published: April 2014

IFN-γ is essential for resistance to many intracellular pathogens, including Mycobacterium tuberculosis. Transcription of the IFN-γ gene in activated T cells is controlled by the proximal promoter element (−73 to −48 bp). CREB binds to the IFN-γ proximal promoter, and binding is enhanced by phosphorylation of CREB. Studies in human T cell lines and in transgenic mice have yielded conflicting results about whether CREB is a positive or a negative regulator of IFN-γ transcription. To determine the role of CREB in mediating IFN-γ production in response to a microbial pathogen, we evaluated the peripheral blood T cell response to M. tuberculosis in healthy tuberculin reactors. EMSAs, chromatin immunoprecipitation, and Western blotting demonstrated that stimulation of PBMC with M. tuberculosis induced phosphorylation and enhanced binding of CREB to the IFN-γ proximal promoter. Neutralization of CREB with intracellular Abs or down-regulation of CREB levels with small interfering RNA decreased M. tuberculosis-induced production of IFN-γ and IFN-γ mRNA expression. In addition, M. tuberculosis-stimulated T cells from tuberculosis patients, who have ineffective immunity, showed diminished IFN-γ production, reduced amounts of CREB binding to the IFN-γ proximal promoter, and absence of phosphorylated CREB. These findings demonstrate that CREB positively regulates IFN-γ production by human T cells that respond to M. tuberculosis.
Estradiol Is Required for a Proper Immune Response to Bacterial and Viral Pathogens in the Female Brain
Authors: Soucy Geneviève , Boivin Guy , Labrie Fernand , Rivest Serge
Published: April 2014

Although the neuroprotective effects of estrogens are well recognized, the exact mechanisms involved in the ability of these sex steroids to protect the cerebral tissue still remain unclear. We tested in our study the hypothesis that estradiol (E2) modulates the innate immune response and expression of genes encoding proteins that a provide survival signal to neurons during infection. Mice received a single systemic or cerebral injection of LPS to trigger a robust but transient inflammatory reaction in the brain. The endotoxin increased transcriptional activation of genes encoding TLR2, TNF-α, and IL-12 in microglial cells. Expression of these transcripts was largely inhibited in the brain of ovariectomized mice at time 24 h postchallenge. E2 replacement therapy totally rescued the ability of the endotoxin to trigger microglial cells and these permissive effects of E2 are mediated via the estrogen receptor (ER)α. Indeed, ERα-deficient mice exhibited an inappropriate reaction to LPS when compared with ERβ-deficient and wild-type mice. This defective innate immune response was also associated with a widespread viral replication and neurodegeneration in ovariectomized mice inoculated intranasally with HSV-2. These data provide evidence that interaction of E2 with their nuclear ERα plays a critical role in the control of cytokines involved in the transfer from the innate to adaptive immunity. This transfer is deviant in mice lacking E2, which allows pathogens to hide from immune surveillance and exacerbates neuronal damages during viral encephalitis.
Neutrophil-Derived Heparin-Binding Protein (HBP/CAP37) Deposited on Endothelium Enhances Monocyte Arrest under Flow Conditions
Authors: Soehnlein Oliver , Xie Xun , Ulbrich Holger , Kenne Ellinor , Rotzius Pierre , Flodgaard Hans , Eriksson Einar E. , Lindbom Lennart
Published: April 2014

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca2+ that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.
Junctional Adhesion Molecule-C Regulates the Early Influx of Leukocytes into Tissues during Inflammation
Authors: Aurrand-Lions Michel , Lamagna Chrystelle , Dangerfield John P. , Wang Shijun , Herrera Pedro , Nourshargh Sussan , Imhof Beat A.
Published: April 2014

Leukocyte recruitment from blood to inflammatory sites occurs in a multistep process that involves discrete molecular interactions between circulating and endothelial cells. Junctional adhesion molecule (JAM)-C is expressed at different levels on endothelial cells of lymphoid organs and peripheral tissues and has been proposed to regulate neutrophil migration by its interaction with the leukocyte integrin Mac-1. In the present study, we show that the accumulation of leukocytes in alveoli during acute pulmonary inflammation in mice is partially blocked using neutralizing Abs against JAM-C. To confirm the function of JAM-C in regulating leukocyte migration in vivo, we then generated a strain of transgenic mice overexpressing JAM-C under the control of the endothelial specific promotor Tie2. The transgenic animals accumulate more leukocytes to inflammatory sites compared with littermate control mice. Intravital microscopy shows that this is the result of increased leukocyte adhesion and transmigration, whereas rolling of leukocytes is not significantly affected in transgenic mice compared with littermates. Thus, JAM-C participates in the later steps of the leukoendothelial adhesion cascade.
Questioning Current Concepts in Acute Pancreatitis: Endotoxin Contamination of Porcine Pancreatic Elastase Is Responsible for Experimental Pancreatitis-Associated Distant Organ Failure
Authors: Geisler Fabian , Algül Hana , Riemann Marc , Schmid Roland M.
Published: April 2014

