bluuebunny/crossref_metadata_2025 huggingface dataset:
Fraktalkine Produced by Airway Smooth Muscle Cells Contributes to Mast Cell Recruitment in Asthma
Authors: El-Shazly Amr , Berger Patrick , Girodet Pierre-Olivier , Ousova Olga , Fayon Michael , Vernejoux Jean-Marc , Marthan Roger , Tunon-de-Lara J. Manuel
Published: April 2014

Human airway smooth muscle cells (HASMC) secrete fractalkine (FKN), a chemokine the concentration of which is increased in asthmatic patients. HASMC also induce mast cell chemotaxis, as a component of asthma inflammation. We therefore evaluated the role of smooth muscle-derived FKN in mast cell migration. We assessed the capacity of recombinant FKN to induce human mast cell chemotaxis. This effect implicates a calcium-independent pathway involving actin reorganization and protein kinase C-δ. We found that HASMC constitutively produce FKN, the synthesis of which is reinforced upon proinflammatory stimulation. Under basal experimental conditions, FKN production by HASMC is not sufficient to induce mast cell chemotaxis. However, pretreatment of mast cells with the neuropeptide vasoactive intestinal peptide (VIP) increases FKN potency to attract mast cells. Since we observed, in asthmatic patients, an increase in both FKN and VIP expression by airway smooth muscle and a positive correlation between VIP staining and mast cell infiltration of the smooth muscle layer, we conclude that HASMC-derived FKN may contribute to mast cell recruitment in asthma.
Corticotropin-Releasing Factor and the Urocortins Induce the Expression of TLR4 in Macrophages via Activation of the Transcription Factors PU.1 and AP-1
Authors: Tsatsanis Christos , Androulidaki Ariadne , Alissafi Themis , Charalampopoulos Ioannis , Dermitzaki Erini , Roger Thierry , Gravanis Achille , Margioris Andrew N.
Published: April 2014

Corticotropin-releasing factor (CRF) augments LPS-induced proinflammatory cytokine production from macrophages. The aim of the present study was to determine the mechanism by which CRF and its related peptides urocortins (UCN) 1 and 2 affect LPS-induced cytokine production. We examined their role on TLR4 expression, the signal-transducing receptor of LPS. For this purpose, the murine macrophage cell line RAW 264.7 and primary murine peritoneal macrophages were used. Exposure of peritoneal macrophages and RAW 264.7 cells to CRF, UCN1, or UCN2 up-regulated TLR4 mRNA and protein levels. To study whether that effect occurred at the transcriptional level, RAW 264.7 cells were transfected with a construct containing the proximal region of the TLR4 promoter linked to the luciferase gene. CRF peptides induced activation of the TLR4 promoter, an effect abolished upon mutation of a proximal PU.1-binding consensus or upon mutation of an AP-1-binding element. Indeed, all three peptides promoted PU.1 binding to the proximal PU.1 site and increased DNA-binding activity to the AP-1 site. The effects of CRF peptides were inhibited by the CRF2 antagonist anti-sauvagine-30, but not by the CRF1 antagonist antalarmin, suggesting that CRF peptides mediated the up-regulation of TLR4 via the CRF2 receptor. Finally, CRF peptides blocked the inhibitory effect of LPS on TLR4 expression. In conclusion, our data suggest that CRF peptides play an important role on macrophage function. They augment the effect of LPS by inducing Tlr4 gene expression, through CRF2, via activation of the transcription factors PU.1 and AP-1.
Lipoxin A4 Redistributes Myosin IIA and Cdc42 in Macrophages: Implications for Phagocytosis of Apoptotic Leukocytes
Authors: Reville Keira , Crean John K. , Vivers Sharon , Dransfield Ian , Godson Catherine
Published: April 2014

