bluuebunny/crossref_metadata_2025 huggingface dataset:
Global Analysis of Smad2/3-Dependent TGF-β Signaling in Living Mice Reveals Prominent Tissue-Specific Responses to Injury
Authors: Lin Amy H. , Luo Jian , Mondshein Lauren H. , ten Dijke Peter , Vivien Denis , Contag Christopher H. , Wyss-Coray Tony
Published: April 2014

Smad2 and Smad3 (Smad2/3) proteins are key signaling molecules for TGF-β and some related family members regulating the transcription of several hundred genes. TGF-β have key roles in development, tissue homeostasis, and the pathogenesis of many human diseases, including cancer, fibrotic disorders, developmental defects, and neurodegeneration. To study the temporal and spatial patterns of Smad2/3-dependent signaling in normal and pathological conditions in the living organism, we engineered transgenic mice with a Smad-responsive luciferase reporter construct (SBE-luc mice). Using bioluminescent imaging, we assessed Smad2/3 signaling activity noninvasively in living mice. At baseline, this activity was highest in brain, intestine, heart, and skin, and correlated with biochemical measurements of reporter activity. Primary astrocytes cultured from SBE-luc mice showed specific activation of the reporter in response to Smad2/3-activating TGF-β family members. Treatment of mice with the endotoxin LPS resulted in a fast and vigorous, but transient activation of the reporter in the intestine. Although the response was similarly rapid in brain, it remained increased, indicating important but different cellular responses to endotoxin challenge in these organs. Traumatic brain injury with a needle stab resulted in local activation of Smad2/3-dependent genes and a severalfold increase in bioluminescence in living mice. SBE-luc mice can therefore be used to study temporal, tissue-specific activation of Smad2/3-dependent signaling in living mice in normal or pathological conditions as well as for the identification of endogenous or synthetic modulators of this pathway.
X Chromosome Monosomy: A Common Mechanism for Autoimmune Diseases
Authors: Invernizzi Pietro , Miozzo Monica , Selmi Carlo , Persani Luca , Battezzati Pier Maria , Zuin Massimo , Lucchi Simona , Meroni Pier Luigi , Marasini Bianca , Zeni Silvana , Watnik Mitchell , Grati Francesca R. , Simoni Giuseppe , Gershwin M. Eric , Podda Mauro
Published: April 2014

The majority of human autoimmune diseases are characterized by female predominance. Although sex hormone influences have been suggested to explain this phenomenon, the mechanism remains unclear. In contrast to the role of hormones, it has been suggested, based on pilot data in primary biliary cirrhosis, that there is an elevation of monosomy X in autoimmune disease. Using peripheral white blood cells from women with systemic sclerosis (SSc), autoimmune thyroid disease (AITD), or healthy age-matched control women, we studied the presence of monosomy X rates using fluorescence in situ hybridization. We also performed dual-color fluorescence in situ hybridization analysis with a chromosome Y α-satellite probe to determine the presence of the Y chromosome in the monosomic cells. In subsets of patients and controls, we determined X monosomy rates in white blood cell subpopulations. The rates of monosomy X increased with age in all three populations. However, the rate of monosomy X was significantly higher in patients with SSc and AITD when compared with healthy women (6.2 ± 0.3% and 4.3 ± 0.3%, respectively, vs 2.9 ± 0.2% in healthy women, p < 0.0001 in both comparisons). Importantly, X monosomy rate was more frequent in peripheral T and B lymphocytes than in the other blood cell populations, and there was no evidence for the presence of male fetal microchimerism. These data highlight the thesis that chromosome instability is common to women with SSc and AITD and that haploinsufficiency for X-linked genes may be a critical factor for the female predominance of autoimmune diseases.
Response of Memory CD8+ T Cells to Severe Acute Respiratory Syndrome (SARS) Coronavirus in Recovered SARS Patients and Healthy Individuals
Authors: Chen Huabiao , Hou Jinlin , Jiang Xiaodong , Ma Shiwu , Meng Minjie , Wang Baomei , Zhang Minghui , Zhang Mingxia , Tang Xiaoping , Zhang Fuchun , Wan Tao , Li Nan , Yu Yizhi , Hu Hongbo , Yang Ruifu , He Wei , Wang Xiaoning , Cao Xuetao
Published: April 2014

