bluuebunny/crossref_metadata_2025 huggingface dataset:
An Anti-Inflammatory Role for Vα14 NK T cells inMycobacterium bovisBacillus Calmette-Guérin-Infected Mice
Authors: Dieli Francesco , Taniguchi Masaru , Kronenberg Mitchell , Sidobre Stephane , Ivanyi Juraj , Fattorini Lanfranco , Iona Elisabetta , Orefici Graziella , De Leo Giacomo , Russo Domenica , Caccamo Nadia , Sireci Guido , Di Sano Caterina , Salerno Alfredo
Published: April 2014

The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the Vα14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of Vα14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1+ Vα14 NKT population preferentially producing IFN-γ predominated at an early stage (day 8), which was substituted by an NK1.1− population preferentially producing IL-4 at later stages (day 30). Despite the fact that Vα14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in Vα14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Vα14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-α in liver and lungs of Vα14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-α mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for Vα14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.
T Cell Costimulatory Molecule Function Determines Susceptibility to Infection withPneumocystis cariniiin Mice
Authors: Beck James M. , Blackmon Michael B. , Rose Christine M. , Kimzey Stephanie L. , Preston Angela M. , Green Jonathan M.
Published: April 2014

Loss of T cell number and function during HIV infection or secondary to pharmacologic immunosuppression renders individuals susceptible to opportunistic infections, including Pneumocystis carinii pneumonia. Because costimulatory receptors are critical for optimal T cell function, we hypothesized that these proteins would regulate susceptibility to opportunistic infections. We found that despite normal T cell numbers, mice deficient in the costimulatory molecules CD2 and CD28 spontaneously developed P. carinii pneumonia. In experiments using intratracheal injection of P. carinii organisms to induce infection, the loss of CD28 alone was sufficient to render mice susceptible to acute infection; however, the organism was eventually cleared. Examination of inflammatory responses to P. carinii revealed that mice deficient in both CD2 and CD28 accumulated CD8+ T cells in their lungs in response to infection and demonstrated markedly reduced specific Ab titers. Analysis of cytokine profiles suggested that regulation of IL-10 and IL-15 may be important elements of the response to this pathogen. Thus, costimulatory molecule function is critical in determining the initial susceptibility to infection with P. carinii. Analysis of immunologic responses in these mice may provide important insights into the defects that render individuals susceptible to opportunistic infection, and provide opportunities for novel immunologically based therapies.
The Fourth Surface-Exposed Region of the Outer Membrane Protein P5-Homologous Adhesin of Nontypable Haemophilus influenzae Is an Immunodominant But Nonprotective Decoying Epitope
Authors: Novotny Laura A. , Bakaletz Lauren O.
Published: April 2014

Nontypable Haemophilus influenzae is a major cause of otitis media and other mucosal infections. After natural disease in children and experimental disease in chinchillas, we found a hierarchical pattern of immunodominance among the four surface-exposed regions of the P5-homologous adhesin, with the greatest response directed to region 4. However, Ab to region 4 is not protective. When this natural but biased response was refocused to region 3 by immunization, augmented bacterial clearance and protection from ascending otitis media was observed. Collectively, the data indicate that region 4 contains a highly immunodominant but nonprotective decoying epitope, the presence of which dampens the immune response to a subdominant but protective epitope in region 3.
Presentation and Binding Affinity of Equine Infectious Anemia Virus CTL Envelope and Matrix Protein Epitopes by an Expressed Equine Classical MHC Class I Molecule
Authors: McGuire Travis C. , Leib Steven R. , Mealey Robert H. , Fraser Darrilyn G. , Prieur David J.
Published: April 2014

Control of a naturally occurring lentivirus, equine infectious anemia virus (EIAV), occurs in most infected horses and involves MHC class I-restricted, virus-specific CTL. Two minimal 12-aa epitopes, Env-RW12 and Gag-GW12, were evaluated for presentation by target cells from horses with an equine lymphocyte Ag-A1 (ELA-A1) haplotype. Fifteen of 15 presented Env-RW12 to CTL, whereas 11 of 15 presented Gag-GW12. To determine whether these epitopes were presented by different molecules, MHC class I genes were identified in cDNA clones from Arabian horse A2152, which presented both epitopes. This horse was selected because it is heterozygous for the SCID trait and is used to breed heterozygous females. Offspring with SCID are used as recipients for CTL adoptive transfer, and normal offspring are used for CTL induction. Four classical and three putative nonclassical full-length MHC class I genes were found. Human 721.221 cells transduced with retroviral vectors expressing each gene had equine MHC class I on their surface. Following peptide pulsing, only cells expressing classical MHC class I molecule 7-6 presented Env-RW12 and Gag-GW12 to CTL. Unlabeled peptide inhibition of 125I-labeled Env-RW12 binding to 7-6-transduced cells demonstrated that Env-RW12 affinity was 15-fold higher than Gag-GW12 affinity. Inhibition with truncated Env-RW12 demonstrated that amino acid positions 1 and 12 were necessary for binding, and single substitutions identified positions 2 and 3 as possible primary anchor residues. Since MHC class I 7-6 presented both epitopes, outbred horses with this allele can be immunized with these epitopes to optimize CTL responses and evaluate their effectiveness against lentiviral challenge.
Nramp1 Functionality Increases Inducible Nitric Oxide Synthase Transcription Via Stimulation of IFN Regulatory Factor 1 Expression
Authors: Fritsche Gernot , Dlaska Margit , Barton Howard , Theurl Igor , Garimorth Katja , Weiss Günter
Published: April 2014