The systemic inflammatory response syndrome is responsible for pancreatitis-associated mortality. Recent in vitro and in vivo studies have suggested that pancreatic elastase is one missing link between the localized inflammatory process in the pancreas and distant organ dysfunction and failure. It has been shown that pancreatic elastase activates transcription factors, including NF-κB, and induces TNF-α secretion in myeloid cells via TLRs. In this study we demonstrate that a highly purified low endotoxin pancreatic elastase preparation (El-UP) failed both to activate NF-κB and to induce TNF-α release in RAW 264.7 cells and bone marrow-derived macrophages. In contrast, a less purified elastase preparation (El-IV) caused activation of NF-κB and was able to induce TNF-α release at very low concentrations. These effects were sensitive to pretreatment of the cells with polymyxin B and were resistant to heat inactivation. Endotoxin activity as determined by the Limulus amebocyte lysate assay was >3 orders of magnitude lower in the low endotoxin elastase preparation (El-UP) compared with less purified elastase preparations (El-IV). In contrast to contaminated elastase or LPS, elastase free of contamination (El-UP) failed to induce elevated serum TNF-α levels or pulmonary neutrophil infiltration after i.p. application in mice and did not induce lethality when coinjected with d-galactosamine. Failure of low endotoxin elastase (El-UP) to induce proinflammatory effects in vivo and in vitro was not due to functional inactivity of the elastase preparation, as determined by elastase activity assay. These results question current concepts of direct proinflammatory effects attributed to pancreatic elastase.
Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2Rγnull Mice Engrafted with Mobilized Human Hemopoietic Stem Cells
Authors: Shultz Leonard D. , Lyons Bonnie L. , Burzenski Lisa M. , Gott Bruce , Chen Xiaohua , Chaleff Stanley , Kotb Malak , Gillies Stephen D. , King Marie , Mangada Julie , Greiner Dale L. , Handgretinger Rupert
Published: April 2014

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common γ-chain deficient NOD/LtSz-scid (NOD-scid IL2Rγnull) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rγnull mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rγnull mice with human HSC generate 6-fold higher percentages of human CD45+ cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rγnull mice contain human Ig+ B cells and lower numbers of human CD3+ T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4+CD8+ thymocytes as well human CD4+CD8− and CD4−CD8+ peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rγnull mice was validated by 1) high levels of TCR excision circles, 2) complex TCRβ repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rγnull mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.
Activation of Discoidin Domain Receptor 1 on CD14-Positive Bronchoalveolar Lavage Fluid Cells Induces Chemokine Production in Idiopathic Pulmonary Fibrosis
Authors: Matsuyama Wataru , Watanabe Masaki , Shirahama Yuko , Oonakahara Ken-ichi , Higashimoto Ikkou , Yoshimura Teizo , Osame Mitsuhiro , Arimura Kimiyoshi
Published: April 2014

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by collagen. We previously reported the functional expression of DDR1 on human monocyte-derived macrophages in vitro; however, information regarding its role in diseases is limited. Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease, and the lesions contain an abundance of collagen. In this study, we examined DDR1 expression on bronchoalveolar lavage fluid (BALF) cells and investigated its functionality using samples obtained from 28 IPF patients, 13 chronic obstructive pulmonary disease patients, and 14 healthy volunteers. The DDR1 expression level in CD14-positive BALF cells was higher in IPF patients than in chronic obstructive pulmonary disease patients or healthy volunteers. The predominant isoform was DDR1b in the IPF group, while DDR1a was predominant in the other two groups. Using immunohistochemical analysis, we also detected DDR1 expression on infiltrating inflammatory cells in the IPF lesion. In IPF patients, DDR1 activation induced the production of MCP-1, IL-8, MIP-1 α, and matrix metalloproteinase-9 (MMP-9) from CD14-positive BALF cells in a p38 MAPK-dependent manner. In contrast, DDR1 activation of CD14-positive BALF cells in the other groups did not induce the production of these chemokines or MMP-9. These chemokines and MMP-9 contribute to the development of IPF and, therefore, we suggest that DDR1 might be associated with the pathogenesis of IPF in the tissue microenvironment.
Absence of Recipient CCR5 Promotes Early and Increased Allospecific Antibody Responses to Cardiac Allografts
Authors: Amano Hiroyuki , Bickerstaff Alice , Orosz Charles G. , Novick Andrew C. , Toma Hiroshi , Fairchild Robert L.
Published: April 2014

