bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-γ Increases the Severity and Accelerates the Onset of Experimental Autoimmune Uveitis in Transgenic Rats
Authors: Egwuagu Charles E. , Sztein Jorge , Mahdi Rashid M. , Li Wenmei , Chao-Chan Chi , Smith Janine A. , Charukamnoetkanok Puwat , Chepelinsky Ana B.
Published: December 2022

Experimental autoimmune uveitis (EAU) is a predominantly Th1-mediated intraocular inflammatory disease that serves as a model for studying the immunopathogenic mechanisms of uveitis and organ-specific autoimmune diseases. Despite the well-documented role of IFN-γ in the activation of inflammatory cells that mediate autoimmune pathology, recent studies in IFN-γ-deficient mice paradoxically show that IFN-γ confers protection from EAU. Because of the implications of these findings for therapeutic use of IFN-γ, we sought to reexamine these results in the rat, another species that shares essential immunopathologic features with human uveitis and is the commonly used animal model of uveitis. We generated transgenic rats (TR) with targeted expression of IFN-γ in the eye and examined whether constitutive ocular expression of IFN-γ would influence the course of EAU. We show here that the onset of rat EAU is markedly accelerated and is severely exacerbated by IFN-γ. In both wild-type and TR rats, we found that the disease onset is preceded by induction of ICAM-1 gene expression and is characterized by selective recruitment of T cells expressing a restricted TCR repertoire in the retina. In addition, these events occur 2 days earlier in TR rats. Thus, in contrast to the protective effects of IFN-γ in mouse EAU, our data clearly show that intraocular secretion of IFN-γ does not confer protection against EAU in the rat and suggest that IFN-γ may activate distinct immunomodulatory pathways in mice and rats during uveitis.
High Frequency of Specific CD8+ T Cells in the Tumor and Blood Is Associated with Efficient Local IL-12 Gene Therapy of Cancer
Authors: Fernandez Nadine C. , Levraud Jean-Pierre , Haddada Hédi , Perricaudet Michel , Kourilsky Philippe
Published: December 2022

Cancer immunotherapy often aims at the reactivation and expansion of tumor-specific CTL. In an attempt to correlate in situ and/or systemic tumor-specific T cell expansion with tumor regression, we investigated the effects of adenovirus-mediated IL-12 or IFN-γ gene transfer into established P815 murine tumors. While IFN-γ was no more potent than the vector alone, IL-12 gene transfer promoted tumor eradication. Despite this antitumor effect, no significant cytolytic activity was detectable using classical cytotoxicity assays from in vitro restimulated splenocytes. Since intratumor gene delivery may induce a localized expansion of CTL, the presence of P815-specific CD8+ T cells in situ was assessed. Using the Immunoscope approach, we found a dramatic increase in clonotypic T cells at the tumor site following IL-12, but not IFN-γ gene delivery. Antitumor CD8+ T cell frequencies were then re-evaluated using this molecular detection technique, which revealed a comparable expansion of specific T cells in the peripheral organs, most strikingly in the blood. These data show that local IL-12 gene transfer, in contrast to IFN-γ, mediates a potent antitumor effect that correlates to clonal tumor-specific T cell expansions in situ and in the periphery.
TNF Recruits TRADD to the Plasma Membrane But Not thetrans-Golgi Network, the Principal Subcellular Location of TNF-R1
Authors: Jones Sally J. , Ledgerwood Elizabeth C. , Prins Johannes B. , Galbraith Jenny , Johnson David R. , Pober Jordan S. , Bradley John R.
Published: December 2022

The subcellular localization of TNF-R1 to the Golgi apparatus, initially observed in endothelial cells, has been confirmed using transfection of bovine aortic endothelial cells with a human TNF-R1 expression plasmid. The subcellular interactions of TNF-R1 and the TRADD (TNFR-associated death domain protein) adaptor protein have been analyzed in the human monocyte cell line U937 and the human endothelial cell line ECV304 by confocal immunofluorescence microscopy and by Western blot analysis of fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but principally localizes to the trans-Golgi network, TRADD is concentrated in the cis- or medial-Golgi region, but separates from the Golgi during cell fractionation. Coimmunoprecipitation studies have shown that TRADD binds to TNF-R1 within 1 min of TNF treatment in a cell fraction-containing plasma membrane. This association is followed by a gradual dissociation, which is prevented if receptor-mediated endocytosis is inhibited by hypertonic medium. In contrast, no association is detected between TRADD and TNF-R1 in the Golgi in response to exogenous TNF at any time examined. These results suggest that although TNF-R1 is predominantly a Golgi-associated protein and TRADD also localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF-R1 only at the plasma membrane.
IgE Hyperproduction Through Enhanced Tyrosine Phosphorylation of Janus Kinase 3 in NC/Nga Mice, a Model for Human Atopic Dermatitis
Authors: Matsumoto Masahiro , Ra Chisei , Kawamoto Keiko , Sato Hiroaki , Itakura Atsuko , Sawada Junko , Ushio Hiroko , Suto Hajime , Mitsuishi Kouichi , Hikasa Yoshiaki , Matsuda Hiroshi
Published: December 2022