Natural-resistance associated macrophage protein 1 (Nramp1) encodes a transmembrane phagolysosomal protein exerting resistance toward infections with intracellular pathogens by a mechanism not fully elucidated so far. We used the murine macrophage cell line RAW264.7, stably transfected with functional (RAW-37) or nonfunctional (RAW-21) Nramp1, to study for differences in the expression of NO, a central antimicrobial effector molecule of macrophages. Following stimulation with IFN-γ and LPS, Nramp1-expressing cells exhibit higher enzymatic activity of inducible NO synthase (iNOS) and increased cytoplasmic iNOS mRNA levels than RAW-21 cells. Time-course experiments showed that iNOS-mRNA levels remain increased in RAW-37 cells after prolonged cytokine stimulation while they decrease in RAW-21 cells. Reporter gene assays with iNOS-promoter luciferase constructs demonstrated an increased and prolonged promoter activity in Nramp1-resistant vs susceptible cells. This was paralleled by increased IFN regulatory factor 1 (IRF-1) expression and binding affinity to the iNOS promoter in RAW-37 cells, which may be related to enhanced STAT-1 binding affinity in these cells. A point mutation within the IRF-1 binding site of the iNOS promoter abolished the differences in iNOS transcription between RAW-21 and RAW-37 cells. Cells carrying functional Nramp1 express increased amounts of NO, which may be related to STAT-1-mediated stimulation of IRF-1 expression with subsequent prolonged activation of iNOS transcription. Enhanced NO expression may partly underlie the protection against infection with intracellular pathogens by Nramp1 functionality.
DNA Vaccines, Combining Form of Antigen and Method of Delivery to Raise a Spectrum of IFN-γ and IL-4-Producing CD4+ and CD8+ T Cells
Authors: Oran Alp E. , Robinson Harriet L.
Published: April 2014

DNA-based immunizations have been used to determine the patterns of type 1 and type 2 cytokines that can be induced in vivo for Ag-specific CD4+ and CD8+ T cells. IL-4 was used as a signature cytokine for a type 2 T cell response and IFN-γ as the signature cytokine for a type 1 response. Gene gun deliveries of secreted Ags were used to bias responses toward type 2 and saline injections of cell-associated Ags to bias responses toward type 1. The studies revealed that gene gun bombardments of DNAs expressing secreted Ags strongly biased responses toward type 2, inducing IL-4-producing CD8+ as well as CD4+ T cells. Saline injections of DNAs expressing cell-associated Ags strongly biased responses toward type 1, inducing IFN-γ-producing CD8+ and CD4+ cells. A mixed type 1/type 2 response of IFN-γ-producing CD8+ T cells and IL-4-producing CD4+ T cells was found for gene gun deliveries of cell-associated Ags. Saline injections of secreted Ags raised a weakly type 1-biased response characterized by only slightly higher frequencies of IFN-γ- than IL-4-producing CD4+ and CD8+ T cells. Studies in B cell knockout and hen egg lysozyme Ig transgenic mice revealed that B cells were required for the generation of IL-4-producing CD8+ T cells.
Protection Against CTL Escape and Clinical Disease in a Murine Model of Virus Persistence
Authors: Kim Taeg S. , Perlman Stanley
Published: April 2014

CTL escape mutations have been identified in several chronic infections, including mice infected with mouse hepatitis virus strain JHM. One outstanding question in understanding CTL escape is whether a CD8 T cell response to two or more immunodominant CTL epitopes would prevent CTL escape. Although CTL escape at multiple epitopes seems intuitively unlikely, CTL escape at multiple CD8 T cell epitopes has been documented in some chronically infected individual animals. To resolve this apparent contradiction, we engineered a recombinant variant of JHM that expressed the well-characterized gp33 epitope of lymphocytic choriomeningitis virus, an epitope with high functional avidity. The results show that the presence of a host response to this second epitope protected mice against CTL escape at the immunodominant JHM-specific CD8 T cell epitope, the persistence of infectious virus, and the development of clinical disease.
Lipomannans, But Not Lipoarabinomannans, Purified fromMycobacterium chelonaeandMycobacterium kansasiiInduce TNF-α and IL-8 Secretion by a CD14-Toll-Like Receptor 2-Dependent Mechanism
Authors: Vignal Cécile , Guérardel Yann , Kremer Laurent , Masson Maryse , Legrand Dominique , Mazurier Joël , Elass Elisabeth
Published: April 2014