To date, the pathogenesis of severe acute respiratory syndrome (SARS) in humans is still not well understood. SARS coronavirus (SARS-CoV)-specific CTL responses, in particular their magnitude and duration of postinfection immunity, have not been extensively studied. In this study, we found that heat-inactivated SARS-CoV elicited recall CTL responses to newly identified spike protein-derived epitopes (SSp-1, S978, and S1202) in peripheral blood of all HLA-A*0201+ recovered SARS patients over 1 year postinfection. Intriguingly, heat-inactivated SARS-CoV elicited recall-like CTL responses to SSp-1 but not to S978, S1202, or dominant epitopes from several other human viruses in 5 of 36 (13.8%) HLA-A*0201+ healthy donors without any contact history with SARS-CoV. SSp-1-specific CTLs expanded from memory T cells of both recovered SARS patients, and the five exceptional healthy donors shared a differentiated effector CTL phenotype, CD45RA+CCR7−CD62L−, and expressed CCR5 and CD44. However, compared with the high avidity of SSp-1-specific CTLs derived from memory T cells of recovered SARS patients, SSp-1-specific CTLs from the five exceptional healthy donors were of low avidity, as determined by their rapid tetramer dissociation kinetics and reduced cytotoxic reactivity, IFN-γ secretion, and intracellular production of IFN-γ, TNF-α, perforin, and granzyme A. These results indicate that SARS-CoV infection induces strong and long-lasting CTL-mediated immunity in surviving SARS patients, and that cross-reactive memory T cells to SARS-CoV may exist in the T cell repertoire of a small subset of healthy individuals and can be reactivated by SARS-CoV infection.
Autoimmune CD4+ T Cell Memory: Lifelong Persistence of Encephalitogenic T Cell Clones in Healthy Immune Repertoires
Authors: Kawakami Naoto , Odoardi Francesca , Ziemssen Tjalf , Bradl Monika , Ritter Thomas , Neuhaus Oliver , Lassmann Hans , Wekerle Hartmut , Flügel Alexander
Published: April 2014

We embedded green fluorescent CD4+ T cells specific for myelin basic protein (MBP) (TMBP-GFP cells) in the immune system of syngeneic neonatal rats. These cells persisted in the animals for the entire observation period spanning >2 years without affecting the health of the hosts. They maintained a memory phenotype with low levels of L-selectin and CD45RC, but high CD44. Although persisting in low numbers (0.01–0.1% of lymph node cells) they were sufficient to raise susceptibility toward clinical autoimmune disease. Immunization with MBP in IFA induced CNS inflammation and overt clinical disease in animals carrying neonatally transferred TMBP-GFP cells, but not in controls. The onset of the clinical disease coincided with mass infiltration of TMBP-GFP cells into the CNS. In the periphery, following the amplification phase a rapid contraction of the T cell population was observed. However, elevated numbers of fully reactive TMBP-GFP cells remained in the peripheral immune system after acute experimental autoimmune encephalomyelitis mediating reimmunization-induced disease relapses.
Cutting Edge: Murine Vascular Endothelium Activates and Induces the Generation of Allogeneic CD4+25+Foxp3+ Regulatory T Cells
Authors: Krupnick Alexander Sasha , Gelman Andrew E. , Barchet Winfried , Richardson Steve , Kreisel Friederike H. , Turka Laurence A. , Colonna Marco , Patterson G. Alexander , Kreisel Daniel
Published: April 2014

Unlike graft-resident donor-derived hemopoietic APCs, which decrease in number over time after transplantation, vascular endothelial cells are lifelong residents of a vascularized allograft. Endothelial cells are potent APCs for allogeneic CD8+ T lymphocytes but are unable to induce proliferation of allogeneic CD4+ T lymphocytes. Although the reason for this differential response has been poorly understood, here we report that alloantigen presentation by vascular endothelium to CD4+ T lymphocytes activates and induces CD4+25+Foxp3+ regulatory T cells, which can inhibit proliferation of alloreactive T cells both in vitro and in vivo. This process occurs independently of B7.1 costimulation but is dependent on programmed death ligand 1 (B7-H1). This finding may have important implications for tolerance induction in transplantation.
Permanent Survival of Fully MHC-Mismatched Islet Allografts by Targeting a Single Chemokine Receptor Pathway
Authors: Wang Liqing , Han Rongxiang , Lee Iris , Hancock Aidan S. , Xiong Guoxiang , Gunn Michael D. , Hancock Wayne W.
Published: April 2014

Chemokine receptor blockade can diminish the recruitment of host effector cells and prolong allograft survival, but little is known of the role of chemokine receptors in promoting host sensitization. We engrafted fully allogeneic islets into streptozotocin-treated normal mice or mice with the autosomal recessive paucity of lymph node T cell (plt) mutation; the latter lack secondary lymphoid expression of the CCR7 ligands, secondary lymphoid organ chemokine (CCL21) and EBV-induced molecule-1 ligand chemokine (CCL19). plt mice showed permanent survival of islets engrafted under the kidney capsule, whereas controls rejected islet allografts in 12 days (p < 0.001), and consistent with this, plt mice had normal allogeneic T cell responses, but deficient migration of donor dendritic cell to draining lymph nodes. Peritransplant i.v. injection of donor splenocytes caused plt recipients to reject their allografts by 12 days, and sensitization at 60 days posttransplant of plt mice with well-functioning allografts restored acute rejection. Finally, islet allografts transplanted intrahepatically in plt mice were rejected ∼12 days posttransplant, like controls, as were primarily revascularized cardiac allografts. These data show that the chemokine-directed homing of donor dendritic cell to secondary lymphoid tissues is essential for host sensitization and allograft rejection. Interruption of such homing can prevent T cell priming and islet allograft rejection despite normal T and B cell functions of the recipient, with potential clinical implications.
Ultrasensitive Detection and Phenotyping of CD4+ T Cells with Optimized HLA Class II Tetramer Staining
Authors: Scriba Thomas J. , Purbhoo Marco , Day Cheryl L. , Robinson Nicola , Fidler Sarah , Fox Julie , Weber Jonathan N. , Klenerman Paul , Sewell Andrew K. , Phillips Rodney E.
Published: April 2014

HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.
Control of Genotypic Allelic Inclusion through TCR Surface Expression
Authors: Warmflash Aryeh , Weigert Martin , Dinner Aaron R.
Published: April 2014

To gain insight into the molecular causes and functional consequences of allelic inclusion of TCR α-chains, we develop a computational model for thymocyte selection in which the signal that determines cell fate depends on surface expression. Analysis of receptor pairs on selected dual TCR cells reveals that allelic inclusion permits both autoreactive TCR and receptors not in the single TCR cell repertoire to be selected. However, in comparison with earlier theoretical studies, relatively few dual TCR cells display receptors with high avidity for thymic ligands because their α-chains compete aggressively for the β-chain, which hinders rescue from clonal deletion. This feature of the model makes clear that allelic inclusion does not in itself compromise central tolerance. A specific experiment based on modulation of TCR surface expression levels is proposed to test the model.
Phagocytosis of Hemozoin Enhances Matrix Metalloproteinase-9 Activity and TNF-α Production in Human Monocytes: Role of Matrix Metalloproteinases in the Pathogenesis of Falciparum Malaria
Authors: Prato Mauro , Giribaldi Giuliana , Polimeni Manuela , Gallo Valentina , Arese Paolo
Published: April 2014

Matrix metalloproteinase-9 (MMP-9), secreted by activated monocytes, degrades matrix proteins, disrupts basal lamina, and activates TNF-α from its precursors. In turn, TNF-α enhances synthesis of MMP-9 in monocytes. We show here that trophozoite-parasitized RBCs/hemozoin-fed adherent human monocytes displayed increased MMP-9 activity and protein/mRNA expression, produced TNF-α time-dependently, and showed higher matrix invasion ability. MMP-9 activation was specific for trophozoite/hemozoin-fed monocytes, was dependent on TNF-α production, and abrogated by anti-TNF-α Ab and by a specific inhibitor of MMP-9/MMP-13 activity. Hemozoin-induced enhancement of MMP-9 and TNF-α production would have a 2-fold effect: to start and feed a cyclic reinforcement loop in which hemozoin enhances production of TNF-α, which in turn induces both activation of MMP-9 and shedding of TNF-α into the extracellular compartment; and, second, to disrupt the basal lamina of endothelia. Excess production of TNF-α and disruption of the basal lamina with extravasation of blood cells into perivascular tissues are hallmarks of severe malaria. Pharmacological inhibition of MMP-9 may offer a new chance to control pathogenic mechanisms in malaria.
IL-1β-Mediated Proinflammatory Responses Are Inhibited by Estradiol via Down-Regulation of IL-1 Receptor Type I in Uterine Epithelial Cells
Authors: Schaefer Todd M , Wright Jacqueline A. , Pioli Patricia A. , Wira Charles R.
Published: April 2014

The objective of this study was to examine the effects of sex hormones on IL-1β-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human β-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1β in the presence of estradiol or progesterone. Estradiol inhibited the IL-1β-mediated mRNA expression and secretion of human β-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1β-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10−7 to 10−10 M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1β-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell’s ability to respond to IL-1β. These results suggest that the inhibitory effect of estradiol on IL-1β-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.
Prostaglandin D2 Causes Preferential Induction of Proinflammatory Th2 Cytokine Production through an Action on Chemoattractant Receptor-Like Molecule Expressed on Th2 Cells
Authors: Xue Luzheng , Gyles Shân L. , Wettey Frank R. , Gazi Lucien , Townsend Elizabeth , Hunter Michael G. , Pettipher Roy
Published: April 2014