IgE hyperproduction frequently observed in patients with atopic dermatitis (AD) may greatly contribute to the pathogenesis of AD, but its mechanisms are still unclear. NC/Nga mice raised in nonsterile circumstances spontaneously suffered from AD-like skin lesions with elevation of plasma IgE levels. We investigated mechanisms of the IgE hyperproduction in NC/Nga mice. Splenic T cells from SPF NC/Nga mice had a level of CD40 ligand (CD40L) expression comparable to that of BALB/c mice. Although there was no difference in the expression of CD40 on B cells between NC/Nga and BALB/c mice, B cells of NC/Nga mice produced much more IgE in the presence of soluble CD40L and IL-4. The stimulation with CD40L and/or IL-4 resulted in tyrosine phosphorylation of Janus kinase 3 (JAK3) in B cells, which was more strongly inducible in NC/Nga mice than in BALB/c mice. In B cells isolated from PBMC of AD patients with high serum IgE levels, JAK3 was constitutively phosphorylated at the tyrosine residue, and its phosphorylation was enhanced by the treatment with CD40L and/or IL-4 as was that in splenic B cells of NC/Nga mice with dermatitis and high IgE levels. Thus, it is suggested that constitutive and enhanced JAK3 phosphorylation in B cells highly sensitive to CD40L and IL-4 may be attributable to IgE hyperproduction in NC/Nga mice and patients with AD.
Regulation of Acute Lung Inflammatory Injury by Endogenous IL-13
Authors: Lentsch Alex B. , Czermak Boris J. , Jordan Jacqueline A. , Ward Peter A.
Published: December 2022

Using IgG immune complex deposition to trigger acute lung inflammation in rats, we have previously shown that exogenously administered IL-13 suppresses the acute inflammatory response. In the same model, expression of both mRNA and protein for IL-13 has now been detected. Treatment of rats with Ab to IL-13 accentuated the inflammatory response, with significant increases in lung vascular permeability and in the number of neutrophils in bronchoalveolar lavage fluids. In the presence of anti-IL-13, activation of the transcription factor, NF-κB, was significantly increased in lung. In addition, anti-IL-13 caused significant increases in bronchoalveolar lavage levels of TNF-α, macrophage inflammatory protein-2, and cytokine-inducible neutrophil chemoattractant but no changes in lung vascular ICAM-1. These data suggest that during lung inflammation endogenous IL-13 regulates NF-κB activation and related cytokine/chemokine generation, all of which determines the intensity of the lung inflammatory response.
IL-18 Is Produced by Articular Chondrocytes and Induces Proinflammatory and Catabolic Responses
Authors: Olee Tsaiwei , Hashimoto Sanshiro , Quach Jacqueline , Lotz Martin
Published: December 2022

IL-18, a cytokine originally identified as IFN-γ-inducing factor, is a member of the IL-1 family of proteins. Because IL-1α and IL-1β are important mediators in the pathogenesis of arthritis, the present study addresses the expression of IL-18 and its role in regulating in articular chondrocytes. IL-18 mRNA was induced by IL-1β in chondrocytes. Chondrocytes produced the IL-18 precursor and in response to IL-1 stimulation secreted the mature form of IL-18. Studies on IL-18 effects on chondrocytes showed that it inhibits TGF-β-induced proliferation and enhances nitric oxide production. IL-18 stimulated the expression of several genes in normal human articular chondrocytes including inducible nitric oxide synthase, inducible cyclooxygenase, IL-6, and stromelysin. Gene expression was associated with the synthesis of the corresponding proteins. Treatment of normal human articular cartilage with IL-18 increased the release of glycosaminoglycans. These finding identify IL-18 as a cytokine that regulates chondrocyte responses and contributes to cartilage degradation.
Immunogenicity and Protective Efficacy of Tuberculosis DNA Vaccines Encoding Putative Phosphate Transport Receptors
Authors: Tanghe Audrey , Lefèvre Philippe , Denis Olivier , D’Souza Sushila , Braibant Martine , Lozes Evelyne , Singh Mahavir , Montgomery Donna , Content Jean , Huygen Kris
Published: December 2022

