bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of mRNA Turnover in the Regulation of Tristetraprolin Expression: Evidence for an Extracellular Signal-Regulated Kinase-Specific, AU-Rich Element-Dependent, Autoregulatory Pathway
Authors: Brooks Seth A. , Connolly John E. , Rigby William F. C.
Published: April 2014

Tristetraprolin (TTP) is a regulator of TNF-α mRNA stability and is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal. Using the THP-1 myelomonocytic cell line, we demonstrated for the first time that TTP is encoded by an mRNA with a short half-life under resting conditions. Using pharmacologic inhibitors of the mitogen-activated protein kinase pathways, we show that the induction of TTP by LPS activation is mediated through changes in transcription, mRNA stability, and translation. A coordinate increase in both TTP and TNF-α mRNA stability occurs within 15 min of LPS treatment, but is transduced through different mitogen-activated protein kinase pathways. This regulation of TTP and TNF-α mRNA stability is associated with the finding that TTP binds these mRNA under both resting and LPS-activated conditions in vivo. Finally, we demonstrate that TTP can regulate reporter gene expression in a TTP 3′ untranslated region-dependent manner and identify three distinct AU-rich elements necessary to mediate this effect. Thus, TTP regulates its own expression in a manner identical to that seen with the TNF-α 3′ untranslated region, indicating that this autoregulation is mediated at the level of mRNA stability. In this manner, TTP is able to limit the production of its own proteins as well as that of TNF-α and thus limit the response of the cell to LPS.
Tumor-Derived MUC1 Mucins Interact with Differentiating Monocytes and Induce IL-10highIL-12low Regulatory Dendritic Cell
Authors: Monti Paolo , Leone Biagio Eugenio , Zerbi Alessandro , Balzano Gianpaolo , Cainarca Silvia , Sordi Valeria , Pontillo Marina , Mercalli Alessia , Di Carlo Valerio , Allavena Paola , Piemonti Lorenzo
Published: April 2014

Dendritic cells (DC) initiate immunity by the activation of naive T cells and control immunity through their ability to induce unresponsiveness of lymphocytes by mechanisms that include deletion and induction of regulatory cells. An inadequate presentation to T cells by tumor-induced “regulatory” DC, among several mechanisms, can explain tolerance to tumor-associated Ags. In this study, we show that tumor-derived mucin profoundly affects the cytokine repertoire of monocyte-derived DC and switch them into IL-10highIL-12low regulatory APCs with a limited capacity to trigger protective Th1 responses. In fact, DC cocultured with pancreatic tumor cell lines in a Transwell system did not reach full maturation, had low immunostimulatory functions, did not produce IL-12, and released high levels of IL-10. The involvement of known tumor-derived immune-suppressive factors (e.g., vascular endothelial growth factor, TGF-β, IL-6, and IL-10) was considered and excluded. We provide evidence that tumor-derived MUC1 mucins are responsible for the impaired DC maturation and function. DC obtained in the presence of tumor microenvironment preferentially polarized IL-4+ response. Moreover, T cells primed by these regulatory DC became anergic and behaved as suppressor/regulatory cells. These findings identify mucin secretion as a novel mechanism of tumor escape from immune surveillance and provide the basis for the generation of potentially tolerogenic DC.
CD4−CD8αα Subset of CD1d-Restricted NKT Cells Controls T Cell Expansion
Authors: Ho Ling-Pei , Urban Britta C. , Jones Louise , Ogg Graham S. , McMichael Andrew J.
Published: April 2014

Vα24 invariant (Vα24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4−CD8αα+ subset of Vα24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4− and CD4+ Vα24i NKT clones and demonstrate that a CD4−CD8αα+ subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.
On the Relevance of TCR Rearrangement Circles as Molecular Markers for Thymic Output during Experimental Graft-versus-Host Disease
Authors: Krenger Werner , Schmidlin Heike , Cavadini Gionata , Holländer Georg A.
Published: April 2014

Efficient reconstitution of the pool of peripheral T cells after hemopoietic stem cell transplantation (HSCT) is dependent on normal thymic function. However, the development of graft-vs-host disease (GVHD) in the context of allogeneic HSCT is associated with injurious effects on thymocyte development. In this study, we examined in models of syngeneic and allogeneic murine HSCT whether actual posttransplant thymic output is accurately reflected by analysis of signal-joint TCR rearrangement excision circles (sjTRECs). Our data demonstrate that the de novo generation of T cells following syngeneic HSCT of T cell-deficient B6.RAG2−/− (recombination-activating gene 2−/−) mice correlates firmly with an increase of sjTRECs in the thymus and spleen. However, the altered homeostasis of naive peripheral T cells in the presence of GVHD necessitates the combined analysis of cell division in vivo and determinations of sjTREC contents and total sjTREC numbers to draw informative conclusions. From our data, we substantiate that thymic output and peripheral division of newly generated T cells are diminished in the presence of acute GVHD in an experimental radiation/allogeneic HSCT model.
A Two-Step Model of Acute CD4 T-Cell Mediated Cardiac Allograft Rejection
Authors: Grazia Todd J. , Pietra Biagio A. , Johnson Zachary A. , Kelly Brian P. , Plenter Robert J. , Gill Ronald G.
Published: April 2014