PGD2, produced by mast cells, has been detected in high concentrations at sites of allergic inflammation. It can stimulate vascular and other inflammatory responses by interaction with D prostanoid receptor (DP) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) receptors. A significant role for PGD2 in mediating allergic responses has been suggested based on the observation that enhanced eosinophilic lung inflammation and cytokine production is apparent in the allergen-challenged airways of transgenic mice overexpressing human PGD2 synthase, and PGD2 can enhance Th2 cytokine production in vitro from CD3/CD28-costimulated Th2 cells. In the present study, we investigated whether PGD2 has the ability to stimulate Th2 cytokine production in the absence of costimulation. At concentrations found at sites of allergic inflammation, PGD2 preferentially elicited the production of IL-4, IL-5, and IL-13 by human Th2 cells in a dose-dependent manner without affecting the level of the anti-inflammatory cytokine IL-10. Gene transcription peaked within 2 h, and protein release peaked ∼8 h after stimulation. The effect of PGD2 was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD2 but not by the selective DP agonist BW245C, suggesting that the stimulation is mediated by CRTH2 and not DP. Ramatroban, a dual CRTH2/thromboxane-like prostanoid receptor antagonist, markedly inhibited Th2 cytokine production induced by PGD2, while the selective thromboxane-like prostanoid receptor antagonist SQ29548 was without effect. These data suggest that PGD2 preferentially up-regulates proinflammatory cytokine production in human Th2 cells through a CRTH2-dependent mechanism in the absence of any other costimulation and highlight the potential utility of CRTH2 antagonists in the treatment of allergic diseases.
The Cooperation between Two CD4 T Cells Induces Tumor Protective Immunity in MUC.1 Transgenic Mice
Authors: Gerloni Mara , Castiglioni Paola , Zanetti Maurizio
Published: April 2014

Immunity and tumor protection in mice transgenic for human MUC.1, a glycoprotein expressed in the majority of cancers of epithelial origin in humans, were induced by vaccination with B lymphocytes genetically programmed to activate MUC.1-specific CD4 T cells. Their activation required a functional cooperation between two Th cells, one specific for a self (MUC.1) and the other for a nonself T cell determinant. The immunological switch provided by Th-Th cooperation was sufficient to induce MUC.1-specific CD4 and CD8 T cell responses in MUC.1-transgenic mice, and protect them permanently from tumor growth. CD4 T cells specific for MUC.1 lacked cytolytic function, but produced IFN-γ upon restimulation with Ag. We conclude that immunity against tumor self-Ags and tumor protection can be regulated exploiting an inherent property of the immune system.
Activation and Inactivation of Antiviral CD8 T Cell Responses during Murine Pneumovirus Infection
Authors: Claassen Erwin A. W. , van der Kant Patrick A. A. , Rychnavska Zuzana S. , van Bleek Grada M. , Easton Andrew J. , van der Most Robbert G.
Published: April 2014

Pneumonia virus of mice (PVM) is a natural pathogen of mice and has been proposed as a tractable model for the replication of a pneumovirus in its natural host, which mimics human infection with human respiratory syncytial virus (RSV). PVM infection in mice is highly productive in terms of virus production compared with the situation seen with RSV in mice. Because RSV suppresses CD8 T cell effector function in the lungs of infected mice, we have investigated the nature of PVM-induced CD8 T cell responses to study pneumovirus-induced T cell responses in a natural virus-host setting. PVM infection was associated with a massive influx of activated CD8 T cells into the lungs. After identification of three PVM-specific CD8 T cell epitopes, pulmonary CD8 T cell responses were enumerated. The combined frequency of cytokine-secreting CD8 T cells specific for the three epitopes was much smaller than the total number of activated CD8 T cells. Furthermore, quantitation of the CD8 T cell response against one of these epitopes (residues 261–270 from the phosphoprotein) by MHC class I pentamer staining and by in vitro stimulation followed by intracellular IFN-γ and TNF-α staining indicated that the majority of pulmonary CD8 specific for the P261 epitope were deficient in cytokine production. This deficient phenotype was retained up to 96 days postinfection, similar to the situation in the lungs of human RSV-infected mice. The data suggest that PVM suppresses T cell effector functions in the lungs.
Impaired Assembly Results in the Accumulation of Multiple HLA-C Heavy Chain Folding Intermediates
Authors: Sibilio Leonardo , Martayan Aline , Setini Andrea , Fraioli Rocco , Fruci Doriana , Shabanowitz Jeffrey , Hunt Donald F. , Giacomini Patrizio
Published: April 2014

Class I MHC H chains assemble with β2-microglobulin (β2m) and are loaded with peptide Ags through multiple folding steps. When free of β2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, β2m-free H chains with calnexin in β2m-defective cells, and with calreticulin and TAP in β2m-expressing cells. In β2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the α1 domain α helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the α1 domain α helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.
Francisella tularensis Induces Aberrant Activation of Pulmonary Dendritic Cells
Authors: Bosio Catharine M. , Dow Steven W.
Published: April 2014