Using culture filtrate Ag-specific mAbs generated from mycobacteria-infected H-2b haplotype mice, we have previously identified three genes in the Mycobacterium tuberculosis genome, encoding proteins homologous to the periplasmic ATP-binding cassette phosphate-binding receptor PstS of the phosphate-specific transport system of E. coli. To define the potential vaccinal properties of these phosphate-binding proteins, female C57BL/6 mice were injected i.m. with plasmid DNA encoding PstS-1, PstS-2, or PstS-3 proteins from M. tuberculosis and immunogenicity and protective efficacy against i.v. challenge with M. tuberculosis H37Rv was analyzed. Significant levels of highly Ag-specific Abs and Th1-type cytokines IL-2 and IFN-γ could be detected following vaccination with each of the three genes. However, only mice vaccinated with PstS-3 DNA demonstrated significant and sustained reduction in bacterial CFU numbers in spleen and lungs for 3 mo after M. tuberculosis challenge, as compared with CFU counts in mice vaccinated with control DNA. Vaccination with PstS-2 DNA induced a modest reduction in CFU counts in spleen only, whereas vaccination with PstS-1 DNA was completely ineffective in reducing bacterial multiplication. In conclusion, our results indicate that DNA vaccination is a powerful and easy method for comparative screening of potentially protective Ags from M. tuberculosis and that the PstS-3 protein is a promising new subunit vaccine candidate.
Cutting Edge: Protective Effects of Notch-1 on TCR-Induced Apoptosis
Authors: Jehn Birgit M. , Bielke Wolfgang , Pear Warren S. , Osborne Barbara A.
Published: December 2022

The Notch receptor protein was originally identified in Drosophila and is known to mediate cell to cell communication and influence cell fate decisions. Members of this family have been isolated from invertebrates as well as vertebrates. We isolated mouse Notch-1 in a yeast two-hybrid screen with Nur77, which is a protein that has been shown previously to be required for apoptosis in T cell lines. The data presented below indicate that Notch-1 expression provides significant protection to T cell lines from TCR-mediated apoptosis. These data demonstrate a new antiapoptotic role for Notch-1, providing evidence that, in addition to regulating cell fate decisions, Notch-1 can play a critical role in controlling levels of cell death in T cells.
Differential Requirement of IFN Consensus Sequence Binding Protein for the Production of IL-12 and Induction of Th1-Type Cells in Response to IFN-γ
Authors: Wu Chang-You , Maeda Haruko , Contursi Cristina , Ozato Keiko , Seder Robert A.
Published: December 2022

IFN-γ exerts multiple biological activities in the modulation of immune responses by the induction of transcription factors. One transcriptional factor of the IFN regulatory factor family found to be critical in regulating IL-12-dependent IFN-γ production in vivo following infectious challenge has been designated IFN consensus sequence-binding protein (ICSBP). In this study, the role of ICSBP in regulating type 1 responses to T cell-specific stimulation in vitro was assessed. Total splenocytes from ICSBP−/− mice stimulated with soluble anti-CD3 were markedly impaired in the production of IFN-γ compared with similarly stimulated cells from ICSBP+/+ mice. Consistent with the decrease in IFN-γ production, splenocytes from ICSBP−/− mice stimulated with anti-CD3 in the presence or absence of IFN-γ or a soluble CD40 ligand agonist failed to produce IL-12 p40 and IL-12 p70 protein; however, the deficient production of IFN-γ from ICSBP−/− mice could be restored by the addition of anti-CD28 Ab in an IL-12-independent manner. In contrast to the previous data, production of IFN-γ from naive CD4+/LECAM-1high cells of ICSBP−/− mice that had been primed in vitro with anti-CD3 was similar to or greater than that of ICSBP+/+ controls. In addition, the presence of IFN-γ in priming cultures enhanced both priming for IFN-γ and IL-12 responsiveness from ICSBP−/− CD4+ T cells. Overall, these results provide evidence that ICSBP is differentially required for the ability of IFN-γ to regulate type 1 cytokine responses from APCs and CD4+ T cells.
Persistence of Autoreactive T Cell Drive Is Required to Elicit Anti-Chromatin Antibodies in a Murine Model of Drug-Induced Lupus
Authors: Kretz-Rommel Anke , Rubin Robert L.
Published: December 2022