CD4 T cells are both necessary and sufficient to mediate acute cardiac allograft rejection in mice. This process requires “direct” engagement of donor MHC class II molecules. That is, acute rejection by CD4+ T cells requires target MHC class II expression by the donor and not by the host. However, it is unclear whether CD4+ T cell rejection requires MHC class II expression on donor hemopoietic cells, nonhemopoietic cells, or both. To address this issue, bone marrow transplantation in mice was used to generate chimeric heart donors in which MHC class II was expressed either on somatic or on hemopoietic cells. We report that direct recognition of hemopoietic and nonhemopoietic cells are individually rate limiting for CD4+ T cell-mediated rejection in vivo. Importantly, active immunization with MHC class II+ APCs triggered acute rejection of hearts expressing MHC class II only on the somatic compartment. Thus, donor somatic cells, including endothelial cells, are not sufficient to initiate acute rejection; but they are necessary as targets of direct alloreactive CD4 T cells. Taken together, results support a two-stage model in which donor passenger leukocytes are required to activate the CD4 response while direct interaction with the somatic compartment is necessary for the efferent phase of acute graft rejection.
The Electrostatic Nature of C3d-Complement Receptor 2 Association
Authors: Morikis Dimitrios , Lambris John D.
Published: April 2014

The association of complement component C3d with B or T cell complement receptor 2 (CR2 or CD21) is a link between innate and adaptive immunity. It has been recognized in experimental studies that the C3d-CR2 association is pH- and ionic strength-dependent. This led us to perform electrostatic calculations to obtain a theoretical understanding of the mechanism of C3d-CR2 association. We used the crystallographic structures of human free C3d, free CR2 (short consensus repeat (SCR)1–2), and the C3d-CR2(SCR1–2) complex, and continuum solvent representation, to obtain a detailed atomic-level picture of the components of the two molecules that contribute to association. Based on the calculation of electrostatic potentials for the free and bound species and apparent pKa values for each ionizable residue, we show that C3d-CR2(SCR1–2) recognition is electrostatic in nature and involves not only the association interface, but also the whole molecules. Our results are in qualitative agreement with experimental data that measured the ionic strength and pH dependence of C3d-CR2 association. Also, our results for the native molecules and a number of theoretical mutants of C3d explain experimental mutagenesis studies of amino acid replacements away from the association interface that modulate binding of iC3b with full-length CR2. Finally, we discuss the packing of the two SCR domains. Overall, our data provide global and site-specific explanations of the physical causes that underlie the ionic strength dependence of C3d-CR2 association in a unified model that accounts for all experimental data, some of which were previously thought to be contradictory.
Akt Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis
Authors: Bommhardt Ursula , Chang Katherine C. , Swanson Paul E. , Wagner Tracey H. , Tinsley Kevin W. , Karl Irene E. , Hotchkiss Richard S.
Published: April 2014

Sepsis induces extensive death of lymphocytes that may contribute to the immunosuppression and mortality of the disorder. The serine/threonine kinase Akt is a key regulator of cell proliferation and death. The purpose of this study was to determine whether overexpression of Akt would prevent lymphocyte apoptosis and improve survival in sepsis. In addition, given the important role of Akt in cell signaling, T cell Th1 and Th2 cytokine production was determined. Mice that overexpress a constitutively active Akt in lymphocytes were made septic, and survival was recorded. Lymphocyte apoptosis and cytokine production were determined at 24 h after surgery. Mice with overexpression of Akt had a marked improvement in survival compared with wild-type littermates, i.e., 94 and 47% survival, respectively, p < 0.01. In wild-type littermates, sepsis caused a marked decrease in IFN-γ production, while increasing IL-4 production >2-fold. In contrast, T cells from Akt transgenic mice had an elevated production of IFN-γ at baseline that was maintained during sepsis, while IL-4 had little change. Akt overexpression also decreased sepsis-induced lymphocyte apoptosis via a non-Bcl-2 mechanism. In conclusion, Akt overexpression in lymphocytes prevents sepsis-induced apoptosis, causes a Th1 cytokine propensity, and improves survival. Findings from this study strengthen the concept that a major defect in sepsis is impairment of the adaptive immune system, and suggest that strategies to prevent lymphocyte apoptosis represent a potential important new therapy.
Following Immunization Antigen Becomes Concentrated in a Limited Number of APCs Including B Cells
Authors: Byersdorfer Craig A. , DiPaolo Richard J. , Petzold Shirley J. , Unanue Emil R.
Published: April 2014