Acute rejection is mediated by T cell infiltration of allografts, but mechanisms mediating the delayed rejection of allografts in chemokine receptor-deficient recipients remain unclear. The rejection of vascularized, MHC-mismatched cardiac allografts by CCR5−/− recipients was investigated. Heart grafts from A/J (H-2a) donors were rejected by wild-type C57BL/6 (H-2b) recipients on day 8–10 posttransplant vs day 8–11 by CCR5−/− recipients. When compared with grafts from wild-type recipients, however, significant decreases in CD4+ and CD8+ T cells and macrophages were observed in rejecting allografts from CCR5-deficient recipients. These decreases were accompanied by significantly lower numbers of alloreactive T cells developing to IFN-γ-, but not IL-4-producing cells in the CCR5−/− recipients, suggesting suboptimal priming of T cells in the knockout recipients. CCR5 was more prominently expressed on activated CD4+ than CD8+ T cells in the spleens of allograft wild-type recipients and on CD4+ T cells infiltrating the cardiac allografts. Rejecting cardiac allografts from wild-type recipients had low level deposition of C3d that was restricted to the graft vessels. Rejecting allografts from CCR5−/− recipients had intense C3d deposition in the vessels as well as on capillaries throughout the graft parenchyma similar to that observed during rejection in donor-sensitized recipients. Titers of donor-reactive Abs in the serum of CCR5−/− recipients were almost 20-fold higher than those induced in wild-type recipients, and the high titers appeared as early as day 6 posttransplant. These results suggest dysregulation of alloreactive Ab responses and Ab-mediated cardiac allograft rejection in the absence of recipient CCR5.
Human 60-kDa Heat Shock Protein Is a Target Autoantigen of T Cells Derived from Atherosclerotic Plaques
Authors: Benagiano Marisa , D’Elios Mario M. , Amedei Amedeo , Azzurri Annalisa , van der Zee Ruurd , Ciervo Alessandra , Rombolà Gianni , Romagnani Sergio , Cassone Antonio , Del Prete Gianfranco
Published: April 2014

Epidemiological studies suggest the potential importance of an inflammatory component in atherosclerosis and support the hypothesis that immune responses to Ags of pathogens cross-react with homologous host proteins due to molecular mimicry. Protein candidates involved may be the stress-induced proteins known as heat shock proteins (HSP). In this study, we report that atherosclerotic plaques harbor in vivo-activated CD4+ T cells that recognize the human 60-kDa HSP. Such in vivo-activated 60-kDa HSP-specific T cells are not detectable in the peripheral blood. In patients with positive serology and PCR for Chlamydia pneumoniae DNA, but not in patients negative for both, most of plaque-derived T cells specific for human 60-kDa HSP also recognized the C. pneumoniae 60-kDa HSP. We characterized the submolecular specificity of such 60-kDa HSP-specific plaque-derived T cells and identified both the self- and cross-reactive epitopes of that autoantigen. On challenge with human 60-kDa HSP, most of the plaque-derived T cells expressed Th type 1 functions, including cytotoxicity and help for monocyte tissue factor production. We suggest that arterial endothelial cells, undergoing classical atherosclerosis risk factors and conditioned by Th type 1 cytokines, express self 60-kDa HSP, which becomes target for both autoreactive T cells and cross-reactive T cells to microbial 60-kDa HSP via a mechanism of molecular mimicry. This hypothesis is in agreement with the notion that immunization with HSP exacerbates atherosclerosis, whereas immunosuppression and T cell depletion prevent the formation of arteriosclerotic lesions in experimental animals.
Nucleotide-Binding Oligomerization Domain-2 Modulates Specific TLR Pathways for the Induction of Cytokine Release
Authors: Netea Mihai G. , Ferwerda Gerben , de Jong Dirk J. , Jansen Trees , Jacobs Liesbeth , Kramer Matthijs , Naber Ton H. J. , Drenth Joost P. H. , Girardin Stephen E. , Jan Kullberg Bart , Adema Gosse J. , Van der Meer Jos W. M.
Published: April 2014