Lipoarabinomannans (LAMs) are glycolipids from the mycobacterial cell wall that exhibit various biological activities, including proinflammatory and anti-inflammatory responses. However, little is known about the properties of lipomannans (LMs), considered to be precursors of LAMs. In this study, we provide evidence that LMs purified from Mycobacterium chelonae and a clinical strain of Mycobacterium kansasii stimulated mRNA expression and secretion of TNF-α and IL-8 from human macrophage-like differentiated THP-1 cells. In contrast to LMs, LAMs were not able to induce a significant cytokine-inducing effect. The mechanism of activation by LMs was investigated using various Abs raised against surface receptors for multiple bacterial products. The presence of anti-CD14 or anti-Toll-like receptor 2 (TLR2) Abs profoundly affected production of TNF-α and IL-8, suggesting that both CD14 and TLR2 participate in the LM-mediated activation process. Furthermore, stimulation of cells was dependent on the presence of the LPS-binding protein, a plasma protein that transfers glycolipids to CD14. Chemical degradation of the arabinan domain of mannose-capped LAM from M. kansasii, which presented no cytokine-eliciting effect, restored the cytokine-inducing activity at a level similar to those of LMs. These results support the hypothesis that the presence of an arabinan in LAMs prevents the interaction of these glycolipids with TLR2/CD14 receptors. In addition, we found that phosphatidylinositol dimannosides isolated from M. kansasii did not induce cytokine secretion. This study suggests that LMs isolated from different mycobacterial species participate in the immunomodulation of the infected host and that the d-mannan core of this glycolipid is essential for this function.
Protracted Protection to Plasmodium berghei Malaria Is Linked to Functionally and Phenotypically Heterogeneous Liver Memory CD8+ T Cells
Authors: Berenzon Dmitri , Schwenk Robert J. , Letellier Lisa , Guebre-Xabier Mimi , Williams Jackie , Krzych Urszula
Published: April 2014

We previously demonstrated that protection induced by radiation-attenuated (γ) Plasmodium berghei sporozoites is linked to MHC class I-restricted CD8+ T cells specific for exoerythrocytic-stage Ags, and that activated intrahepatic memory CD8+ T cells are associated with protracted protection. In this study, we further investigated intrahepatic memory CD8+ T cells to elucidate mechanisms required for their maintenance. Using phenotypic markers indicative of activation (CD44, CD45RB), migration (CD62L), and IFN-γ production, we identified two subsets of intrahepatic memory CD8+ T cells: the CD44highCD45RBlowCD62LlowCD122low phenotype, representing the dominant effector memory set, and the CD44highCD45RBhighCD62Llow/highCD122high phenotype, representing the central memory set. Only the effector memory CD8+ T cells responded swiftly to sporozoite challenge by producing sustained IFN-γ; the central memory T cells responded with delay, and the IFN-γ reactivity was short-lived. In addition, the subsets of liver memory CD8+ T cells segregated according to the expression of CD122 (IL-15R) in that only the central memory CD8+ T cells were CD122high, whereas the effector memory CD8+ T cells were CD122low. Moreover, the effector memory CD8+ T cells declined as protection waned in mice treated with primaquine, a drug that interferes with the formation of liver-stage Ags. We propose that protracted protection induced by P. berghei radiation-attenuated sporozoites depends in part on a network of interactive liver memory CD8+ T cell subsets, each representing a different phase of activation or differentiation, and the balance of which is profoundly affected by the repository of liver-stage Ag and IL-15.
Helicobacter pyloriInfection Interferes with Epithelial Stat6-Mediated Interleukin-4 Signal Transduction Independent ofcagA,cagE, or VacA
Authors: Ceponis Peter J. M. , McKay Derek M. , Menaker Rena J. , Galindo-Mata Esther , Jones Nicola L.
Published: April 2014

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA+/cagE+/VacA+), LC20 (type 2; cagA−, cagE−, VacA−), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain α or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.
Mycobacterium tuberculosisExerts Gene-Selective Inhibition of Transcriptional Responses to IFN-γ Without Inhibiting STAT1 Function
Authors: Kincaid Eleanor Z. , Ernst Joel D.
Published: April 2014