Immunization with the hen egg-white lysozyme (HEL) protein induces T cells to various of its peptide determinants. The distribution of such T cells, however, does not correlate with the peptide level of each epitope on class II molecules. For this reason, we sought information on the cells responsible for Ag presentation following immunization, hoping to understand the lack of immunodominance in this system. By tracking HEL, and the ensuing peptide/MHC complexes, we find the following: 1) that HEL in the draining lymph node gets concentrated in a limited number of APC, particularly in dendritic cells and macrophages, 2) that these APC are functionally capable of presenting both major and minor determinants of HEL over a 100-fold range of Ag dose, and 3) that B cells present Ag gained at early times after immunization, but only following higher dose immunization. These data indicate that the breadth of a response is maintained over a wide dosage range by concentration of Ag in a limited number of cells presenting high levels and a great diversity of epitopes.
Immunity to the Extracellular Domain of Nogo-A Modulates Experimental Autoimmune Encephalomyelitis
Authors: Fontoura Paulo , Ho Peggy P. , DeVoss Jason , Zheng Binhai , Lee Byung J. , Kidd Brian A. , Garren Hideki , Sobel Raymond A. , Robinson William H. , Tessier-Lavigne Marc , Steinman Lawrence
Published: April 2014

Nogo-66, the extracellular 66 aa loop of the Nogo-A protein found in CNS myelin, interacts with the Nogo receptor and has been proposed to mediate inhibition of axonal regrowth. It has been shown that immunization with Nogo-A promotes recovery in animal models of spinal cord injury through induction of Ab production. In this report, studies were performed to characterize the immune response to Nogo-66 and to determine the role of Nogo in experimental autoimmune encephalomyelitis (EAE). Immunization of EAE-susceptible mouse strains with peptides derived from Nogo-66 induced a CNS immune response with clinical and pathological similarities to EAE. The Nogo-66 peptides elicited strong T cell responses that were not cross-reactive to other encephalitogenic myelin Ags. Using a large scale spotted microarray containing proteins and peptides derived from a wide spectrum of myelin components, we demonstrated that Nogo-66 peptides also generated a specific Ab response that spreads to several other encephalitogenic myelin Ags following immunization. Nogo-66-specific T cell lines ameliorated established EAE, via Nogo-66-specific Th2 cells that entered the CNS. These results indicate that some T cell and B cell immune responses to Nogo-66 are associated with suppression of ongoing EAE, whereas other Nogo-66 epitopes can be encephalitogenic.
CXC Chemokine Ligand 10 Neutralization Suppresses the Occurrence of Diabetes in Nonobese Diabetic Mice through Enhanced β Cell Proliferation without Affecting Insulitis
Authors: Morimoto Jiro , Yoneyama Hiroyuki , Shimada Akira , Shigihara Toshikatsu , Yamada Satoru , Oikawa Yoichi , Matsushima Kouji , Saruta Takao , Narumi Shosaku
Published: April 2014

We have shown that neutralization of IFN-inducible protein 10/CXCL10, a chemokine for Th1 cells, breaks Th1 retention in the draining lymph nodes, resulting in exacerbation in Th1-dominant autoimmune disease models induced by immunization with external Ags. However, there have been no studies on the role of CXCL10 neutralization in Th1-dominant disease models induced by constitutive intrinsic self Ags. So, we have examined the effect of CXCL10 neutralization using a type 1 diabetes model initiated by developmentally regulated presentation of β cell Ags. CXCL10 neutralization suppressed the occurrence of diabetes after administration with cyclophosphamide in NOD mice, although CXCL10 neutralization did not significantly inhibit insulitis and gave no influence on the trafficking of effector T cells into the islets. Because both CXCL10 and CXCR3 were, unexpectedly, coexpressed on insulin-producing cells, CXCL10 was considered to affect mature and premature β cells in an autocrine and/or paracrine fashion. In fact, CXCL10 neutralization enhanced proliferative response of β cells and resultantly increased β cell mass without inhibiting insulitis. Thus, CXCL10 neutralization can be a new therapeutic target for β cell survival, not only during the early stage of type 1 diabetes, but also after islet transplantation.
Cutting Edge: Estrogen Drives Expansion of the CD4+CD25+ Regulatory T Cell Compartment
Authors: Polanczyk Magdalena J. , Carson Bryan D. , Subramanian Sandhya , Afentoulis Michael , Vandenbark Arthur A. , Ziegler Steven F. , Offner Halina
Published: April 2014