The recognition of peptidoglycan by cells of the innate immune system has been controversial; both TLR2 and nucleotide-binding oligomerization domain-2 (NOD2) have been implicated in this process. In the present study we demonstrate that although NOD2 is required for recognition of peptidoglycan, this leads to strong synergistic effects on TLR2-mediated production of both pro- and anti-inflammatory cytokines. Defective IL-10 production in patients with Crohn’s disease bearing loss of function mutations of NOD2 may lead to overwhelming inflammation due to a subsequent Th1 bias. In addition to the potentiation of TLR2 effects, NOD2 is a modulator of signals transmitted through TLR4 and TLR3, but not through TLR5, TLR9, or TLR7. Thus, interaction between NOD2 and specific TLR pathways may represent an important modulatory mechanism of innate immune responses.
Cutting Edge: Atopy Promotes Th2 Responses to Alloantigens and Increases the Incidence and Tempo of Corneal Allograft Rejection
Authors: Beauregard Clay , Stevens Christina , Mayhew Elizabeth , Niederkorn Jerry Y.
Published: April 2014

A large body of evidence suggests that corneal allograft rejection is mediated by a type 1 Th cell response and that deviation toward type 2 immunity favors graft survival. However, clinical observations indicate that patients with severe ocular allergies have increased risk of corneal allograft rejection. We used a mouse model of atopic conjunctivitis to evaluate the effects of Th2 immune deviation on corneal allograft survival and possible mechanisms of graft rejection. Our results reveal the following novel findings: 1) atopic conjunctivitis promotes systemic Th2 immune responses to corneal graft donor alloantigens; 2) corneal allografts in atopic host eyes have an increased incidence and swifter tempo of rejection; 3) increased rejection is associated with alterations in systemic T cell-mediated responses to donor alloantigens; and 4) corneal allograft rejection in atopic hosts does not require the direct involvement of infiltrating eosinophils.
Cutting Edge: Silencing Suppressor of Cytokine Signaling 3 Expression in Dendritic Cells Turns CD28-Ig from Immune Adjuvant to Suppressant
Authors: Orabona Ciriana , Belladonna Maria Laura , Vacca Carmine , Bianchi Roberta , Fallarino Francesca , Volpi Claudia , Gizzi Stefania , Fioretti Maria Cristina , Grohmann Ursula , Puccetti Paolo
Published: April 2014

CTLA-4-Ig and CD28-Ig are both agonist ligands of B7 coreceptor molecules on mouse dendritic cells (DCs), yet they bias the downstream response in opposite directions, and CTLA-4-Ig promotes tolerance, whereas CD28-Ig favors the onset of immunity. Although B7 engagement by either ligand leads to a mixed cytokine response, a dominant IL-6 production in response to CD28-Ig prevents the IFN-γ-driven induction of immunosuppressive tryptophan catabolism mediated by IDO. In the present study, we show that silencing the expression of suppressor of cytokine signaling 3 (SOCS3) in DCs by RNA interference renders CD28-Ig capable of activating IDO, likely as a result of unrestrained IFN-γ signaling and IFN-γ-like actions of IL-6. Thus, in the absence of SOCS3, CD28-Ig becomes immunosuppressive and mimics the action of CTLA-4-Ig on tryptophan catabolism.
Cutting Edge: Expression of IL-1 Receptor-Associated Kinase-4 (IRAK-4) Proteins with Mutations Identified in a Patient with Recurrent Bacterial Infections Alters Normal IRAK-4 Interaction with Components of the IL-1 Receptor Complex
Authors: Medvedev Andrei E. , Thomas Karen , Awomoyi Agnes , Kuhns Douglas B. , Gallin John I. , Li Xiaoxia , Vogel Stefanie N.
Published: April 2014