Francisella tularensis is an obligate intracellular bacterium that induces severe, acute, often fatal disease when acquired by the respiratory route. Despite the seriousness of this pathogen, very little is understood about its interaction with key target cells in the airways and lungs (alveolar macrophages and airway dendritic cells (DC)) after inhalation. In this study we demonstrate replication of F. tularensis in primary DC. Early after infection, F. tularensis induced increased expression of MHC class II and CD86 on DC, but not macrophages. This was followed by depletion of DC from the airways and lungs. Despite logarithmic replication and phenotypic maturation of DC, F. tularensis failed to induce production of several key proinflammatory cytokines, including TNF-α and IL-6, from DC. However, F. tularensis infection did elicit production of the potent immunosuppressive cytokine, TGF-β. Furthermore, F. tularensis actively suppressed the ability of DC to secrete cytokines in response to specific TLR agonists. Finally, we also found that infection of DC and macrophages in the lungs appears to actually increase the severity of pulmonary infection with F. tularensis. For example, depletion of airway DC and alveolar macrophages before infection resulted in significantly prolonged survival times. Together, these data suggest F. tularensis is able to selectively uncouple Ag-presenting functions from proinflammatory cytokine secretion by critical APCs in the lungs, which may serve to create a relatively immunosuppressive environment favorable to replication and dissemination of the organism.
Positive and Negative Regulation of Mast Cell Activation by Lyn via the FcεRI
Authors: Xiao Wenbin , Nishimoto Hajime , Hong Hong , Kitaura Jiro , Nunomura Satoshi , Maeda-Yamamoto Mari , Kawakami Yuko , Lowell Clifford A. , Ra Chisei , Kawakami Toshiaki
Published: April 2014

Aggregation of the high affinity receptor for IgE (FcεRI) induces activation of mast cells. In this study we show that upon low intensity stimulation of FcεRI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with FcεRI β subunit, whereas high intensity stimulation enhanced Lyn activity and its association with FcεRI β. The latter induced much higher levels of FcεRI β phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with FcεRI β, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant FcεRI β showed that FcεRI β subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of FcεRI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the FcεRI β-ITAM.
The Role of Complement in Cryoglobulin-Induced Immune Complex Glomerulonephritis
Authors: Trendelenburg Marten , Fossati-Jimack Liliane , Cortes-Hernandez Josefina , Turnberg Daniel , Lewis Margarita , Izui Shozo , Cook H. Terence , Botto Marina
Published: April 2014

Many forms of glomerulonephritis are triggered by Ab localization in the glomerulus, but the mechanisms by which this induces glomerular inflammation are not fully understood. In this study we investigated the role of complement in a mouse model of cryoglobulin-induced immune complex glomerulonephritis. Several complement-deficient mice on a C57BL/6 and BALB/c genetic background were used and compared with strain-matched, wild-type controls. Cryoglobulinemia was induced by i.p. injection of 6-19 hybridoma cells producing an IgG3 cryoglobulin with rheumatoid factor activity against IgG2a of allotype a present in BALB/c, but not C57BL/6, mice. Thus, the cryoprecipitate in C57BL/6 mice consisted of the IgG3 cryoglobulin only (type I cryoglobulinemia) compared with IgG3-IgG2a complexes in BALB/c (type II cryoglobulinemia). The survival of mice was not affected by complement deficiency. Glomerular influx of neutrophils was significantly less in C3-, factor B-, and C5-deficient mice compared with wild-type and C1q-deficient mice. It did not correlate with C3 deposition, but did correlate with the amount of C6 deposited. Deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration, suggesting that the generation of C5a accounts for the effects observed. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Our results suggest that in this model of cryoglobulin-induced glomerulonephritis the neutrophil influx was mediated by C5 activation with the alternative pathway playing a prominent role in its cleavage. Thus, blocking C5 is a potential therapeutic strategy for preventing renal injury in cryoglobulinemia.
A Mimic of p21WAF1/CIP1 Ameliorates Murine Lupus
Authors: Goulvestre Claire , Chéreau Christiane , Nicco Carole , Mouthon Luc , Weill Bernard , Batteux Frédéric
Published: April 2014

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by the production of high levels of affinity-matured IgG autoantibodies to dsDNA and, possibly, visceral involvement. Pathogenic autoantibodies result from the activation and proliferation of autoreactive T and B lymphocytes stimulated by epitopes borne by nucleosomal histones. To inhibit the proliferation of autoreactive cells and abrogate the development of SLE, a novel tool, cell cycle inhibiting peptide therapy, was used. Thus, a peptidyl mimic of p21WAF1/CIP1 that inhibits the interaction between cyclin-dependent kinase 4 and type D cyclins abrogated the in vitro proliferative response of T cells to histones and T-independent and T-dependent proliferative responses of B cells. The WAF1/CIP1 mimic also abrogated the in vitro production of total and anti-dsDNA IgG Abs by B cells. Similarly, the p21WAF1/CIP1 construct inhibited the ex vivo T and B cell proliferative responses to histones and decreased the numbers of activated/memory B and T spleen cells. The alterations in the balance of spleen cell subsets resulted from proapoptotic effects of the p21WAF1/CIP1 construct on activated splenocytes. Finally, in vivo, four i.v. injections of the p21WAF1/CIP1 mimic were sufficient to inhibit the progression of the lupus-like syndrome in (NZB × NZW)F1 mice. The levels of anti-dsDNA IgG autoantibodies and the incidence and severity of renal involvement were lower in treated mice than in nontreated mice. Those observations open new avenues for the treatment of SLE and prompt us to evaluate the potential interest of peptidic therapy in human SLE.
Proteomic Scan for Tyrosinase Peptide Antigenic Pattern in Vitiligo and Melanoma: Role of Sequence Similarity and HLA-DR1 Affinity
Authors: Lucchese Alberta , Willers Jörg , Mittelman Abraham , Kanduc Darja , Dummer Reinhard
Published: April 2014

Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95–104FMGFNCGNCK; aa175–182 LFVWMHYY; aa176–190FVWMHYYVSMDALLG; aa222–236IQKLTGDENFTIPYW, and aa233–247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95–104FMGFNCGNCK, aa222–236IQKLTGDENFTIPYW, and aa233–247 IPYWDWRDAEKCDIC, but did not react with the aa175–182LFVWMHYY and aa176–190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.
Cutting Edge: Murine CD59a Modulates Antiviral CD4+ T Cell Activity in a Complement-Independent Manner
Authors: Longhi M. Paula , Sivasankar Baalasubramanian , Omidvar Nader , Morgan B. Paul , Gallimore Awen
Published: April 2014

CD59 blocks formation of the membrane attack complex of complement by inhibiting binding of C9 to the C5b-8 complex. To investigate a role for CD59 in promoting T cell responses, we compared T cell activation in CD59a-deficient (Cd59a−/−) and wild-type (WT) mice after in vitro stimulation and after infection with rVV. Virus-specific CD4+ T cell responses were significantly enhanced in Cd59a−/− mice compared with WT mice. Similarly, Cd59a−/− T cells responded more vigorously to in vitro stimulation with CD3-specific Abs compared with WT mice. This effect of CD59a on T cell proliferation was found to be complement-independent. Collectively, these results demonstrate that CD59a down-modulates CD4+ T cell activity in vitro and in vivo, thereby revealing another link between complement regulators and T cell activation.
Glucosamine Abrogates the Acute Phase of Experimental Autoimmune Encephalomyelitis by Induction of Th2 Response
Authors: Zhang Guang-Xian , Yu Shuo , Gran Bruno , Rostami Abdolmohamad
Published: April 2014

Glucosamine, a natural glucose derivative and an essential component of glycoproteins and proteoglycans, has been safely used to relieve osteoarthritis in humans. Recent studies have shown that glucosamine also possesses immunosuppressive properties and is effective in prolonging graft survival in mice. Whether this reagent is effective in human multiple sclerosis (MS), an inflammatory demyelination in the CNS, is not known. We thus investigated the therapeutic effect of glucosamine on experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We demonstrated that oral, i.p., or i.v. administration of glucosamine significantly suppressed acute EAE, with reduced CNS inflammation and demyelination. A significant, albeit not strong, blockade of Th1 response and an up-regulation of Th2 cytokines (IL-5 and IL-10) are observed in the splenocytes of glucosamine-treated mice. Glucosamine also regulates IL-5 and IL-10 in vitro. As glucosamine is able to effectively suppress acute EAE, has low or absent toxicity, and has been safely used in humans orally, our study suggests a potential use for this drug alone or in combination with other disease-modifying immunotherapies to enhance their efficacy and reduce their doses in MS and possibly other autoimmune disorders. Furthermore, because glucosamine functions not simply as an immunosuppressant, but as a mild immunomodulator, administration of glucosamine provides a novel immunoregulatory approach for autoimmune disorders.
A Distal Regulatory Region Is Required for Constitutive and IFN-β-Induced Expression of Murine TLR9 Gene
Authors: Guo Zhu , Garg Sanjay , Hill Karen M. , Jayashankar Lakshmi , Mooney Myesha R. , Hoelscher Mary , Katz Jacqueline M. , Boss Jeremy M. , Sambhara Suryaprakash
Published: April 2014

TLR9 is critical for the recognition of unmethylated CpG DNA in innate immunity. Accumulating evidence suggests distinct patterns of TLR9 expression in various types of cells. However, the molecular mechanism of TLR9 expression has received little attention. In the present study, we demonstrate that transcription of murine TLR9 is induced by IFN-β in peritoneal macrophages and a murine macrophage cell line RAW264.7. TLR9 is regulated through two cis-acting regions, a distal regulatory region (DRR) and a proximal promoter region (PPR), which are separated by ∼2.3 kbp of DNA. Two IFN-stimulated response element/IFN regulatory factor-element (ISRE/IRF-E) sites, ISRE/IRF-E1 and ISRE/IRF-E2, at the DRR and one AP-1 site at the PPR are required for constitutive expression of TLR9, while only the ISRE/IRF-E1 motif is essential for IFN-β induction. In vivo genomic footprint assays revealed constitutive factor occupancy at the DRR and the PPR and an IFN-β-induced occupancy only at the DRR. IRF-2 constitutively binds to the two ISRE/IRF-E sites at the DRR, while IRF-1 and STAT1 are induced to bind to the two ISRE/IRF-E sites and the ISRE/IRF-E1, respectively, only after IFN-β treatment. AP-1 subunits, c-Jun and c-Fos, were responsible for the constitutive occupancy at the proximal region. Induction of TLR9 by IFN-β was absent in STAT1−/− macrophages, while the level of TLR9 induction was decreased in IRF-1−/− cells. This study illustrates the crucial roles for AP-1, IRF-1, IRF-2, and STAT1 in the regulation of murine TLR9 expression.
Stat1-Dependent Synergistic Activation of T-bet for IgG2a Production during Early Stage of B Cell Activation
Authors: Xu Weifeng , Zhang J. Jillian
Published: April 2014