Lipoxins (LXs) are endogenously produced anti-inflammatory agents that modulate leukocyte trafficking and stimulate nonphlogistic macrophage phagocytosis of apoptotic neutrophils, thereby promoting the resolution of inflammation. Previous data suggest a role for altered protein phosphorylation and cytoskeletal rearrangement in LX-stimulated phagocytosis but the exact mechanisms remain unclear. In this study we examine the effects of LXA4 on the protein phosphorylation pattern of THP-1 cells differentiated into a macrophage-like phenotype. THP-1 cells stimulated with LXA4 (1 nM) exhibit dephosphorylation of a 220-kDa protein. Using mass spectrometry, this protein was identified as MYH9, a nonmuscle myosin H chain II isoform A, which is involved in cytoskeleton rearrangement. THP-1 cells treated with LXA4 adopt a polarized morphology with activated Cdc42 localized toward the leading edge and MYH9 localized at the cell posterior. Polarized distribution of Cdc42 is associated with Akt/PKB-mediated Cdc42 activation. Interestingly, the annexin-derived peptide Ac2–26, a recently described agonist for the LXA4 receptor, also stimulates macrophage phagocytosis, MYH9 dephosphorylation, and MYH9 redistribution. In addition, we demonstrate that LXA4 stimulates the phosphorylation of key polarity organization molecules: Akt, protein kinase Cζ, and glycogen synthase kinase-3β. Inhibition of LXA4-induced Akt and protein kinase Cζ activity with specific inhibitors prevented LXA4-stimulated phagocytosis of both apoptotic polymorphonuclear neutrophils and lymphocytes, highlighting a potential use for LXA4 in the treatment of autoimmune diseases. Furthermore, phosphorylation and subsequent inactivation of glycogen synthase kinase-3β resulted in an increase in phagocytosis similar to that of LXA4. These data highlight an integrated mechanism whereby LXA4 regulates phagocytosis through facilitative actin cytoskeleton rearrangement and cell polarization.
Proinflammatory Effects of Tweak/Fn14 Interactions in Glomerular Mesangial Cells
Authors: Campbell Sean , Burkly Linda C. , Gao Hua-Xin , Berman Joan W. , Su Lihe , Browning Beth , Zheng Timothy , Schiffer Lena , Michaelson Jennifer S. , Putterman Chaim
Published: April 2014

TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, CXCL10/IFN-γ-induced protein 10 kDa, and CXCL1/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-α + IL-1β. CXCL11/interferon-inducible T cell α chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Iκ-B, while pretreatment with an Iκ-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-κB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.
Essential Role of MAPK Phosphatase-1 in the Negative Control of Innate Immune Responses
Authors: Salojin Konstantin V. , Owusu Iris B. , Millerchip Karen A. , Potter Mark , Platt Ken A. , Oravecz Tamas
Published: April 2014

TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1−/− mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-α, IL-6, IL-12, MCP-1, IFN-γ, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1−/− macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.
In Vitro and In Situ Expression of IL-23 by Keratinocytes in Healthy Skin and Psoriasis Lesions: Enhanced Expression in Psoriatic Skin
Authors: Piskin Gamze , Sylva-Steenland Regien M. R. , Bos Jan D. , Teunissen Marcel B. M.
Published: April 2014

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-γ-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-γ production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-γ-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-γ, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.
Circulating Progenitor Epithelial Cells Traffic via CXCR4/CXCL12 in Response to Airway Injury
Authors: Gomperts Brigitte N. , Belperio John A. , Rao P. Nagesh , Randell Scott H. , Fishbein Michael C. , Burdick Marie D. , Strieter Robert M.
Published: April 2014

Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases.
Suppression of Discoidin Domain Receptor 1 by RNA Interference Attenuates Lung Inflammation
Authors: Matsuyama Wataru , Watanabe Masaki , Shirahama Yuko , Hirano Ryuki , Mitsuyama Hideo , Higashimoto Ikkou , Osame Mistuhiro , Arimura Kimiyoshi
Published: April 2014

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase whose ligand is collagen. Recently, we have reported the association of DDR1 in the cytokine production of human leukocytes in in vitro and in vivo expression in idiopathic pulmonary fibrosis. However, its role in in vivo inflammation has not been fully elucidated. Small interference RNA (siRNA) can induce specific suppression of in vitro and in vivo gene expression. In this study, using a bleomycin-induced pulmonary fibrosis mouse model, we administered siRNA against DDR1 transnasally and evaluated histological changes, cytokine expression, and signaling molecule activation in the lungs. Histologically, siRNA against DDR1 successfully reduced in vivo DDR1 expression and attenuated bleomycin-induced infiltration of inflammatory cells. Furthermore, it significantly reduced inflammatory cell counts and concentrations of cytokines such as MCP-1, MIP-1α, and MIP-2 in bronchoalveolar lavage fluid. Subsequently, bleomycin-induced up-regulation of TGF-β in bronchoalveolar lavage fluid was significantly inhibited, and collagen deposition in the lungs was reduced. Furthermore, siRNA against DDR1 significantly inhibited bleomycin-induced P38 MAPK activation in the lungs. Considered together, we propose that DDR1 contributes to the development of bleomycin-induced pulmonary inflammation and fibrosis.
Deletion of TLR3 Alters the Pulmonary Immune Environment and Mucus Production during Respiratory Syncytial Virus Infection
Authors: Rudd Brian D. , Smit Jetse J. , Flavell Richard A. , Alexopoulou Lena , Schaller Matthew A. , Gruber Achim , Berlin Aaron A. , Lukacs Nicholas W.
Published: April 2014