Mycobacterium tuberculosis is a highly successful human pathogen. A major component of this success is the pathogen’s ability to avoid eradication by the innate and adaptive immune responses throughout the course of infection. IFN-γ, a potent activator of the microbicidal activities of macrophages, is essential for control of M. tuberculosis infection, but is unable to stimulate macrophages to kill M. tuberculosis. We have found that infection of the human monocytic cell line, THP-1, resulted in reduced cellular responses to IFN-γ, manifested as impaired induction of CD64 surface expression and transcription. This defect in transcription occurred despite normal activation of STAT1 in infected macrophages: there was no decrease in STAT1 tyrosine or serine phosphorylation, nuclear translocation, or binding of a minimal IFN-γ response sequence. Assays of STAT1 function in M. tuberculosis-treated cells also revealed no defect in activation of a minimal γ-activated sequence construct or STAT1 recruitment to and binding at the endogenous CD64 promoter. In addition, M. tuberculosis did not affect histone acetylation at the CD64 promoter. The inhibition of transcription was gene selective: while transcription of CD64 and class II transactivator were decreased, certain other IFN-γ-responsive genes either were unaffected or were increased by M. tuberculosis. These results indicate that M. tuberculosis inhibits the response to IFN-γ by a mechanism distinct from either suppressor of cytokine signaling-1 inhibition of STAT1 phosphorylation or protein inhibitor of activated STAT interference with DNA binding, and indicate that other mechanisms of inhibition of IFN-γ responses remain to be discovered.
Expression and Function of Formyl Peptide Receptors on Human Fibroblast Cells
Authors: VanCompernolle Scott E. , Clark Krista L. , Rummel Kevin A. , Todd Scott C.
Published: April 2014

The migration of polymorphonuclear leukocytes from the blood to sites of infection in tissues is a hallmark of the innate immune response. Formylated peptides produced as a byproduct of bacterial protein synthesis are powerful chemoattractants for leukocytes. Formyl peptides bind to two different G protein-coupled receptors (formyl peptide receptor (FPR) and the low affinity formyl peptide receptor-like-1 (FPRL1)) to initiate a signal transduction cascade leading to cell activation and migration. Our analysis of expressed sequences from many cDNA libraries draws attention to the fact that FPRs are widely expressed in nonlymphoid tissues. Here we demonstrate that FPRs are expressed by normal human lung and skin fibroblasts and the human fibrosarcoma cell line HT-1080. The expression on fibroblasts of receptors for bacteria-derived peptides raises questions about the possible function of these receptors in nonleukocyte cells. We studied the function of FPRs on fibroblasts and find that stimulation with fMLP triggers dose-dependent migration of these cells. Furthermore, fMLP induces signal transduction including intracellular calcium flux and a transient increase in F-actin. The fMLP-induced adhesion and motility of fibroblasts on fibronectin require functional protein kinase C and phosphatidylinositol 3-kinase. This first report of a functional formyl peptide receptor in cells of fibroblast origin opens new possibilities for the role of fibroblasts in innate immune responses.
Plasminogen Activator Inhibitor-1 Supports IL-8-Mediated Neutrophil Transendothelial Migration by Inhibition of the Constitutive Shedding of Endothelial IL-8/Heparan Sulfate/Syndecan-1 Complexes
Authors: Marshall Lindsay J. , Ramdin Lara S. P. , Brooks Teresa , DPhil Peter Charlton , Shute Janis K.
Published: April 2014

The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.
Inflammatory Chemoreceptor Cross-Talk Suppresses Leukotriene B4 Receptor 1-Mediated Neutrophil Calcium Mobilization and Chemotaxis After Trauma
Authors: Tarlowe Michael H. , Kannan K. B. , Itagaki Kiyoshi , Adams John M. , Livingston David H. , Hauser Carl J.
Published: April 2014

G protein-coupled chemoattractants recruit neutrophils (PMN) to sites of injury and infection. The leukotrienes (LT) and CXC chemokines (CXC) and their receptors (BLT1/BLT2 and CXCR1/CXCR2) are all known to play roles in these responses. Each system has been studied separately in vitro, but in vivo they act concurrently, and the clinical interactions between the two systems are unstudied. We prospectively studied calcium mobilization and chemotactic responses to LTB4 in PMN from major trauma patients. The responses of the high affinity BLT1 receptor were suppressed at the 3-day postinjury time point, but recovered by 1 wk. Trauma patients had transient elevations of plasma LT and CXC levels. Functional deficits identical with those in trauma PMN were reproduced in vitro by exposing healthy PMN to CXCs at the elevated plasma concentrations found. Functional responses to LTB4 were suppressed by cross-talk with CXC and BLT2 receptors that desensitize BLT1. Since the suppression of intracellular calcium mobilization was prominent, we also studied the role of suppressed cell calcium mobilization in the defective chemotactic responses to LTB4. We noted that PMN chemotaxis to LTB4 showed far more dependence on store-operated calcium entry than on the release of cellular calcium stores, and that store-operated calcium responses to BLT1 activation were markedly inhibited during the same time period as was chemotaxis. The intermittent release of inflammatory mediators after injury can blunt PMN responses to LTs by suppressing BLT1 as well as downstream calcium entry. Diminished LT receptor activity due to cross-talk with CXC receptors can inhibit PMN recruitment to infective sites. This may predispose injured patients to septic complications.
Functional Expression of Neurokinin 1 Receptors on Mast Cells Induced by IL-4 and Stem Cell Factor
Authors: van der Kleij Hanneke P. M. , Ma Donglai , Redegeld Frank A. M. , Kraneveld Aletta D. , Nijkamp Frans P. , Bienenstock John
Published: April 2014