In a patient with recurrent bacterial infections and profound hyporesponsiveness to LPS and IL-1, we previously identified two mutations in IL-1R-associated kinase-4 (IRAK-4) that encoded proteins with truncated kinase domains. Overexpression of either of these mutant IRAK-4 variants in HEK293 cells failed to activate endogenous IRAK-1 and suppressed IL-1-induced IRAK-1 kinase activity, in contrast to wild-type (WT) IRAK-4. In this study, interactions of WT and mutant IRAK-4 species with IL-1R, IRAK-1, and MyD88 in HEK293 transfectants were compared. IL-1 induced a strong interaction among the IL-1R, activated IRAK-1, MyD88, and WT, but not mutant, IRAK-4. Truncated IRAK-4 proteins constitutively interacted more strongly with MyD88 and blunted IL-1-induced recruitment of IRAK-1 and MyD88 to the IL-1R. Thus, decreased IL-1-induced association of IRAK-1 and MyD88 with the IL-1RI may result from sequestration of cytoplasmic MyD88 by IRAK-4 mutant proteins. Therefore, mimetics of these truncated IRAK-4 proteins may represent a novel approach to mitigating hyperinflammatory states.
Cutting Edge: Generation of Splenic CD8+ and CD8− Dendritic Cell Equivalents in Fms-Like Tyrosine Kinase 3 Ligand Bone Marrow Cultures
Authors: Naik Shalin H. , Proietto Anna I. , Wilson Nicholas S. , Dakic Aleksandar , Schnorrer Petra , Fuchsberger Martina , Lahoud Mireille H. , O’Keeffe Meredith , Shao Qi-xiang , Chen Wei-feng , Villadangos José A. , Shortman Ken , Wu Li
Published: April 2014

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8+ and CD8− conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RAhigh plasmacytoid DC, two distinct CD24high and CD11bhigh cDC subsets were present, and these subsets showed equivalent properties to splenic CD8+ and CD8− cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-α; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-α, MIP-1α, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24high subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.
EBV and Systemic Lupus Erythematosus: A New Perspective
Authors: Gross Andrew J. , Hochberg Donna , Rand William M. , Thorley-Lawson David A.
Published: April 2014

We have proposed that EBV uses mature B cell biology to access memory B cells as a site of persistent infection. A central feature of this model is that EBV adapts its gene expression profile to the state of the B cell it resides in and that the level of infection is stable over time. This led us to question whether changes in the behavior or regulation of mature B cells would alter the state of EBV persistence. To investigate this, we studied the impact of systemic lupus erythematosus (SLE), a disease characterized by immune dysfunction, on EBV infection. We show that patients with SLE have abnormally high frequencies of EBV-infected cells in their blood, and this is associated with the occurrence of SLE disease flares. Although patients with SLE have frequencies of infected cells comparable to those seen in immunosuppressed patients, in SLE the effect was independent of immunosuppressive therapy. Aberrant expression of viral lytic (BZLF1) and latency (latency membrane proteins 1 and 2a) genes was also detected in the blood of SLE patients. We conclude that the abnormal regulation of EBV infection in SLE patients reflects the sensitivity of the virus to perturbation of the immune system.
Cocaine Modulates Dendritic Cell-Specific C Type Intercellular Adhesion Molecule-3-Grabbing Nonintegrin Expression by Dendritic Cells in HIV-1 Patients
Authors: Nair Madhavan P. N. , Mahajan Supriya D. , Schwartz Stanley A. , Reynolds Jessica , Whitney Robert , Bernstein Zail , Chawda Ram P. , Sykes Don , Hewitt Ross , Hsiao Chiu Bin
Published: April 2014

We report that cocaine may act as cofactor in HIV pathogenesis by increasing dendritic cell-specific C type ICAM-3-grabbing nonintegrin (DC-SIGN) expression on dendritic cells (DC). Our results show that cocaine-using, long-term nonprogressors and normal progressors of HIV infection manifest significantly higher levels of DC-SIGN compared with cocaine-nonusing long-term nonprogressors and normal progressors, respectively. Furthermore, in vitro HIV infection of MDC from normal subjects cultured with cocaine and/or HIV peptides up-regulated DC-SIGN, confirming our in vivo finding. Cocaine, in synergy with HIV peptides, also up-regulates DC-SIGN gene expression by MDC. Furthermore, the cocaine-induced effects were reversed by a D1 receptor antagonist demonstrating the specificity of the reaction. Our results indicate that cocaine exacerbates HIV infection by up-regulating DC-SIGN on DC and these effects are mediated via dysregulation of MAPKs. These data are the first evidence that cocaine up-regulates the expression of DC-SIGN on DC. A better understanding of the role of DC-SIGN in HIV infection may help to design novel therapeutic strategies against the progression of HIV disease in the drug-using population.