The detection of a viral infection by pattern recognition receptors (PAMPs) is an integral part of antiviral immunity. In these studies we have investigated the role of TLR3, which recognizes dsRNA, in Respiratory Syncytial virus (RSV) infection using B6 background mice with a TLR3 deletion. Although we observed no changes in viral growth, we did find that TLR3−/− mice demonstrated significant increases in mucus production in the airways of RSV-infected mice. The qualitative assessment was observed by examining differentially stained lungs, followed by immunohistochemical staining for gob5, a mucus-associated protein. The histopathologic observations were verified using quantitative gene expression analyses examining gob5 gene expression. Changes in pulmonary mucus production were accompanied by an increase in pulmonary IL-13 as well as IL-5 expression and eosinophils in the airways of TLR3−/− mice. Examining leukocytes in the airway indicated an accumulation of eosinophils in TLR3−/− mice, but not wild-type mice, after RSV infection. Isolated lung draining lymph node cells from TLR3−/− mice produced significant increases in Th2-type cytokines, IL-5, and IL-13, compared with wild-type TLR3+/+ mice only after RSV infection. To demonstrate a causative link, we depleted TLR3−/− mice of IL-13 during RSV infection and found that mucus and gob5 expression in the lungs was attenuated. Together, these studies highlight that although TLR3 may not be required for viral clearance, it is necessary to maintain the proper immune environment in the lung to avoid developing pathologic symptoms of disease.
Inhibition of Chronic Airway Inflammation and Remodeling by Galectin-3 Gene Therapy in a Murine Model
Authors: López Esther , del Pozo Victoria , Miguel Teresa , Sastre Beatriz , Seoane Carmen , Civantos Esther , Llanes Elena , Baeza M. Luisa , Palomino Pilar , Cárdaba Blanca , Gallardo Soledad , Manzarbeitia Félix , Zubeldia José M. , Lahoz Carlos
Published: April 2014

We previously demonstrated that treatment of acute asthmatic rats with gene therapy using plasmid-encoding Galectin-3 (Gal-3) resulted in an improvement of cellular and functional respiratory parameters. The next question that we wanted to clarify was if in a chronic situation where the treated animal continues to inhale the Ag, does this procedure prevent the chronicity and the remodeling? Chronic inflammation was induced by intranasal administration of OVA over a period of 12 wk. In the treated group, the Gal-3 gene was introduced by intranasal instillation in 50 μl of plasmid-encoding Gal-3. Noninvasive airway responsiveness to methacholine was tested at different times. Cells were obtained by bronchoalveolar lavage and used for RNA extraction and cytometric studies. Eosinophils were counted in blood and bronchoalveolar lavage fluid. Real-time PCR was used to measure Gal-3 and cytokine mRNA expression in lung. Lungs were paraffined and histologic analyses were performed (H&E, periodic acid-Schiff, and Masson Trichrome stain). Our results showed that 12 wk after the first intranasal Ag instillation in chronically asthmatic mice, treatment with the Gal-3 gene led to an improvement in the eosinophil count and the normalization of hyperresponsiveness to methacholine. Concomitantly, this treatment resulted in an improvement in mucus secretion and subepithelial fibrosis in the chronically asthmatic mice, with a quantitatively measured reduction in lung collagen, a prominent feature of airway remodeling. Plasmid-encoding Gal-3 acts as a novel treatment for chronic asthma in mice producing nearly complete blockade of Ag responses with respect to eosinophil airway accumulation, airway hyperresponsiveness, and remodeling.
Redundancy in Antigen-Presenting Function of the HLA-DR and -DQ Molecules in the Multiple Sclerosis-Associated HLA-DR2 Haplotype
Authors: Sospedra Mireia , Muraro Paolo A. , Stefanová Irena , Zhao Yingdong , Chung Katherine , Li Yili , Giulianotti Marc , Simon Richard , Mariuzza Roy , Pinilla Clemencia , Martin Roland
Published: April 2014

The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction.
Effacing of the T Cell Compartment by Cardiac Transplantation in Infancy
Authors: Ogle Brenda M. , West Lori J. , Driscoll David J. , Strome Scott E. , Razonable Raymund R. , Paya Carlos V. , Cascalho Marilia , Platt Jeffrey L.
Published: April 2014