During the adaptive phase of an immune response, naive B cells receive multiple signals to become activated. Among them are the engagement of the B cell Ag receptor and stimulation by cytokines. Specifically for an anti-microbial response, the recognition of viral or bacterial Ags by the BCR and the stimulation of IFN-γ result in the predominant production of IgG2a. The T-bet protein has been shown to be required for class switching to IgG2a. In this report we further investigated the regulation of T-bet gene expression during the early stage of B cell activation. We show that there is a striking synergistic activation of T-bet in primary B cells when both the BCR and IFN-γ signaling pathways are activated. The synergistic activation of T-bet correlates with a 100% increase in the number of B cells that produce IgG2a. This transcription synergy on T-bet is transient in the first 24 h of B cell activation. Furthermore, we demonstrate that the synergistic activation of T-bet is dependent on Stat1 and that Stat1 is required for the IgG2a germline transcription and the production of IgG2a in response to the simultaneous signaling of BCR and IFN-γ. Finally, we show that Stat1 directly regulates the expression of T-bet by binding to the T-bet promoter. These results reveal the mechanism of regulation of T-bet expression and uncover a novel physiological function of Stat1 for B cell activation.
The Complex Mechanism of Antibody-Mediated Clearance of Bordetella from the Lungs Requires TLR4
Authors: Kirimanjeswara Girish S. , Mann Paul B. , Pilione Mylisa , Kennett Mary J. , Harvill Eric T.
Published: April 2014

Although the antibacterial effects of Abs are well studied in in vitro systems, the in vivo effects of Abs cannot always be accurately predicted. Complicated cross-talk between different effector functions of Abs and various arms of the immune system can affect their activities in vivo. Using the mouse respiratory pathogen Bordetella bronchiseptica, we examined the mechanisms of Ab-mediated clearance of bacteria from the respiratory tract. Interestingly, although TLR4 was not necessary for protective immunity following infection, it was required for rapid bacterial clearance in mice that were vaccinated or adoptively transferred Abs. TLR4 was important for the rapid recruitment of neutrophils that are necessary for Ab-mediated bacterial clearance via a mechanism that requires both FcγR and CR3. These data are consistent with a model in which TLR4-mediated inflammatory responses aid in the recruitment of neutrophils, which phagocytose Ab- and complement-opsonized bacteria via FcγRs and CR3. Although pattern recognition receptors are known to be involved in innate immunity and the generation of adaptive immunity, their contributions to specific adaptive immune functions should be considered in ongoing efforts to improve vaccine-induced protective immunity.
Staphylococcal Protein A Deletes B-1a and Marginal Zone B Lymphocytes Expressing Human Immunoglobulins: An Immune Evasion Mechanism
Authors: Viau Muriel , Longo Nancy S. , Lipsky Peter E. , Zouali Moncef
Published: April 2014

Protein A (SpA) of Staphylococcus aureus is endowed with the capacity to interact with the H chain variable region (VH) of human Abs and to target >40% of B lymphocytes. To investigate whether this property represents a virulence factor and to determine the in vivo consequences of the confrontation of SpA with B lymphocytes, we used transgenic mice expressing fully human Abs. We found that administration of soluble SpA reduces B-1a lymphocytes of the peritoneal cavity and marginal zone B lymphocytes of the spleen, resulting in a markedly deficient type 2 humoral response. Single-cell PCR analysis and sequencing of the Ab VH gene repertoire revealed a significant reduction of VH3+ marginal zone B cells. Since the two B lymphocyte subsets targeted are involved in innate immune functions, our data suggest that crippling of humoral immunity by S. aureus represents an immune evasion mechanism that may aggravate recurrent infections.
Signal Regulatory Proteins in the Immune System
Authors: van Beek Ellen M. , Cochrane Fiona , Barclay A. Neil , van den Berg Timo K.
Published: April 2014