It is widely accepted that neurokinin 1 (NK1) receptors are not generally expressed on mast cells but little is known about their expression in inflammation. The present study shows expression of NK1 receptors on bone marrow-derived mast cells (BMMC) under the influence of IL-4 or stem cell factor (SCF). Highest expression was found when both cytokines are present. Six days of coculture with the cytokines IL-4 and SCF showed significant expression of NK1 receptors (NK1 receptor+/c-kit+ BMMC; control: 7%, IL-4/SCF: 16%), while 12 days of cytokine coculture increased this expression to 37% positive cells. A longer coculture with IL-4 and SCF did not give an additional effect. Increased expression in IL-4/SCF-treated BMMC was further confirmed using Western blot analysis. Next, we demonstrated the functional relevance of NK1 receptor expression for mast cell activation, resulting in an enhanced degranulation upon stimulation by substance P. BMMC activation was significantly diminished by the NK1 receptor antagonist RP67580 (10 μM) when stimulated with low concentrations of substance P. The inactive enantiomer RP65681 had no effect. In addition, BMMC cultured from bone marrow of NK1 receptor knockout mice showed significantly decreased exocytosis to low concentrations of substance P. The present study clearly shows that NK1 receptor-induced activation contributes significantly at low physiological substance P concentrations (<100 μM). In conclusion, BMMC were shown to express NK1 receptors upon IL-4/SCF coculture. This expression of NK1 receptors has been demonstrated to be of functional relevance and leads to an increase in the sensitivity of BMMC to substance P.
Airway IgG Counteracts Specific and Bystander Allergen-Triggered Pulmonary Inflammation by a Mechanism Dependent on FcγR and IFN-γ
Authors: Sehra Sarita , Pynaert Gwenda , Tournoy Kurt , Haegeman Anuschka , Matthys Patrick , Tagawa Yohichi , Pauwels Romain , Grooten Johan
Published: April 2014

Besides IgE, the Ab isotype that gives rise to sensitization and allergic asthma, the immune response to common inhalant allergens also includes IgG. Increased serum titers of allergen-specific IgG, induced spontaneously or by allergen vaccination, have been implicated in protection against asthma. To verify the interference of topical IgG with the allergen-triggered eosinophilic airway inflammation that underlies asthma, sensitized mice were treated by intranasal instillation of specific IgG, followed by allergen challenge. This treatment strongly reduced eosinophilic inflammation and goblet cell metaplasia, and increased Th1 reactivity and IFN-γ levels in bronchoalveolar lavage fluid. In contrast, inflammatory responses were unaffected in IFN-γ-deficient mice or when applying F(ab′)2. Although dependent on specific allergen-IgG interaction, inflammation triggered by bystander allergens was similarly repressed. Perseverance of inflammation repression, apparent after secondary allergen challenge, and increased allergen capture by alveolar macrophages further characterized the consequences of topical IgG application. These results assign a novel protective function to anti-allergen IgG namely at the local level interference with the inflammatory cascade, resulting in repression of allergic inflammation through an FcγR- and IFN-γ-dependent mechanism. Furthermore, these results provide a basis for topical immunotherapy of asthma by direct delivery of anti-allergen IgG to the airways.
Synergism Between Platelet-Activating Factor-Like Phospholipids and Peroxisome Proliferator-Activated Receptor γ Agonists Generated During Low Density Lipoprotein Oxidation That Induces Lipid Body Formation in Leukocytes
Authors: de Assis Edson F. , Silva Adriana R. , Caiado Lara F. C. , Marathe Gopal K. , Zimmerman Guy A. , Prescott Stephen M. , McIntyre Thomas M. , Bozza Patricia T. , de Castro-Faria-Neto Hugo C.
Published: April 2014

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-γ agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-γ agonists generated during LDL oxidation are important mediators in this phenomenon.
Intracellular Domain of Brain Endothelial Intercellular Adhesion Molecule-1 Is Essential for T Lymphocyte-Mediated Signaling and Migration
Authors: Greenwood John , Amos Claire L. , Walters Claire E. , Couraud Pierre-Olivier , Lyck Ruth , Engelhardt Britta , Adamson Peter
Published: April 2014