CD4+CD25+ regulatory T cells are crucial to the maintenance of tolerance in normal individuals. However, the factors regulating this cell population and its function are largely unknown. Estrogen has been shown to protect against the development of autoimmune disease, yet the mechanism is not known. We demonstrate that estrogen (17-β-estradiol, E2) is capable of augmenting FoxP3 expression in vitro and in vivo. Treatment of naive mice with E2 increased both CD25+ cell number and FoxP3 expression level. Further, the ability of E2 to protect against autoimmune disease (experimental autoimmune encephalomyelitis) correlated with its ability to up-regulate FoxP3, as both were reduced in estrogen receptor α-deficient animals. Finally, E2 treatment and pregnancy induced FoxP3 protein expression to a similar degree, suggesting that high estrogen levels during pregnancy may help to maintain fetal tolerance. In summary, our data suggest E2 promotes tolerance by expanding the regulatory T cell compartment.
Cutting Edge: Expression of Chemokine Receptor CXCR1 on Human Effector CD8+ T Cells
Authors: Takata Hiroshi , Tomiyama Hiroko , Fujiwara Mamoru , Kobayashi Naoki , Takiguchi Masafumi
Published: April 2014

IL-8 is a potent inflammatory cytokine that induces chemotaxis of neutrophils expressing CXCR1 and CXCR2, thus indicating its involvement in the migration of these cells to inflammatory sites where bacteria proliferate. Presently, we showed that CXCR1+ cells were predominantly found among CD8+ T cells having effector phenotype, and that the expression of CXCR1 was positively correlated with that of perforin, suggesting that CXCR1 is expressed on effector CD8+ T cells. Indeed, human CMV-specific CD8+ T cells from healthy individuals, which mostly express the effector phenotype and have cytolytic function, expressed CXCR1, whereas EBV-specific CD8+ T cells, which mostly express the memory phenotype and have no cytolytic function, did not express this receptor. The results of a chemotaxis assay showed that the migration of CXCR1+CD8+ T cells was induced by IL-8. These results suggest that the IL-8-CXCR1 pathway plays an important role in the homing of effector CD8+ T cells.
A Vitamin D Analog Down-Regulates Proinflammatory Chemokine Production by Pancreatic Islets Inhibiting T Cell Recruitment and Type 1 Diabetes Development
Authors: Giarratana Nadia , Penna Giuseppe , Amuchastegui Susana , Mariani Roberto , Daniel Kenn C. , Adorini Luciano
Published: April 2014

Type 1 diabetes (T1D) is an autoimmune disease characterized by leukocyte infiltration into the pancreatic islets, and we have previously shown that treatment of adult NOD mice with a vitamin D analog arrests the progression of insulitis, blocks Th1 cell infiltration into the pancreas, and markedly reduces T1D development, suggesting inhibition of chemokine production by islet cells. In this study, we show that all TLRs are expressed by mouse and human islet cells, and their engagement by pathogen-derived ligands markedly enhances proinflammatory chemokine production. The vitamin D analog significantly down-regulates in vitro and in vivo proinflammatory chemokine production by islet cells, inhibiting T cell recruitment into the pancreatic islets and T1D development. The inhibition of islet chemokine production in vivo persists after restimulation with TLR ligands and is associated with up-regulation of IκBα transcription, an inhibitor of NF-κB and with arrest of NF-κBp65 nuclear translocation, highlighting a novel mechanism of action exerted by vitamin D receptor ligands potentially relevant for the treatment of T1D and other autoimmune diseases.
A Structural Basis for the Association of DAP12 with Mouse, but Not Human, NKG2D
Authors: Rosen David B. , Araki Manabu , Hamerman Jessica A. , Chen Taian , Yamamura Takashi , Lanier Lewis L.
Published: April 2014

Prior studies have revealed that alternative mRNA splicing of the mouse NKG2D gene generates receptors that associate with either the DAP10 or DAP12 transmembrane adapter signaling proteins. We report that NKG2D function is normal in human patients lacking functional DAP12, indicating that DAP10 is sufficient for human NKG2D signal transduction. Further, we show that human NKG2D is incapable of associating with DAP12 and provide evidence that structural differences in the transmembrane of mouse and human NKG2D account for the species-specific difference for this immune receptor.
IL-15-Independent Proliferative Renewal of Memory CD8+ T Cells in Latent Gammaherpesvirus Infection
Authors: Obar Joshua J. , Crist Sarah G. , Leung Erica K. , Usherwood Edward J.
Published: April 2014