For cardiac transplantation in infants, T cells are depleted and the thymus is removed. These manipulations should cause profound defects in the T cell compartment. To test this concept, 20 subjects who underwent cardiac transplantation in infancy and healthy age-matched subjects were studied. The number of T cells in the blood was nearly normal in all subjects 1–10 years after surgery. However, newly generated T cells were undetectable in 10 recipients and 10-fold less than controls in 10, suggesting absence of thymic function. TCRβ chain diversity, measured by a novel technique, was ∼100-fold lower than controls. T cell function, deduced from levels of human herpesvirus 7 and response to hepatitis B immunization, were notably impaired. Yet cardiac transplant recipients were generally free of opportunistic infections. Our findings demonstrate a novel approach to measuring lymphocyte diversity and suggest that understanding how these subjects resist infection could yield important insights into immune fitness.
Identification of an Epitope within Human Integrin α6 Subunit for the Binding of Autoantibody and Its Role in Basement Membrane Separation in Oral Pemphigoid
Authors: Rashid Khwaja Aftab , Stern Joel N. H. , Ahmed A. Razzaque
Published: April 2014

Oral pemphigoid (OP) is a rare chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for the binding of OP autoantibodies within the integrin α6 subunit, by cloning four overlapping fragments (A, B, C, and D). Immunoperoxidase studies demonstrated that all of the fragments were present in the oral mucosa. Sera of 20 patients with active OP were studied. All sera bound to integrin α6 in DU145 cell lysate by immunoprecipitation and immunoblot assay. The same sera bound only to fragment A and its subfragment A2 on an immunoblot assay. The specificity of the binding was further characterized by blocking and cross-absorption studies. A 14-aa synthetic peptide A2.1, within fragment A2, bound to all the test sera. The sera in this study bound to only one epitope. Controls were sera samples from 10 healthy volunteers and 40 patients with other variants of mucous membrane pemphigoid and mAb GoH3 and BQ16 to integrin α6. Control sera did not bind to the full-length integrin α6 subunit nor any of the cloned fragments. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera against fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa in organ culture. This study identifies a peptide within the extracellular domain of integrin α6 molecule, to which Abs in the sera from patients with OP bind, and which may play an important role in the pathogenesis of OP.
TLR9 Activation Is Defective in Common Variable Immune Deficiency
Authors: Cunningham-Rundles Charlotte , Radigan Lin , Knight Adina K. , Zhang Li , Bauer Laura , Nakazawa Atsushi
Published: April 2014

Common variable immune deficiency (CVID) is a primary immune deficiency characterized by low levels of serum immune globulins, lack of Ab, and reduced numbers of CD27+ memory B cells. Although T, B, and dendritic cell defects have been described, for the great majority, genetic causes have not been identified. In these experiments, we investigated B cell and plasmacytoid dendritic cell activation induced via TLR9, an intracellular recognition receptor that detects DNA-containing CpG motifs from viruses and bacteria. CpG-DNA activates normal B cells by the constitutively expressed TLR9, resulting in cytokine secretion, IgG class switch, immune globulin production, and potentially, the preservation of long-lived memory B cells. We found that CpG-DNA did not up-regulate expression of CD86 on CVID B cells, even when costimulated by the BCR, or induce production of IL-6 or IL-10 as it does for normal B cells. TLR9, found intracytoplasmically and on the surface of oligodeoxynucleotide-activated normal B cells, was deficient in CVID B cells, as was TLR9 mRNA. TLR9 B cell defects were not related to proportions of CD27+ memory B cells. CpG-activated CVID plasmacytoid dendritic cells did not produce IFN-α in normal amounts, even though these cells contained abundant intracytoplasmic TLR9. No mutations or polymorphisms of TLR9 were found. These data show that there are broad TLR9 activation defects in CVID which would prevent CpG-DNA-initiated innate immune responses; these defects may lead to impaired responses of plasmacytoid dendritic cells and loss of B cell function.
Allelic Variation in Key Peptide-Binding Pockets Discriminates between Closely Related Diabetes-Protective and Diabetes-SusceptibleHLA-DQB1*06Alleles
Authors: Ettinger Ruth A. , Papadopoulos George K. , Moustakas Antonis K. , Nepom Gerald T. , Kwok William W.
Published: April 2014