To examine the role of the ICAM-1 C-terminal domain in transendothelial T lymphocyte migration and ICAM-1-mediated signal transduction, mutant human (h)ICAM-1 molecules were expressed in rat brain microvascular endothelial cells. The expression of wild-type hICAM-1 resulted in a significant increase over basal levels in both adhesion and transendothelial migration of T lymphocytes. Endothelial cells (EC) expressing ICAM-1 in which the tyrosine residue at codon 512 was substituted with phenylalanine (hICAM-1Y512F) also exhibited increased lymphocyte migration, albeit less than that with wild-type hICAM-1. Conversely, the expression of truncated hICAM-1 proteins, in which either the intracellular domain was deleted (hICAM-1ΔC) or both the intracellular and transmembrane domains were deleted through construction of a GPI anchor (GPI-hICAM-1), did not result in an increase in lymphocyte adhesion, and their ability to increase transendothelial migration was attenuated. Truncated hICAM-1 proteins were also unable to induce ICAM-1-mediated Rho GTPase activation. EC treated with cell-permeant penetratin-ICAM-1 peptides comprising human or rat ICAM-1 intracellular domain sequences inhibited transendothelial lymphocyte migration, but not adhesion. Peptides containing a phosphotyrosine residue were equipotent in inhibiting lymphocyte migration. These data demonstrate that the intracellular domain of ICAM-1 is essential for transendothelial migration of lymphocytes, and that peptidomimetics of the ICAM-1 intracellular domain can also inhibit this process. Such competitive inhibition of transendothelial lymphocyte migration in the absence of an affect on adhesion further implicates ICAM-1-mediated signaling events in the facilitation of T lymphocyte migration across brain EC. Thus, agents that mimic the ICAM-1 intracellular domain may be attractive targets for novel anti-inflammatory therapeutics.
Prevention of Collagen-Induced Arthritis in Mice Transgenic for the Complement Inhibitor Complement Receptor 1-Related Gene/Protein y
Authors: Banda Nirmal K. , Kraus Damian M. , Muggli Michele , Bendele Alison , Holers V. Michael , Arend William P.
Published: April 2014

The objective of these studies was to examine collagen-induced arthritis (CIA) in C57BL/6 mice transgenic for the rodent complement regulatory protein complement receptor 1-related gene/protein y (Crry) (Crry-Tg), a C3 convertase inhibitor. The scores for clinical disease activity and for histological damage in the joints were both significantly decreased in Crry-Tg mice in comparison to wild-type (WT) littermates. The production of both IgG1 and IgG2a anti-collagen Abs was reduced in the Crry-Tg mice, although spleen cell proliferation in response to collagen type II was not altered. The production of IFN-γ, TNF-α, and IL-1β by LPS-stimulated spleen cells was decreased, and IL-10 was increased, in cells from Crry-Tg mice in comparison to WT. The steady-state mRNA levels for IFN-γ, TNF-α, and IL-1β were all decreased in the joints of Crry-Tg mice in comparison to WT. The synovium from Crry-Tg mice without CIA contained the mRNA for the Crry transgene, by RT-PCR, and the synovium from transgenic mice with CIA exhibited little deposition of C3 protein by immunohistological analysis. These results suggest that suppression of CIA in Crry-Tg mice may be due to enhanced synthesis of Crry locally in the joint with decreased production of proinflammatory cytokines.
A Plant-Based Allergy Vaccine Suppresses Experimental Asthma Via an IFN-γ and CD4+CD45RBlow T Cell-Dependent Mechanism
Authors: Smart Vanessa , Foster Paul S. , Rothenberg Marc E. , Higgins T. J. V. , Hogan S. P.
Published: April 2014

Allergic asthma is currently considered a chronic airway inflammatory disorder associated with the presence of activated CD4+ Th2-type lymphocytes, eosinophils, and mast cells. Interestingly, therapeutic strategies based on immune deviation and suppression have been shown to successfully attenuate the development of the asthma phenotype. In this investigation, we have for the first time used a genetically modified (GM) plant, narrow leaf lupin (Lupinus angustifolius L.), expressing a gene for a potential allergen (sunflower seed albumin) (SSA-lupin) to examine whether a GM plant/food-based vaccine strategy can be used to suppress the development of experimental asthma. We show that oral consumption of SSA-lupin promoted the induction of an Ag-specific IgG2a Ab response. Furthermore, we demonstrate that the plant-based vaccine attenuated the induction of delayed-type hypersensitivity responses and pathological features of experimental asthma (mucus hypersecretion, eosinophilic inflammation, and enhanced bronchial reactivity (airways hyperreactivity). The suppression of experimental asthma by SSA-lupin was associated with the production of CD4+ T cell-derived IFN-γ and IL-10. Furthermore, we show that the specific inhibition of experimental asthma was mediated via CD4+CD45RBlow regulatory T cells and IFN-γ. Thus, our data demonstrate that a GM plant-based vaccine can promote a protective immune response and attenuate experimental asthma, suggesting that plant-based vaccines may be potentially therapeutic for the protection against allergic diseases.
A Novel Therapeutic Approach Targeting Articular Inflammation Using the Filarial Nematode-Derived Phosphorylcholine-Containing Glycoprotein ES-62
Authors: McInnes Iain B. , Leung Bernard P. , Harnett Margaret , Gracie J. Alastair , Liew Foo Y. , Harnett William
Published: April 2014