IL-15 is known to be critical in the homeostasis of Ag-specific memory CD8+ T cells following acute viral infection. However, little is known about the homeostatic requirements of memory CD8+ T cells during a latent viral infection. We have used the murine gammaherpesvirus-68 (MHV-68) model system to investigate whether IL-15 is necessary for the maintenance of memory CD8+ T cells during a latent viral infection. IL-15 is not essential either for the initial control of MHV-68 infection or for the maintenance of MHV-68-specific memory CD8+ T cells. Even at 140 days postinfection, the proportion of CD8+ T cells recognizing the MHV-68 epitopes were the same as in control mice. The maintenance of these memory CD8+ T cells was attributable to their ability to turn over in vivo, probably in response to the presence of low levels of Ag. IL-15−/− mice had a significantly higher turnover rate within the virus-specific memory CD8+ T cell population, which was the result of increased levels of viral gene expression rather than an increase in viral load. These cells did not accumulate in the spleens of the IL-15−/− mice due to an increased sensitivity to apoptosis as a result of decreased Bcl-2 levels. Intriguingly, memory CD8+ T cells from latently infected mice failed to undergo homeostatic proliferation in a naive secondary host. These data highlight fundamental differences between memory CD8+ T cells engaged in active immune surveillance of latent viral infections vs memory CD8+ T cells found after acute viral infections.
Tolerance Induced by the Lipopeptide Pam3Cys Is Due to Ablation of IL-1R-Associated Kinase-1
Authors: Siedlar Maciej , Frankenberger Marion , Benkhart Elke , Espevik Terje , Quirling Martina , Brand Korbinian , Zembala Marek , Ziegler-Heitbrock Loems
Published: April 2014

Stimulation of the human monocytic cell line Mono Mac 6 with the synthetic lipopeptide (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride (Pam3Cys) at 10 μg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam3Cys at 1 μg/ml leads to a reduced response after subsequent stimulation with Pam3Cys at 10 μg/ml, indicating that the cells have become tolerant to Pam3Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-κB binding in Pam3Cys-tolerant cells shows a failure to mobilize NF-κB-p50p65 heterodimers, while NF-κB-p50p50 homodimers remain unchanged. Pam3Cys-tolerant cells showed neither IκBα-Ser32 phosphorylation nor IκBα degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam3Cys-induced tolerance, while IRAK-1 mRNA was still detectable at 30% compared with untreated cells. In contrast, in LPS-tolerized cells, p50p50 homodimers were induced, IRAK-1 protein level was only partially decreased, and p50p65 mobilization remained intact. It is concluded that in Mono Mac 6 monocytic cells, inhibition of IRAK-1 expression at the mRNA and protein levels is the main TLR-2-dependent mechanism responsible for Pam3Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.
Toll IL-1 Receptors Differ in Their Ability to Promote the Stabilization of Adenosine and Uridine-Rich Elements Containing mRNA
Authors: Datta Shyamasree , Novotny Michael , Li Xiaoxia , Tebo Julie , Hamilton Thomas A.
Published: April 2014

Several ligands for Toll IL-1R (TIR) family are known to promote stabilization of a subset of short-lived mRNAs containing AU-rich elements (AREs) in their 3′ untranslated regions. It is now evident however, that members of the TIR family may use distinct intracellular signaling pathways to achieve a spectrum of biological end points. Using human embryonic kidney 293 cells transfected to express different TIRs we now report that signals initiated through IL-1R1 or TLR4 but not TLR3 can promote the stabilization of unstable chemokine mRNAs. Similar results were obtained when signaling from endogenous receptors was examined using a mouse endothelial cell line (H5V). The ability of TIR family members to stabilize ARE-containing mRNAs results from their differential use of signaling adaptors MyD88, MyD88 adaptor-like protein, Toll receptor IFN-inducing factor (Trif), and Trif-related adaptor molecule. Overexpression of MyD88 or MyD88 adaptor-like protein was able to promote enhanced stability of ARE-containing mRNA, whereas Trif and Trif-related adaptor molecule exhibited markedly reduced capacity. Hence the ability of TIRs to signal stabilization of mRNA appears to be linked to the MyD88-dependent signaling pathway.
MHC Class II Isotype- and Allele-Specific Attenuation of Experimental Autoimmune Encephalomyelitis
Authors: de Graaf Katrien L. , Barth Silvia , Herrmann Martin M. , Storch Maria K. , Otto Christoph , Olsson Tomas , Melms Arthur , Jung Günther , Wiesmüller Karl-Heinz , Weissert Robert
Published: April 2014