HLA-DQA1*0102-DQB1*0602 is associated with protection against type 1 diabetes (T1D). A similar allele, HLA-DQA1*0102-DQB1*0604, contributes to T1D susceptibility in certain populations but differs only at seven amino acids from HLA-DQA1*0102-DQB1*0602. Five of these polymorphisms are found within the peptide-binding groove, suggesting that differences in peptide binding contribute to the mechanism of their association with T1D. In this study, we determine the peptide-binding motif for HLA-DQA1*0102-DQB1*0604 allelic protein (DQ0604) in comparison to the established HLA-DQA1*0102-DQB1*0602 (DQ0602) motif using binding assays with model peptides from T1D autoantigens and homology modeling using the coordinates of the DQ0602-hypocretin 1–13 crystal structure. The peptide binding preferences were deduced with a peptide from insulin that bound both with a 2- to 3-fold difference in avidity using the same amino acids in the peptide as anchors. Peptide binding differences directly influenced by the polymorphisms in or nearby pockets 1, 6, and 9 were observed. In pocket 1, DQ0604 was better able to accommodate aromatic residues due to the β86 and β87 polymorphisms. A negatively charged amino acid was preferred by DQ0604 in pocket 6 due to the positively charged β30His. In pocket 9, DQ0604 preferred aromatic amino acids due to the β9 and β30 polymorphisms and had low tolerance of acidic residues. β57Val in DQ0604 functions differently than β57Ala, in that it pushes α76Arg outside of the pocket, preventing the formation of a salt bridge with an acidic amino acid in the peptide. This study furthers our understanding of the structure-function relationships of MHC class II polymorphisms.
Predicting Tumor Outcome following Cancer Vaccination by Monitoring Quantitative and Qualitative CD8+ T Cell Parameters
Authors: Rosato Antonio , Zoso Alessia , Santa Silvia Dalla , Milan Gabriella , Del Bianco Paola , De Salvo Gian Luca , Zanovello Paola
Published: April 2014

Identification of reliable surrogate predictors for evaluation of cancer vaccine efficacy is a critical issue in immunotherapy. We analyzed quantitative and qualitative CD8+ T cell parameters in a large pool of BALB/c mice that were DNA-vaccinated against P1A self tumor-specific Ag. After immunization, mice were splenectomized and kept alive for a subsequent tumor challenge to correlate results of immune monitoring assays with tumor regression or progression in each individual animal, and to assess the prognostic value of the assays. The parameters tested were 1) percentage of in vivo vaccine-induced tumor-specific CD8+ T cells; 2) results of ELISPOT tests from fresh splenocytes; 3) percentage of tumor-specific CD8+ T cells in culture after in vitro restimulation; 4) in vitro increase of tumor-specific CD8+ T cell population expressed as fold of expansion; and 5) antitumor lytic activity of restimulated cultures. Except for the ELISPOT assay, each parameter tested was shown by univariate statistical analysis to correlate with tumor regression. However, multivariate analysis revealed that only in vitro percentage of Ag-specific CD8+ T cells was an independent prognostic factor that predicted tumor outcome. These findings should be considered in the design of new immune monitoring systems used in cancer immunotherapy studies.
Pretreatment Intracerebral and Peripheral Blood Immune Responses in Vietnamese Adults with Tuberculous Meningitis: Diagnostic Value and Relationship to Disease Severity and Outcome
Authors: Simmons Cameron P. , Thwaites Guy E. , Quyen Nguyen Than Ha , Torok Estee , Hoang Dang Minh , Chau Tran Thi Hong , Mai Pham Phuong , Lan Nguyen Thi Ngoc , Dung Nguyen Huy , Quy Hoang Thi , Bang Nguyen Duc , Hien Tran Tinh , Farrar Jeremy
Published: April 2014

Tuberculous meningitis (TBM) is the most devastating form of tuberculosis. Both intracerebral and peripheral blood immune responses may be relevant to pathogenesis, diagnosis, and outcome. In this study, the relationship between pretreatment host response, disease phenotype, and outcome in Vietnamese adults with TBM was examined. Before treatment, peripheral blood IFN-γ ELISPOT responses to the Mycobacterium tuberculosis Ags ESAT-6, CFP-10, and purified protein derivative (PPD) were a poor diagnostic predictor of TBM. Cerebrospinal fluid IL-6 concentrations at presentation were independently associated with severe disease presentation, suggesting an immunological correlate of neurological damage before treatment. Surprisingly however, elevated cerebrospinal fluid inflammatory cytokines were not associated with death or disability in HIV-negative TBM patients at presentation. HIV coinfection attenuated multiple cerebrospinal fluid inflammatory indices. Low cerebrospinal fluid IFN-γ concentrations were independently associated with death in HIV-positive TBM patients, implying that IFN-γ contributes to immunity and survival. Collectively, these results reveal the effect of HIV coinfection on the pathogenesis of TBM and suggest that intracerebral immune responses, at least in HIV-negative cases, may not be as intimately associated with disease outcome as previously thought.
Autoreactive T and B Cells from Bullous Pemphigoid (BP) Patients Recognize Epitopes Clustered in Distinct Regions of BP180 and BP230
Authors: Thoma-Uszynski Sybille , Uter Wolfgang , Schwietzke Susanne , Schuler Gerold , Borradori Luca , Hertl Michael
Published: April 2014