Long-term treatment with procainamide and numerous other medications is occasionally associated with the development of drug-induced lupus. We recently established a murine model for this syndrome by disrupting central T cell tolerance. Two intrathymic injections of procainamide-hydroxylamine (PAHA), a reactive metabolite of procainamide, into (C57BL/6 × DBA/2)F1 mice resulted in the appearance of chromatin-reactive T cells and anti-chromatin autoantibodies. The current study explores in this model the role of autoreactive T cells in autoantibody production and examines why autoantibodies after a single intrathymic drug injection were much more limited in isotype and specificity. Injection of as few as 5000 chromatin-reactive T cells into naive, syngeneic mice induced a rapid IgM anti-denatured DNA response, while injection of at least 100-fold greater number of activated T cells was required for induction of IgG anti-chromatin Abs, suggesting that small numbers of autoreactive T cells can be homeostatically controlled. Mice subjected to a single intrathymic PAHA injection after receiving splenic B cells from an intrathymic PAHA-injected syngeneic donor also developed anti-chromatin Abs, but adoptive transfer of similarly primed T cells or of B cells without intrathymic PAHA injection of the recipient failed to produce an anti-chromatin response. However, anti-chromatin Abs developed after a single intrathymic PAHA injection in Fas-deficient C57BL/6-lpr/lpr mice, suggesting that activation-induced cell death limited autoimmunity in normal mice. Taken together, these results imply that chromatin-reactive T cells produced by intrathymic PAHA created a B cell population primed to somatically mutate and Ig class switch when subjected to a heavy load or second wave of autoreactive T cells.
Cutting Edge: Coordinate Regulation of IFN Regulatory Factor-1 and the Polymeric Ig Receptor by Proinflammatory Cytokines
Authors: Blanch Vincent J. , Piskurich Janet F. , Kaetzel Charlotte S.
Published: December 2022

The polymeric IgR (pIgR) mediates transcytosis of IgA across epithelial barriers of mucous membranes and exocrine glands. Synthesis of pIgR is up-regulated by the proinflammatory cytokines TNF-α, IFN-γ, and IL-1 in HT-29 human colon carcinoma cells. We previously reported that IFN-γ and TNF-α induce production of the transcription factor IFN regulatory factor-1 (IRF-1) in HT-29 cells and that IRF-1 binds to an element in exon 1 of the PIGR gene. We now report that levels of IRF-1 and pIgR mRNA are coordinately regulated in HT-29 cells by TNF-α, IFN-γ, and IL-1β. Furthermore, we demonstrate that in vivo expression of pIgR mRNA is greatly depressed in the intestine and liver of IRF-1-deficient mice. Our findings indicate a major role for the IRF-1 transcription factor in regulation of the PIGR gene and suggest a model for regulation of important genes in the mucosal immune system by proinflammatory cytokines.
CD4-Mediated Inhibiton of IL-2 Production in Activated T Cells
Authors: Bonnard Madeleine , Haughn Loralee , Julius Michael
Published: December 2022

The role of CD4 in T cell activation has been attributed to its capacity to increase the avidity of interaction with APC and to shuttle associated Lck to the TCR/CD3 activation complex. The results presented in this study demonstrate that ligation of CD4 inhibits ongoing responses of preactivated T cells. Specifically, delayed addition of CD4-specific mAb is shown to inhibit Ag- or mAb-induced responses of both primary T cells and T cell clonal variants. The Ag responses of the latter are independent of the adhesion provided by CD4; thus the observed inhibition is not due to blocking CD4-MHC interactions. Further, analysis of the clonal variants demonstrates that CD4-associated Lck is not essential for the inhibition observed, as anti-CD4 inhibits responses of clonal variants, expressing a form of CD4 unable to associate with Lck (double cysteine-mutated CD4). The inhibition is counteracted by the addition of exogenous IL-2, demonstrating that the block is not due to a lesion in IL-2 utilization, rather its production. It is demonstrated that the delayed addition of anti-CD4 results in a rapid reduction in steady-state levels of IL-2 mRNA in both primary T cells and clonal variants.
Isolation of MHC Class I-Restricted Tumor Antigen Peptide and Its Precursors Associated with Heat Shock Proteins hsp70, hsp90, and gp96
Authors: Ishii Tatsuaki , Udono Heiichiro , Yamano Taketoshi , Ohta Hiroyuki , Uenaka Akiko , Ono Toshiro , Hizuta Akio , Tanaka Noriaki , Srivastava Pramod K. , Nakayama Eiichi
Published: December 2022