Most autoimmune diseases are associated with certain MHC class II haplotypes. Autoantigen-based specific immune therapy can lead either to beneficial or, in the context of inflammatory conditions, detrimental outcomes. Therefore, we designed a platform of peptides by combinatorial chemistry selected in a nonbiased Ag-independent approach for strong binding to the rat MHC class II isotype RT1.Dn allelic product of the RT1n haplotype that is presenting autoantigen in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LEW.1N rats. Peptide p17 (Ac-FWFLDNAPL-NH2) was capable of suppressing the induction of and also ameliorated established experimental autoimmune encephalomyelitis. MHC class II isotype and allele specificity of the therapeutic principle were demonstrated in myelin basic protein-induced experimental autoimmune encephalomyelitis in LEW rats bearing the RT1l haplotype. A general immunosuppressive effect of the treatment was excluded by allogeneic heart transplantation studies. In vitro studies demonstrated the blocking effect of p17 on autoantigenic T cell responses. We thus demonstrate a rational design of strong MHC class II-binding peptides with absolute isotype and allele specificity able to compete for autoantigenic sequences presented on disease-associated MHC class II molecules.
Selective Expression of the 21-Kilodalton Tyrosine-Phosphorylated Form of TCR ζ Promotes the Emergence of T Cells with Autoreactive Potential
Authors: Pitcher Lisa A. , Mathis Meredith A. , Subramanian Srividya , Young Jennifer A. , Wakeland Edward K. , Love Paul E. , van Oers Nicolai S. C.
Published: April 2014

T cells undergo negative selection in the thymus to eliminate potentially autoreactive cells. The signals generated through the αβ TCR following receptor interactions with peptide/MHC complexes in the thymus control these selection processes. Following receptor ligation, a fraction of the TCR ζ subunit appears as two distinct tyrosine-phosphorylated forms of 21 and 23 kDa (p21 and p23). Previous data have reported elevated levels of p21 in some murine models of autoimmunity. We have examined the contributions of both the p21 and p23 to T cell negative selection in the HY TCR-transgenic system using ITAM-substituted TCR ζ and CD3 ε transgenic mice. Expression of just p21, in the absence of p23, partially impairs negative selection of self-reactive HY-specific T cells. This results in the emergence of potentially autoreactive peripheral T cells and an elevated population of CD11b+B220+ B cells in the spleen. These data clearly identify a specific and unique role for p21 during negative selection.
Transcriptional Regulation of the HumanCD3γ Gene: The TATA-LessCD3γ Promoter Functions via an Initiator and Contiguous Sp-Binding Elements
Authors: Badran Bassam M. , Kunstman Kevin , Stanton Jennifer , Moschitta Maria , Zerghe Anne , Akl Haidar , Burny Arsène , Wolinsky Steven M. , Willard-Gallo Karen E.
Published: April 2014

Growing evidence that the CD3γ gene is specifically targeted in some T cell diseases focused our attention on the need to identify and characterize the elusive elements involved in CD3γ transcriptional control. In this study, we show that while the human CD3γ and CD3δ genes are oriented head-to-head and separated by only 1.6 kb, the CD3γ gene is transcribed from an independent but weak, lymphoid-specific TATA-less proximal promoter. Using RNA ligase-mediated rapid amplification of cDNA ends, we demonstrate that a cluster of transcription initiation sites is present in the vicinity of the primary core promoter, and the major start site is situated in a classical initiator sequence. A GT box immediately upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these adjacent elements enhanced by the presence of a second GC box 10 nt further upstream. The primary core promoter is limited to a sequence that extends upstream to −15 and contains the initiator and GT box. An identical GT box located ∼50 nt from the initiator functions as a weak secondary core promoter and likely generates transcripts originating upstream from the +1. Finally, we show that two previously identified NFAT motifs in the proximal promoter positively (NFATγ1) or negatively (NFATγ1 and NFATγ2) regulate expression of the human CD3γ gene by their differential binding of NFATc1 plus NF-κB p50 or NFATc2 containing complexes, respectively. These data elucidate some of the mechanisms controlling expression of the CD3γ gene as a step toward furthering our understanding of how its transcription is targeted in human disease.
Human Factor H-Related Protein 5 Has Cofactor Activity, Inhibits C3 Convertase Activity, Binds Heparin and C-Reactive Protein, and Associates with Lipoprotein
Authors: McRae Jennifer L. , Duthy Thomas G. , Griggs Kim M. , Ormsby Rebecca J. , Cowan Peter J. , Cromer Brett A. , McKinstry William J. , Parker Michael W. , Murphy Brendan F. , Gordon David L.
Published: April 2014

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5–7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5–7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.
A Direct Role for C1 Inhibitor in Regulation of Leukocyte Adhesion
Authors: Cai Shenghe , Dole Vandana S. , Bergmeier Wolfgang , Scafidi Jennifer , Feng Hanping , Wagner Denisa D. , Davis Alvin E.
Published: April 2014