Signal regulatory proteins (SIRPs) constitute a family of transmembrane glycoproteins with extracellular Ig-like domains. Several SIRP family members have thus far been identified on myeloid and other cells in man, mouse, rat, and cattle. In the present study, we provide a description of the SIRP multigene family, including a number of previously undescribed SIRP genes, based on the complete genome sequences of various mammalian and bird species. We discuss this information in the context of the known immunological properties of the individual SIRP family members. Our analysis reveals SIRPs as a diverse multigene family of immune receptors, which includes inhibitory SIRPα, activating SIRPβ, nonsignaling SIRPγ, and soluble SIRPδ members. For each species, there appears to be a single inhibitory SIRPα member that, upon interaction with the “self” ligand CD47, controls “homeostatic” innate immune effector functions, such as host cell phagocytosis. The activating SIRPβ proteins show considerable variability in structure and number across species and do not bind CD47. Thus the SIRP family is a rapidly evolving gene family with important roles in immune regulation.
Targeting LFA-1 and CD154 Suppresses the In Vivo Activation and Development of Cytolytic (CD4-Independent) CD8+ T Cells
Authors: Lunsford Keri E. , Koester Mitchel A. , Eiring Anna M. , Horne Phillip H. , Gao Donghong , Bumgardner Ginny L.
Published: April 2014

Short-term immunotherapy targeting both LFA-1 and CD40/CD154 costimulation produces synergistic effects such that long-term allograft survival is achieved in the majority of recipients. This immunotherapeutic strategy has been reported to induce the development of CD4+ regulatory T cells. In the current study, the mechanisms by which this immunotherapeutic strategy prevents CD8+ T cell-dependent hepatocyte rejection in CD4 knockout mice were examined. Combined blockade of LFA-1 and CD40/CD154 costimulation did not influence the overall number or composition of inflammatory cells infiltrating the liver where transplanted hepatocytes engraft. Expression of T cell activation markers CD43, CD69, and adhesion molecule CD103 by liver-infiltrating cells was suppressed in treated mice with long-term hepatocellular allograft survival compared to liver-infiltrating cells of untreated rejector mice. Short-term immunotherapy with anti-LFA-1 and anti-CD154 mAb also abrogated the in vivo development of alloreactive CD8+ cytotoxic T cell effectors. Treated mice with long-term hepatocyte allograft survival did not reject hepatocellular allografts despite adoptive transfer of naive CD8+ T cells. Unexpectedly, treated mice with long-term hepatocellular allograft survival demonstrated prominent donor-reactive delayed-type hypersensitivity responses, which were increased in comparison to untreated hepatocyte rejectors. Collectively, these findings support the conclusion that short-term immunotherapy with anti-LFA-1 and anti-CD154 mAbs induces long-term survival of hepatocellular allografts by interfering with CD8+ T cell activation and development of CTL effector function. In addition, these recipients with long-term hepatocellular allograft acceptance show evidence of immunoregulation which is not due to immune deletion or ignorance and is associated with early development of a novel CD8+CD25high cell population in the liver.
Increased Expression of Genes Linked to FcεRI Signaling and to Cytokine and Chemokine Production in Lyn-Deficient Mast Cells
Authors: Hernandez-Hansen Valerie , Bard Julie D. J. , Tarleton Christy A. , Wilder Julie A. , Lowell Clifford A. , Wilson Bridget S. , Oliver Janet M.
Published: April 2014

Cross-linking the high-affinity IgE receptor, FcεRI, on mast cells activates signaling pathways leading to the release of preformed inflammatory mediators and the production of cytokines and chemokines associated with allergic disorders. Bone marrow-derived mast cells (BMMCs) from Lyn-deficient (Lyn−/−) mice are hyperresponsive to FcεRI cross-linking with multivalent Ag. Previous studies linked the hyperresponsive phenotype in part to increased Fyn kinase activity and reduced SHIP phosphatase activity in the Lyn−/− BMMCs in comparison with wild-type (WT) cells. In this study, we compared gene expression profiles between resting and Ag-activated WT and Lyn−/− BMMCs to identify other factors that may contribute to the hyperresponsiveness of the Lyn−/− cells. Among genes implicated in the positive regulation of FcεRI signaling, mRNA for the tyrosine kinase, Fyn, and for several proteins contributing to calcium regulation are more up-regulated following Ag stimulation in Lyn−/− BMMCs than in WT BMMCs. Conversely, mRNA for the low-affinity IgG receptor (FcγRIIB), implicated in negative regulation of FcεRI-mediated signaling, is more down-regulated in Ag-stimulated Lyn−/− BMMCs than in WT BMMCs. Genes coding for proinflammatory cytokines and chemokines (IL-4, IL-6, IL-13, CSF, CCL1, CCL3, CCL5, CCL7, CCL9, and MIP1β)are all more highly expressed in Ag-stimulated Lyn−/− mast cells than in WT cells. These microarray data identify Lyn as a negative regulator in Ag-stimulated BMMCs of the expression of genes linked to FcεRI signaling and also to the response pathways that lead to allergy and asthma.
The Role of Low Molecular Weight Thiols in T Lymphocyte Proliferation and IL-2 Secretion
Authors: Hadzic Tanja , Li Ling , Cheng Ningli , Walsh Susan A. , Spitz Douglas R. , Knudson C. Michael
Published: April 2014

Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. l-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and l-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular l-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or l-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.