We have previously demonstrated that vaccination with heat shock proteins hsp70, hsp90, and gp96 elicits specific immunity against the tumor from which the hsps were purified. Although the association of tumor Ag peptides with these hsps have been suggested, the identification of the peptides or their precursors stripped from the hsps remained to be resolved. We show in this report that an Ld-restricted cytotoxic T lymphocyte epitope of a mouse leukemia RL♂1 and its precursors are associated with the chaperones hsp90 and hsp70 in the cytosol and gp96 in the lumen of the endoplasmic reticulum. Hsp70 was associated with only final sized octamer, while hsp90 was found to associate with the octamer and two distinct precursor peptides. The gp96 was associated with the octamer and one of the two precursors. Thus, each of the hsps bound a distinct set of peptides. Our results have demonstrated for the first time that the hsps associate not only with final sized tumor Ag peptide but also with its precursors. The implication of this evidence is also discussed in terms of the roles of hsps in MHC class I Ag processing/presentation.
Lipopolysaccharide Inhibits TNF-Induced Apoptosis: Role of Nuclear Factor-κB Activation and Reactive Oxygen Intermediates
Authors: Manna Sunil K. , Aggarwal Bharat B.
Published: December 2022

LPS, a component of the cell wall in Gram-negative bacteria, induces inflammation and septic shock syndrome by stimulating various inflammatory cytokines including TNF. How LPS affects the TNF-mediated cellular responses, however, is not understood. In this study, the effect of LPS on TNF-mediated apoptosis in human histiocytic lymphoma U-937 cells was investigated. We found that treatment of cells with LPS completely abolished TNF-mediated cytotoxicity and activation of caspase-3. LPS-chelating antibiotic, polymyxin B, suppressed the antiapoptotic activity, indicating the specificity of the effect. Within minutes, LPS through CD14 induced the activation of NF-κB, degradation of IκBα (inhibitory subunit of NF-κB) and IκBβ, and nuclear translocation of p65. An antioxidant, pyrrolidine dithiocarbamate, which blocked LPS-induced NF-κB activation, also abolished the antiapoptotic effects of LPS at the same time. Besides TNF, the apoptosis induced by taxol and okadaic acid was also sensitive to LPS-induced NF-κB activation, whereas that induced by H2O2, doxorubicin, daunomycin, vincristine, and vinblastine was NF-κB insensitive. Tumor cells that constitutively expressed NF-κB also showed resistance to the apoptotic effects of TNF, taxol, and okadaic acid, but sensitivity to all other agents, indicating the critical role of NF-κB in blocking apoptosis induced by certain agents. Overall, these results indicate that LPS induces resistance to the apoptotic effects of TNF and other agents, and that NF-κB activation, whether induced or constitutive, inhibits this apoptosis.
Specific Activated T Cells Regulate IL-12 Production by Human Monocytes Stimulated with Cryptococcus neoformans
Authors: Retini Cinzia , Casadevall Arturo , Pietrella Donatella , Monari Claudia , Palazzetti Barbara , Vecchiarelli Anna
Published: December 2022

IL-12 production mediated by a T cell-independent and/or T cell-dependent pathway was investigated in human monocytes responding to Cryptococcus neoformans. The data of this study showed that: 1) appreciable levels of IL-12 were observed when freshly isolated monocytes were exposed to acapsular C. neoformans or Candida albicans and secretion occurred within 24–48 h of incubation; 2) monocytes alone were poor producers of IL-12 when stimulated with encapsulated C. neoformans; 3) the presence of specific anti-glucuronoxylomannan mAb favored IL-12 secretion and Fc cross-linking could play a role; 4) monocytes were able to secrete consistent levels of IL-12 when cultured with activated T cells responding to C. neoformans; 5) the maximum secretion of IL-12 was observed at 5–7 days of culture and was strongly regulated by the presence of endogenous IFN-γ; and 6) the interaction between CD40 on monocytes and CD40 ligand on activated T lymphocytes responding to C. neoformans played a critical role in IL-12 secretion. These data highlight the mechanisms of IL-12 production by human monocytes exposed to C. neoformans, indicating a possible biphasic secretion of IL-12, dependent on the direct effect of fungal insult, and characterized by consistent secretion of IL-12 that is dependent on the interaction of CD40 with the CD40 ligand expressed on activated T cells responding to C. neoformans.
Role of TNF-α in Pulmonary Host Defense in Murine Invasive Aspergillosis
Authors: Mehrad Borna , Strieter Robert M. , Standiford Theodore J.
Published: December 2022