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewisx tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.
Hepatitis C Virus Drives the Unconstrained Monoclonal Expansion of VH1–69-Expressing Memory B Cells in Type II Cryoglobulinemia: A Model of Infection-Driven Lymphomagenesis
Authors: Carbonari Maurizio , Caprini Elisabetta , Tedesco Tiziana , Mazzetta Francesca , Tocco Valeria , Casato Milvia , Russo Giandomenico , Fiorilli Massimo
Published: April 2014

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the VH1–69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of VH1–69+ cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of VH1–69+ B cells: naive (small, IgMhighIgDhighCD38+CD27−CD21highCD95−CD5−), “early memory” (medium-sized, IgMhighIgDlowCD38−CD27+CD21lowCD95+CD5+), and “late memory” (large-sized, IgMlowIgDlow-negCD38−CD27lowCD21low-negCD5−CD95−). The B cells expanded in cryoglobulinemia patients have a “memory” phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated VH1–69+ B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal VH1–69+ B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a VH1–69+ natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.
Dynamic Regulation of IFN-γ Signaling in Antigen-Specific CD8+ T Cells Responding to Infection
Authors: Haring Jodie S. , Corbin Gail A. , Harty John T.
Published: April 2014

IFN-γ plays a critical role in the CD8+ T cell response to infection, but when and if this cytokine directly signals CD8+ T cells during an immune response is unknown. We show that naive Ag-specific CD8+ T cells receive IFN-γ signals within 12 h after in vivo infection with Listeria monocytogenes and then become unresponsive to IFN-γ throughout the ensuing Ag-driven expansion phase. Ag-specific CD8+ T cells regain partial IFN-γ responsiveness throughout the contraction phase, whereas the memory pool exhibits uniform, but reduced, responsiveness that is also modulated during the secondary response. The responsiveness of Ag-specific CD8+ T cells to IFN-γ correlated with modulation in the expression of IFN-γR2, but not with IFN-γR1 or suppressor of cytokine signaling-1. This dynamic regulation suggests that early IFN-γ signals participate in regulation of the primary CD8+ T cell response program, but that evading or minimizing IFN-γ signals during expansion and the memory phase may contribute to appropriate regulation of the CD8+ T cell response.
Neutralizing Anti-IL-10 Antibody Blocks the Protective Effect of Tapeworm Infection in a Murine Model of Chemically Induced Colitis
Authors: Hunter Meaghan M. , Wang Arthur , Hirota Christina L. , McKay Derek M.
Published: April 2014

There is increasing evidence that parasitic helminth infection has the ability to ameliorate other disease conditions. In this study the ability of the rat tapeworm, Hymenolepis diminuta, to modulate dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice is assessed. Mice receiving DNBS (3 mg intrarectally) developed colitis by 72 h after treatment. Mice infected 8 days before DNBS with five H. diminuta larvae were significantly protected from the colitis, as gauged by reduced clinical disease, histological damage scores, and myeloperoxidase levels. This anticolitic effect was dependent on a viable infection and helminth rejection, because no benefit was observed in mice given killed larvae or in infected STAT6 knockout mice or rats, neither of which eliminate H. diminuta. The anticolitic effect of H. diminuta was associated with increased colonic IL-10 mRNA and stimulated splenocytes from H. diminuta- plus DNBS-treated mice produced more IL-10 than splenocytes from DNBS-only treated mice. Coadministration of an anti-IL-10 Ab blocked the anticolitic effect of prophylactic H. diminuta infection. Also, mice infected 48 h after DNBS treatment showed an enhanced recovery response. Finally, using a model of OVA hypersensitivity, we found no evidence of concomitant H. diminuta infection enhancing enteric responsiveness to subsequent ex vivo OVA challenge. The data show that a viable infection of H. diminuta in a nonpermissive system exerts a profound anticolitic effect (both prophylactically and as a treatment) that is mediated at least in part via IL-10 and does not predispose to enhanced sensitivity to bystander proteins.
Probiotics Ameliorate Recurrent Th1-Mediated Murine Colitis by Inducing IL-10 and IL-10-Dependent TGF-β-Bearing Regulatory Cells
Authors: Di Giacinto Claudia , Marinaro Mariarosaria , Sanchez Massimo , Strober Warren , Boirivant Monica
Published: April 2014