Understanding modulation of the host immune system by pathogens offers rich therapeutic potential. Parasitic filarial nematodes are often tolerated in human hosts for decades with little evidence of pathology and this appears to reflect parasite-induced suppression of host proinflammatory immune responses. Consistent with this, we have previously described a filarial nematode-derived, secreted phosphorylcholine-containing glycoprotein, ES-62, with immunomodulatory activities that are broadly anti-inflammatory in nature. We sought to evaluate the therapeutic potential of ES-62 in vitro and in vivo in an autoimmune disease model, namely, collagen-induced arthritis in DBA/1 mice. ES-62 given during collagen priming significantly reduced initiation of inflammatory arthritis. Crucially, ES-62 was also found to suppress collagen-induced arthritis severity and progression when administration was delayed until after clinically evident disease onset. Ex vivo analyses revealed that in both cases, the effects were associated with inhibition of collagen-specific pro-inflammatory/Th1 cytokine (TNF-α, IL-6, and IFN-γ) release. In parallel in vitro human tissue studies, ES-62 was found to significantly suppress macrophage activation via cognate interaction with activated T cells. Finally, ES-62 suppressed LPS-induced rheumatoid arthritis synovial TNF-α and IL-6 production. Evolutionary pressure has promoted the generation by pathogens of diverse mechanisms enabling host immune system evasion and induction of “tolerance.” ES-62 represents one such mechanism. We now provide proof of concept that parasite-derived immunomodulatory strategies offer a novel therapeutic opportunity in inflammatory arthritis.
Differentiation of CD8+ T Cells from Tumor-Invaded and Tumor-Free Lymph Nodes of Melanoma Patients: Role of Common γ-Chain Cytokines
Authors: Anichini Andrea , Scarito Alessia , Molla Alessandra , Parmiani Giorgio , Mortarini Roberta
Published: April 2014

Differentiation of CD8+ T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8+ T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8+ T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7+ CD45RA+/− cytotoxic factor− cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8+ T cell population toward CCR7− CD45RA−/+ perforin+ granzyme B+ differentiation stages. Distinct CD8+ T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7+ perforin− CD8+ T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7+CD45RA− cells, proliferation coupled to differentiation to the CCR7− perforin+ stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8+ T cells differentiated toward CCR7− cytotoxic factor+ stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8+ T cells.
Immunoregulatory Role of CD1d in the Hydrocarbon Oil-Induced Model of Lupus Nephritis
Authors: Yang Jun-Qi , Singh Avneesh K. , Wilson Michael T. , Satoh Minoru , Stanic Aleksandar K. , Park Jang-June , Hong Seokmann , Gadola Stephan D. , Mizutani Akiei , Kakumanu Srinivasa R. , Reeves Westley H. , Cerundolo Vincenzo , Joyce Sebastian , Van Kaer Luc , Singh Ram Raj
Published: April 2014

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is accompanied by the emergence of autoreactive T cells and a reduction in regulatory T cells. Humans and mice with SLE have reduced numbers of CD1d-restricted NK T cells, suggesting a role for these cells in the regulation of SLE. In this study, we show that CD1d deficiency exacerbates lupus nephritis induced by the hydrocarbon oil pristane. This exacerbation in disease is associated with: 1) reduced TNF-α and IL-4 production by T cells, especially during the disease induction phase; and 2) expansion of marginal zone B cells. Strikingly, inoculation of pristane in wild-type mice resulted in reduced numbers and/or functions of NK T cells and CD1d-expressing dendritic cells. These findings suggest that CD1d may play an immunoregulatory role in the development of lupus in the pristane-induced model.
Reconstitution of CD8+ T Cells by Retroviral Transfer of the TCR αβ-Chain Genes Isolated from a Clonally Expanded P815-Infiltrating Lymphocyte
Authors: Tahara Hiroyuki , Fujio Keishi , Araki Yasuto , Setoguchi Keigo , Misaki Yoshikata , Kitamura Toshio , Yamamoto Kazuhiko
Published: April 2014

Gene transfer of TCR αβ-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8+ T cells by transfer of TCR αβ-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vβ subfamily usage of CD8+ TIL in the tumor sites demonstrated that T cells using Vβ10 efficiently infiltrated and expanded clonally. The TCR α- and β-chain sequences derived from a tumor-infiltrating CD8+/Vβ10+ single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR αβ-chain genes were retrovirally introduced into CD8+ T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2Ld mAb. Furthermore, T cells containing both TCR α- and β-chains, but not TCR β-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8+ T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8+ T cells by transfer of TCR αβ-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.
HER-2/neu-Specific Monoclonal Antibodies Collaborate with HER-2/neu-Targeted Granulocyte Macrophage Colony-Stimulating Factor Secreting Whole Cell Vaccination to Augment CD8+ T Cell Effector Function and Tumor-Free Survival in Her-2/neu-Transgenic Mice
Authors: Wolpoe Matthew E. , Lutz Eric R. , Ercolini Anne M. , Murata Satoshi , Ivie Susan E. , Garrett Elizabeth S. , Emens Leisha A. , Jaffee Elizabeth M. , Reilly R. Todd
Published: April 2014