Invasive pulmonary aspergillosis is a common and devastating complication of immunosuppression, whose incidence has increased dramatically in tandem with the increase in the number of immunocompromised patients. Given the role of TNF-α in other pulmonary infections, we hypothesized that TNF-α is an important proximal signal in murine invasive pulmonary aspergillosis. Intratracheal challenge with Aspergillus fumigatus conidia in both neutropenic (cyclophosphamide-treated) and nonneutropenic BALB/c mice resulted in the time-dependent increase in lung TNF-α levels, which correlated with the histologic development of a patchy, peribronchial infiltration of mononuclear and polymorphonuclear cells. Ab-mediated neutralization of TNF-α resulted in an increase in mortality in both normal and cyclophosphamide-treated animals, which was associated with increased lung fungal burden as determined by histology and as quantified by chitin content. Depletion of TNF-α resulted in a reduced lung neutrophil influx in both normal and cyclophosphamide-treated animals, which occurred in association with a decrease in lung levels of the C-X-C chemokine, macrophage inflammatory protein-2 and the C-C chemokines macrophage inflammatory protein-1α and JE. In cyclophosphamide-treated animals, intratracheal administration of a TNF-α agonist peptide (TNF70–80) 3 days before, but not concomitant with, the administration of Aspergillus conidia resulted in improved survival from 9% in control mice to 55% in TNF70–80-treated animals. These studies indicate that TNF-α is a critical component of innate immunity in both immunocompromised and immunocompetent hosts, and that pretreatment with a TNF-α agonist peptide in a compartmentalized fashion can significantly enhance resistance to A. fumigatus in neutropenic animals.
IFN-γ Is Required for Viral Clearance from Central Nervous System Oligodendroglia
Authors: Parra Beatriz , Hinton David R. , Marten Norman W. , Bergmann Cornelia C. , Lin Mark T. , Yang Camillia S. , Stohlman Stephen A.
Published: December 2022

Infection of the central nervous system (CNS) by the JHM strain of mouse hepatitis virus (JHMV) is a rodent model of the human demyelinating disease multiple sclerosis. The inability of effective host immune responses to eliminate virus from the CNS results in a chronic infection associated with ongoing recurrent demyelination. JHMV infects a variety of CNS cell types during the acute phase of infection including ependymal cells, astrocytes, microglia, oligodendroglia, and rarely in neurons. Replication within the majority of CNS cell types is controlled by perforin-dependent virus-specific CTL. However, inhibition of viral replication in oligodendroglia occurs via a perforin-independent mechanism(s). The potential role for IFN-γ as mediator controlling JHMV replication in oligodendroglia was examined in mice deficient in IFN-γ secretion (IFN-γ0/0 mice). IFN-γ0/0 mice exhibited increased clinical symptoms and mortality associated with persistent virus, demonstrating an inability to control replication. Neither antiviral Ab nor CTL responses were diminished in the absence of IFN-γ, although increased IgG1 was detected in IFN-γ0/0 mice. Increased virus Ag in the absence of IFN-γ localized almost exclusively to oligodendroglia and was associated with increased CD8+ T cells localized within white matter. These data suggest that although perforin-dependent CTL control virus replication within astrocytes and microglia, which constitute the majority of infected CNS cells, IFN-γ is critical for control of viral replication in oligodendroglia. Therefore, different mechanisms are used by the host defenses to control virus replication within the CNS, dependent upon the phenotype of the targets of virus replication.
VCAM-1 Expression on Human Dermal Microvascular Endothelial Cells Is Directly and Specifically Up-Regulated by Substance P
Authors: Quinlan Kimberly L. , Song In-Sung , Naik Shubhada M. , Letran Eleanor L. , Olerud John E. , Bunnett Nigel W. , Armstrong Cheryl A. , Caughman S. Wright , Ansel John C.
Published: December 2022

Sensory nerves in skin are capable of releasing multiple neuropeptides, which modulate inflammatory responses by activating specific cutaneous target cells. Extravasation of particular subsets of leukocytes depends upon the regulated expression of cellular adhesion molecules such as VCAM-1 on microvascular endothelial cells. We examined the direct effect of cutaneous neuropeptides on the expression and function of human dermal microvascular endothelial cell (HDMEC) VCAM-1. A significant increase in VCAM-1 immunostaining of microvascular endothelium was observed in vivo following capsaicin application to human skin. Multiple cutaneous sensory C-fiber-released neuropeptides were evaluated for their ability to induce VCAM-1 cell surface expression on HDMEC. Only substance P (SP) was found to be capable of inducing HDMEC VCAM-1 expression. This SP-mediated VCAM-1 induction appeared to be a direct effect that did not require the release of other HDMEC-derived soluble factors. Increased HDMEC VCAM-1 mRNA expression was detected 1 h after the addition of SP, with peak mRNA increase at 6–9 h postinduction. FACS studies demonstrated a 6.5-fold increase in endothelial cell surface VCAM-1 expression detectable 16 h after addition of SP, which was specifically blocked by a neurokinin-1 receptor antagonist. Increased VCAM-1 cell surface expression on SP-treated HDMEC resulted in a 4-fold increase in the functional binding of 51Cr-labeled MOLT-4 T cells. These data indicate that SP is capable of directly and specifically up-regulating functional endothelial VCAM-1 expression and thus may play a key role in modulating certain inflammatory responses in the skin.
IL-18 Up-Regulates Perforin-Mediated NK Activity Without Increasing Perforin Messenger RNA Expression by Binding to Constitutively Expressed IL-18 Receptor
Authors: Hyodo Yasuko , Matsui Kiyoshi , Hayashi Nobuki , Tsutsui Hiroko , Kashiwamura Shin-ichiro , Yamauchi Hiroshi , Hiroishi Keisai , Takeda Kiyoshi , Tagawa Yoh-ichi , Iwakura Yoichiro , Kayagaki Nobuhiko , Kurimoto Masashi , Okamura Haruki , Hada Toshikazu , Yagita Hideo , Akira Shizuo , Nakanishi2 Kenji , Higashino Kazuya
Published: December 2022