Recent studies of murine models of mucosal inflammation suggest that, whereas some kinds of bacterial microflora are inducers of disease, others, known as probiotics, prevent disease. In the present study, we analyzed the regulatory cytokine and cell response to probiotic (VSL#3) administration in the context of the Th1 T cell colitis induced by trinitrobenzene sulfonic acid treatment of SJL/J mice. Daily administration of probiotics for 3 wk to mice during a remission period between a first and second course of colitis induced by trinitrobenzene sulfonic acid, resulted in a milder form of recurrent colitis than observed in mice administered PBS during this same period. This protective effect was attributable to effects on the lamina propria mononuclear cell (LPMC) population, because it could be transferred by LPMC from probiotic-treated mice to naive mice. Probiotic administration was associated with an early increase in the production of IL-10 and an increased number of regulatory CD4+ T cells bearing surface TGF-β in the form of latency-associated protein (LAP) (LAP+ T cells). The latter were dependent on the IL-10 production because administration of anti-IL-10R mAb blocked their appearance. Finally, the LAP+ T cells were essential to the protective effect of probiotics because administration of anti-IL-10R or anti-TGF-β at the initiation of recurrent colitis induction or depletion of LAP+ T cells from LPMC abolished the latter’s capacity to transfer protection to naive recipients. These studies show that probiotic (VSL#3) administration during a remission period ameliorates the severity of recurrent colitis by inducing an immunoregulatory response involving TGF-β-bearing regulatory cells.
Neisserial Porin-Induced Dendritic Cell Activation Is MyD88 and TLR2 Dependent
Authors: Singleton Theresa E. , Massari Paola , Wetzler Lee M.
Published: April 2014

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin’s ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin’s immune stimulatory activity.
IL-22 Inhibits Epidermal Differentiation and Induces Proinflammatory Gene Expression and Migration of Human Keratinocytes
Authors: Boniface Katia , Bernard François-Xavier , Garcia Martine , Gurney Austin L. , Lecron Jean-Claude , Morel Franck
Published: April 2014

IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.
Activation of the Prostaglandin D2 Receptor DP2/CRTH2 Increases Allergic Inflammation in Mouse
Authors: Spik Isabelle , Brénuchon Céline , Angéli Véronique , Staumont Delphine , Fleury Sébastien , Capron Monique , Trottein François , Dombrowicz David
Published: April 2014

Allergic pathologies are often associated with IgE production, mast cell activation, and eosinophilia. PGD2 is the major eicosanoid, among several inflammatory mediators, released by mast cells. PGD2 binds to two membrane receptors, D prostanoid receptor (DP)1 and DP2, endowed with antagonistic properties. In humans, DP2 is preferentially expressed on type 2 lymphocytes, eosinophils, and basophils and mediates chemotaxis in vitro. Although not yet supported by in vivo studies, DP2 is thought to be important in the promotion of Th2-related inflammation. Herein, we demonstrate that mouse eosinophils express both DP1 and DP2 and that PGD2 exerts in vitro chemotactic effects on eosinophils through DP2 activation. Furthermore, 13,14-dihydro-15-keto-PGD2, a specific DP2 agonist not only increases eosinophil recruitment at inflammatory sites but also the pathology in two in vivo models of allergic inflammation: atopic dermatitis and allergic asthma. By contrast, DP1 activation tends to ameliorate the pathology in asthma. Taken together, these results support the hypothesis that DP2 might play a critical role in allergic diseases and underline the interest of DP2 antagonists in human therapy.
Unconjugated Bilirubin Inhibits VCAM-1-Mediated Transendothelial Leukocyte Migration
Authors: Keshavan Pavitra , Deem Tracy L. , Schwemberger Sandy J. , Babcock George F. , Cook-Mills Joan M. , Zucker Stephen D.
Published: April 2014

During lymphocyte migration, engagement of VCAM-1 stimulates the generation of endothelial cell-derived reactive oxygen species (ROS) and activation of matrix metalloproteinases, facilitating endothelial retraction. Because bilirubin is a potent antioxidant, we examined the hypothesis that this bile pigment inhibits VCAM-1-dependent cellular events. The migration of isolated murine splenic lymphocytes across monolayers of murine endothelial cell lines (which constitutively express VCAM-1) is significantly inhibited by physiological concentrations of bilirubin, in the absence of an effect on lymphocyte adhesion. Bilirubin administration also suppresses VCAM-1-stimulated ROS generation and reduces endothelial cell matrix metalloproteinase activity. In a murine asthma model characterized by VCAM-1-dependent airway inflammation, treatment of C57BL6/J mice with i.p. bilirubin decreases the total leukocyte count in the lung parenchyma and lavage fluid, through specific inhibition of eosinophil and lymphocyte infiltration. Blood eosinophil counts were increased in bilirubin-treated animals, while VCAM-1 expression in the capillary endothelium and cytokine levels in both lung lavage and supernatants from cultured lymph node lymphocytes were unchanged, suggesting that bilirubin inhibits leukocyte migration. Conclusion: bilirubin blocks VCAM-1-dependent lymphocyte migration in vitro and ameliorates VCAM-1-mediated airway inflammation in vivo, apparently through the suppression of cellular ROS production. These findings support a potential role for bilirubin as an endogenous immunomodulatory agent.