Bullous pemphigoid (BP) is a well-characterized model of autoantibody-mediated autoimmunity, which presumably depends on autoreactive Th cells that promote the activation of autoreactive B cells. The two major autoantigens of BP are BP180 and BP230, two components of dermoepidermal adhesion complexes. Both, autoreactive Th cell responses and autoantibody profiles were characterized in 35 patients with acute onset BP using BP180 and BP230 proteins. Our findings indicate the following: 1) autoreactive Th cells recognized epitopes within the NH2-terminal (77.1%), COOH-terminal (65.7%), and central portion (57.1%) of the BP180 ectodomain; 2) IgG autoantibodies were found to exhibit similar or identical reactivity against the NH2-terminal (82.8%), COOH-terminal (77.1%), and central portion (37.1%) of the BP180 ectodomain; 3) T and B cell reactivity with the NH2-terminal portion of the BP180 ectodomain was associated with extensive BP, whereas the central portion was more frequently recognized in limited BP; 4) only 7 of 16 (43.7%) and 6 of 16 (37.5%) BP patients showed a Th cellular response against the COOH- and NH2-terminal regions of BP230, respectively, whereas 5) IgG reactivity against the COOH- and NH2-termini of BP230 was detected in 5 of 16 (31.3%) and 6 of 16 (37.5%) patients, respectively. These results demonstrate that Th and B cell reactivities against BP180, are, in contrast to BP230 reactivity, almost constantly detectable in BP patients, and differential epitope recognition of BP180 seems to be associated with distinct clinical severity. These observations support the concept that BP180, but not BP230, is the primary autoantigen of BP critical for disease development.
Mechanisms of Immunopathology in Murine Models of Central Nervous System Demyelinating Disease
Authors: Ercolini Anne M. , Miller Stephen D.
Published: April 2014

Many disorders of the CNS, such as multiple sclerosis (MS), are characterized by the loss of the myelin sheath surrounding nerve axons. MS is associated with infiltration of inflammatory cells into the brain and spinal cord, which may be the primary cause of demyelination or which may be induced secondary to axonal damage. Both the innate and adaptive arms of the immune system have been reported to play important roles in myelin destruction. Numerous murine demyelinating models, both virus-induced and/or autoimmune, are available, which reflect the clinical and pathological variability seen in human disease. This review will discuss the immunopathologic mechanisms involved in these demyelinating disease models.
Cutting Edge: Anti-CD25 Monoclonal Antibody Injection Results in the Functional Inactivation, Not Depletion, of CD4+CD25+ T Regulatory Cells
Authors: Kohm Adam P. , McMahon Jeffrey S. , Podojil Joseph R. , Begolka Wendy Smith , DeGutes Mathew , Kasprowicz Deborah J. , Ziegler Steven F. , Miller Stephen D.
Published: April 2014

CD4+CD25+ T regulatory (TR) cells are an important regulatory component of the adaptive immune system that limit autoreactive T cell responses in various models of autoimmunity. This knowledge was generated by previous studies from our lab and others using TR cell supplementation and depletion. Contrary to dogma, we report here that injection of anti-CD25 mAb results in the functional inactivation, not depletion, of TR cells, resulting in exacerbated autoimmune disease. Supporting this, mice receiving anti-CD25 mAb treatment display significantly lower numbers of CD4+CD25+ T cells but no change in the number of CD4+FoxP3+ TR cells. In addition, anti-CD25 mAb treatment fails to both reduce the number of Thy1.1+ congenic CD4+CD25+ TR cells or alter levels of CD25 mRNA expression in treatment recipients. Taken together, these findings have far-reaching implications for the interpretation of all previous studies forming conclusions about CD4+CD25+ TR cell depletion in vivo.
Cutting Edge: CD28 Controls Dominant Regulatory T Cell Activity during Active Immunization
Authors: Lyddane Clay , Gajewska Beata U. , Santos Elmer , King Philip D. , Furtado Glaucia C. , Sadelain Michel
Published: April 2014

Ligation of CD28 during Ag recognition plays an important role in the generation of effective T cell responses. However, its peripheral control of regulatory T cell function remains obscure. In this study, we show that naive wild-type or CD28−/− CD4+CD25− T cells exposed to peptide in vivo develop regulatory activity that suppresses the response of adoptively transferred naive T cells to a subsequent immunogenic challenge. We find that although CD28 is engaged during the initial peptide-priming event and is essential to sustain T cell survival, it is not sufficient to prevent the dominance of regulatory T cell function. Immunization with adjuvant abrogates regulatory dominance, reducing overall Foxp3 expression in a CD28-dependent manner. We conclude that CD28 licenses active immunization by regulating Ag-induced immunoregulation.
Cutting Edge: Hormonal Milieu, Not Antigenic Specificity, Determines the Mature Phenotype of Autoreactive B Cells
Authors: Venkatesh Jeganathan , Peeva Elena , Xu Xiaonan , Diamond Betty
Published: April 2014