IL-18 is a powerful inducer of IFN-γ production, particularly in collaboration with IL-12. IL-18, like IL-12, also augments NK activity. Here we investigated the molecular mechanism underlying the up-regulation of killing activity of NK cells by IL-18. IL-18, like IL-12, dose dependently enhanced NK activity of splenocytes. This action was further enhanced by costimulation with IL-12. Treatment with anti-IL-2R Ab did not affect IL-18- and/or IL-12-augmented NK activity, and splenocytes from IFN-γ-deficient mice showed enhanced NK activity following stimulation with IL-12 and/or IL-18. Splenocytes from the mice deficient in both IL-12 and IL-18 normally responded to IL-18 and/or IL-12 with facilitated NK activity, suggesting that functional NK cells develop in the absence of IL-12 and IL-18. IL-18R, as well as IL-12R mRNA, was constitutively expressed in splenocytes from SCID mice, which lack T cells and B cells but have intact NK cells, and in those from IL-12 and IL-18 double knockout mice. NK cells isolated from SCID splenocytes expressed IL-18R on their surface. IL-18, in contrast to IL-12, did not enhance mRNA expression of perforin, a key molecule for exocytosis-mediated cytotoxicity. However, pretreatment with concanamycin A completely inhibited this IL-18- and/or IL-12-augmented NK activity. Furthermore, IL-18, like IL-12, failed to enhance NK activity of splenocytes from perforin-deficient mice. These data suggested that NK cells develop and express IL-12R and IL-18R in the absence of IL-12 or IL-18, and that both IL-18 and IL-12 directly and independently augment perforin-mediated cytotoxic activity of NK cells.
Frequency of Class I HLA-Restricted Anti-HIV CD8+ T Cells in Individuals Receiving Highly Active Antiretroviral Therapy (HAART)
Authors: Gray Clive M. , Lawrence Jody , Schapiro Jonathan M. , Altman John D. , Winters Mark A. , Crompton Meg , Loi Muoi , Kundu Smriti K. , Davis Mark M. , Merigan Thomas C.
Published: December 2022

Peptide/MHC tetrameric complexes were used to enumerate the frequency of HLA class I-restricted epitope-specific CD8+ T cells in 18 HLA-A*0201 HIV type 1-infected asymptomatic patients. HLA-A*0201 molecules were complexed to HIV Gag p17 (amino acids 77–85) and reverse transcriptase (amino acids 464–472) peptides, biotinylated, and bound to streptavidin-phycoerythrin to form tetramers. We show in this study that 17 of 18 HIV-1-infected asymptomatic patients have circulating frequencies of 1/50–1/1000 CD8+ T cells that recognize both Gag and Pol CTL epitopes or either epitope alone. The functional nature of these cells is open to interpretation, as we show that despite relatively high frequencies of fresh epitope-specific CD8+ T cells, variant epitope sequences in viral plasma progeny were rare. In addition, the majority of tetramer-positive cells did not display discernible fresh CTL activity; only after restimulation with specific peptide in culture was there an expansion of epitope-specific CD8+ cells, correlating with high CTL activity. These data suggest that fresh tetramer-stained cells probably represent memory precursors; we demonstrate, with the application of highly active antiretroviral therapy, that the interruption of chronic antigenic stimulation causes significant reductions in the frequency of these cells in five of six patients. In conclusion, this study provides evidence that persistently replicating viral populations are probably required to maintain high frequencies of HIV-1 epitope-specific CD8+ T cells in asymptomatic chronically infected individuals
A Re-Evaluation of the Frequency of CD8+ T Cells Specific for EBV in Healthy Virus Carriers
Authors: Tan Linda C. , Gudgeon Nancy , Annels Nicola E. , Hansasuta Pokrath , O’Callaghan Chris A. , Rowland-Jones Sarah , McMichael Andrew J. , Rickinson Alan B. , Callan Margaret F. C.
Published: December 2022