HER-2/neu is overexpressed in several cancers including 30% of breast carcinomas, and correlates with a poor outcome. HER-2/neu-transgenic (neu-N) mice that overexpress the non-transforming rat neu develop spontaneous mammary carcinomas and demonstrate immunotolerance to the neu protein similar to that observed in patients with neu-expressing cancers. In neu-N mice, neu-targeted vaccination induces weak T cell and negligible Ab responses sufficient to delay but not eradicate transplanted neu-expressing tumor. Here we demonstrate that passive infusion of neu-specific mAbs in sequence with whole cell vaccination significantly improves tumor-free survival over either modality alone. Importantly, treatment of neu-N mice with vaccine in combination with two distinct neu-specific Abs is particularly efficacious, preventing tumor in 70% and eradicating established tumor in 30% of neu-N mice. In vivo lymphocyte subpopulation depletion experiments demonstrate that the efficacy of Ab, alone or combined with vaccine, is dependent on both CD4+ and CD8+ T cells. Furthermore, the in vivo antitumor effects of vaccine and Ab are associated with a significant increase in the number and function of neu-specific CD8+ T cells. Collectively, these observations suggest that similarly increased efficacy could be obtained by combining neu-targeted vaccination and neu-specific Abs such as trastuzumab (Herceptin) in patients with neu-expressing cancers.
Cutting Edge: Requirement for Growth Hormone-Releasing Hormone in the Development of Experimental Autoimmune Encephalomyelitis
Authors: Ikushima Hideto , Kanaoka Masaharu , Kojima Shinichi
Published: April 2014

Growth hormone (GH)-releasing hormone (GHRH) is a neuropeptide that stimulates secretion of GH from the pituitary gland. Although GHRH and its receptor (GHRHR) are expressed in leukocytes, physiological function of GHRH in the immune system remains unclear. To study the influence of GHRH in autoimmunity, susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined in C57BL/6J-Ghrhrlit/lit (lit/lit), mice deficient in the GHRHR gene. We found that lit/lit mice were resistant to myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Splenocytes from MOG-immunized lit/lit mice proliferated normally in response to MOG peptide, suggesting that activation of MOG-specific T cells in GHRHR-deficient mice is not impaired. Our data strongly suggest that GHRH plays a crucial role in the development of EAE and may provide the basis for a novel therapeutic approach protecting from autoimmune diseases.
Cutting Edge: A Possible Role for CD4+ Thymic Macrophages as Professional Scavengers of Apoptotic Thymocytes
Authors: Esashi Eiji , Sekiguchi Takashi , Ito Hiroaki , Koyasu Shigeo , Miyajima Atsushi
Published: April 2014

A vast majority of thymocytes are eliminated during T cell development by apoptosis. However, apoptotic thymocytes are not usually found in the thymus, indicating that apoptotic thymocytes must be eliminated rapidly by scavengers. Although macrophages and dendritic cells are believed to play such role, little is known about scavengers in the thymus. We found that CD4+/CD11b+/CD11c− cells were present in the thymus and that they expressed costimulatory molecules for T cell selection and possessed Ag-presenting activity. Moreover, these CD4+/CD11b+ cells phagocytosed apoptotic thymocytes much more efficiently than thymic CD4−/CD11b+ cells as well as activated peritoneal macrophages. CD4+/CD11b+ cells became larger along with thymus development, while no such change was observed in CD4−/CD11b+ cells. Finally, engulfed nuclei were frequently found in CD4+/CD11b+ cells. These results strongly suggest that thymic CD4+/CD11b+ cells are major scavengers of apoptotic thymocytes.
Cutting Edge: Multiple Autoimmune Pathways in kd/kd Mice
Authors: Hancock Wayne W. , Tsai Tsai-Lung , Madaio Michael P. , Gasser David L.
Published: April 2014

The kidney disease (kd) mutation was transferred to a C57BL/6 (B6) background by selection for closely linked microsatellite markers. The resulting congenic strain, B6.kd, was mated with partners homozygous for targeted mutations of CD4, CD8, CD28, IL-2, recombinase-activating gene-1 (Rag-1), ICAM-1, or β2-microglobulin. In most of the resulting double mutants, kidney disease occurred as readily and as severely as in the B6.kd controls, although disease occurred somewhat less frequently in age-matched CD28−/− kd/kd mice. Immunohistology demonstrated a predominance of macrophages in the lesions of B6.kd and most of the double mutants, with the remaining cells consisting of T cells and variable numbers of NK cells. In Rag-1−/− kd/kd, ∼50% of infiltrating cells were macrophages, and ∼50% were NK cells. These results suggest that the initial lesion caused by the mutant gene is intrinsic to the kidney and that the immune response that subsequently occurs can involve any one of several different cellular compositions.