Although both marginal zone and follicular B cells produce anti-DNA Abs in murine models of systemic lupus erythematosus, it has been unclear whether these distinct B cell subsets make identical or different Abs. Single-cell analysis demonstrates that the same DNA-reactive B cells can mature to either subset, depending on the hormonal environment. Anti-DNA B cells in estradiol-treated mice become marginal zone cells while identical cells from prolactin-treated mice become follicular cells. The B cell receptor signaling pathway is influenced by hormonal milieu. Thus, hormonal milieu and perhaps B cell receptor signaling, but not antigenic specificity, correlates with the differentiation pathway. These observations have implications for the pathogenesis and treatment of autoimmune disease.
Cutting Edge: The Direct Action of Type I IFN on CD4 T Cells Is Critical for Sustaining Clonal Expansion in Response to a Viral but Not a Bacterial Infection
Authors: Havenar-Daughton Colin , Kolumam Ganesh A. , Murali-Krishna Kaja
Published: April 2014

The action of type I IFN (IFN-I) on APCs is well studied, but their direct effect on CD4 T cells is unclear. To address this, we transferred IFN-I receptor-deficient (IFN-IR0) and -sufficient (wild-type, WT) TCR-transgenic CD4 T cells into WT mice and analyzed their response to immunization. In response to lymphocytic choriomeningitis virus immunization, WT CD4 T cells expanded ∼100-fold, whereas IFN-IR0 CD4 T cells expanded <10-fold. However, both WT and IFN-IR0 CD4 T cells expanded ∼10-fold after Listeria monocytogenes immunization. Poor expansion of IFN-IR0 CD4 T cells after lymphocytic choriomeningitis virus immunization was not due to a defect in proliferation or initial activation but to poor survival of the daughter cells. Thus, direct IFN-I signals can play either a critical or minimal role in CD4 T cell clonal expansion depending on the specific pathogen.
TGF-β Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+ Regulatory Cells
Authors: Zheng Song Guo , Wang Ju Hua , Stohl William , Kim Kyoung Soo , Gray J. Dixon , Horwitz David A.
Published: April 2014

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-β could not induce CD4+CD25− cells from CTLA-4−/− mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-β to induce FoxP3-expressing mouse suppressor cells. TGF-β accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-β to induce CD4+CD25− cells to express FoxP3 and develop suppressor activity. TGF-β also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4−/− mice, the lack of TGF-β-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.
Dendritic Cell Synthesis of C3 Is Required for Full T Cell Activation and Development of a Th1 Phenotype
Authors: Peng Qi , Li Ke , Patel Hetal , Sacks Steven H. , Zhou Wuding
Published: April 2014

Previous studies have found that deficiency of complement component C3 is associated with reduced T cell responses in several disease models including viral infection, autoimmune disease, and transplantation. However, the underlying mechanism is unclear. In this study, we demonstrate that dendritic cells (DCs) are able to synthesize C3 and this synthesis is required for the capacity of DCs to stimulate alloreactive T cell responses in vitro and in vivo. Compared with C3-producing DCs, C3-nonproducing DCs exhibit reduced potency to stimulate an alloreactive T cell response, favor the polarization of CD4+ T cells toward Th2 phenotype, and have regulatory T cell-driving capacity. In addition, priming mice with C3-deficient DCs compared with wild-type DCs led to delayed skin allograft rejection. Our findings that nonproduction of C3 by DCs significantly reduced T cell stimulation and impaired allograft rejection provide a potentially important explanation of how C3-deficient mice develop reduced T cell responses and of how C3-deficient donor kidney is protected from T cell-mediated graft rejection.
In Vitro Suppression of CD8+ T Cell Function by Friend Virus-Induced Regulatory T Cells
Authors: Robertson Shelly J. , Messer Ronald J. , Carmody Aaron B. , Hasenkrug Kim J.
Published: April 2014

Regulatory T cell (Treg)-mediated suppression of CD8+ T cells has been implicated in the establishment and maintenance of chronic viral infections, but little is known about the mechanism of suppression. In this study an in vitro assay was developed to investigate the suppression of CD8+ T cells by Friend retrovirus (FV)-induced Tregs. CD4+CD25+ T cells isolated from mice chronically infected with the FV suppressed the development of effector function in naive CD8+ T cells without affecting their ability to proliferate or up-regulate activation markers. In vitro restimulation was not required for suppression by FV-induced Tregs, correlating with their high activation state in vivo. Suppression was mediated by direct T cell-T cell interactions and occurred in the absence of APCs. Furthermore, suppression occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, FV-induced Tregs were able to suppress the function of CD8+ effector T cells that had been physiologically activated during acute FV infection. The ability to suppress the effector function of activated CTLs is likely a requisite role for Tregs in limiting immunopathology by CD8+ T cells during antiviral immune responses. Such activity may also have adverse consequences by allowing viruses to establish and maintain chronic infections if suppression of antiviral immune responses occurs before virus eradication.
Attenuation of IL-7 Receptor Signaling Is Not Required for Allelic Exclusion
Authors: Will Wynette M. , Aaker Joshua D. , Burchill Matthew A. , Harmon Ian R. , O’Neil Jennifer J. , Goetz Christine A. , Hippen Keli L. , Farrar Michael A.
Published: April 2014

Allelic exclusion prevents pre-B cells from generating more than one functional H chain, thereby ensuring the formation of a unique pre-BCR. The signaling processes underlying allelic exclusion are not clearly understood. IL-7R-dependent signals have been clearly shown to regulate the accessibility of the Ig H chain locus. More recent work has suggested that pre-BCR-dependent attenuation of IL-7R signaling returns the H chain loci to an inaccessible state; this process has been proposed to underlie allelic exclusion. Importantly, this model predicts that preventing pre-BCR-dependent down-regulation of IL-7R signaling should interfere with allelic exclusion. To test this hypothesis, we made use of transgenic mice that express a constitutively active form of STAT5b (STAT5b-CA). STAT5b-CA expression restores V(D)J recombination in IL-7R−/− B cells, demonstrating that IL-7 regulates H chain locus accessibility and V(D)J recombination via STAT5 activation. To examine the effects of constitutively active STAT5b on allelic exclusion, we crossed STAT5b-CA mice (which express the IgMb allotype) to IgMa allotype congenic mice. We found no difference in the percentage of IgMa/IgMb-coexpressing B cells in STAT5b-CA vs littermate control mice; identical results were observed when crossing STAT5b-CA mice with hen egg lysozyme (HEL) H chain transgenic mice. The HEL transgene enforces allelic exclusion, preventing rearrangement of endogenous H chain genes; importantly, rearrangement of endogenous H chain genes was suppressed to a similar degree in STAT5b-CA vs HEL mice. Thus, attenuation of IL-7R/STAT5 signaling is not required for allelic exclusion.
Soluble MHC-Peptide Complexes Containing Long Rigid Linkers Abolish CTL-Mediated Cytotoxicity
Authors: Angelov Georgi S. , Guillaume Philippe , Cebecauer Marek , Bosshard Giovanna , Dojcinovic Danijel , Baumgaertner Petra , Luescher Immanuel F.
Published: April 2014

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and coengage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (≥80 Å) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long Db-gp33 complexes strongly and specifically inhibited the Db-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.
Loss of the Surface Antigen 3G11 Characterizes a Distinct Population of Anergic/Regulatory T Cells in Experimental Autoimmune Encephalomyelitis
Authors: Zhang Guang-Xian , Yu Shuo , Calida Divina , Zhao Zhao , Gran Bruno , Kamoun Malek , Rostami Abdolmohamad
Published: April 2014

T cell anergy is an important mechanism in the induction of peripheral tolerance against autoimmune diseases, yet no surface marker unique to anergic T cells in these diseases has been identified. In this study we induced in vivo anergy by i.v. tolerance against experimental autoimmune encephalomyelitis in myelin basic protein TCR transgenic mice, and showed that the hyporesponsiveness of autoantigen-reactive T cells from tolerized mice was associated with a dramatic loss of 3G11, a cell surface molecule on the surface of CD4+ T cells. Purified 3G11−CD4+ T cells lost autoantigen-induced proliferation and IL-2 production, whereas 3G11+CD4+ T cells retained responsiveness. Furthermore, 3G11− T cells actively suppressed proliferation and Th1 cytokine production of 3G11+ T cells and splenocytes of nontolerized mice. Active suppression by 3G11− T cells was at least partially due to soluble immunoregulatory factors, including IL-10. The T regulatory property of 3G11− T cells was confirmed in vivo because the transfer of purified 3G11− T cells effectively suppressed clinical experimental autoimmune encephalomyelitis. We conclude that loss of the surface molecule 3G11 characterizes a distinct population of anergic/regulatory T cells. This is the first demonstration of the ability to identify and purify anergic T cells by a distinct cell surface marker in an autoimmune disease and paves the way for a better understanding of the mechanism of tolerance in autoimmune diseases.