EBV is a gammaherpesvirus that can establish both nonproductive (latent) and productive (lytic) infections within the cells of its host. Although T cell responses to EBV latent proteins have been well characterized, little is known about the importance of responses to lytic proteins in long term virus carriers. Here we have compared the frequencies of CD8+ T cells specific for EBV latent and lytic Ags in healthy virus carriers, using three techniques: limiting dilution analysis, enzyme-linked immunospot assay, and FACS staining with tetrameric MHC-peptide complexes. T cells specific for EBV lytic protein epitopes were readily detectable in all donors and were usually more abundant than those specific for latent epitopes. We infer that direct T cell control of viral replicative lesions is maintained in long term carriers of EBV and is an important component of the immune response to this virus. Estimates of CD8+ T cell frequencies varied considerably according to methodology; values obtained from MHC-peptide tetramer staining were, on the average, 4.4-fold higher than those obtained from enzyme-linked immunospot assays, which were, in turn, on the average, 5.3-fold higher than those obtained from limiting dilution analysis. Tetramer staining showed that as many as 5.5% circulating CD8+ T cells in a virus carrier were specific for a single EBV lytic protein epitope. Such values are much greater than previously imagined and illustrate how antigenic challenge from a persistent herpesvirus can influence the composition of the host’s CD8+ T cell pool.
An Altered Peptide Ligand Specifically Inhibits Th2 Cytokine Synthesis by Abrogating TCR Signaling
Authors: Faith Alexander , Akdis Cezmi A. , Akdis Mübeccel , Joss Andrea , Wymann Daniel , Blaser Kurt
Published: December 2022

Altered peptide ligands (APL) can modify T cell effector function by their diversity in binding to the TCR or MHC class II-presenting molecules. The capacity to inhibit Th2 cytokine production by allergen-specific T cells would contribute to combating allergic inflammation. The presence of APL generated by Ala-substitutions in a synthetic dodeca-peptide spanning an immunodominant epitope of bee venom phospholipase A2 (PLA) was investigated in human T cells. Four of five substituted peptides reduced proliferation, IL-4, and IFN-γ production by cloned PLA-specific Th0 cells proportionately. However, one APL, PLA-F82A, inhibited IL-4 but had no effect on IFN-γ production. This uncoupling of IL-4 from IFN-γ production was also observed on immunogenic restimulation of the cloned T cells pre-exposed to the APL/APCs. It appeared to result from lower affinity of binding to MHC class II by the APL compared with the native peptide. The APL also inhibited IL-4 production by polyclonal T cells. In consequence of the change in cytokine secretion, the production of IgG4 in vitro increased by PLA-F82A stimulation, compared with the native peptide. Exposure of the cloned T cells to either the APL or the native peptide, in the absence of professional APC, induced anergy such that proliferation and production of IL-4, IL-5, and IL-13 was abrogated on immunogenic rechallenge. Defective T cell activation appeared to result from alterations in transmembrane signaling through the TCR, specifically to lack of tyrosine phosphorylation of the tyrosine kinase, ZAP-70.
Clonal Expansion of CD4+ TCRββ+ T Cells in TCR α-Chain- Deficient Mice by Gut-Derived Antigens
Authors: Takahashi Ichiro , Iijima Hideki , Katashima Rumi , Itakura Mitsuo , Kiyono Hiroshi
Published: December 2022

A population of CD4+ α−β+ T cells increases in the mucosal and peripheral lymphoid tissues of TCRα-chain-deficient mice with inflammatory bowel disease. The α−β+ T cells, which produce predominantly IL-4, mediate the proliferation of colonic epithelial crypts and the infiltration of large numbers of IgA-producing plasma cells into the lamina propria of the colon. To examine whether enteric Ags were recognized by a population of monoclonal α−β+ T cells leading to the intestinal inflammation, we examined the usage and clonotypes of TCR expressed by the α−β+ T cells in TCRα-chain-deficient mice with inflammatory bowel disease. Analyses of immunoprecipitates by two dimensional electrophoresis and single-cell RT-PCR revealed that TCR of the α−β+ T cells was a homodimer of β-chains that was capable of recognizing luminal bacterial Ags. PCR single-strand conformation polymorphism analysis of TCR Vβ transcripts revealed monoclonal accumulation of the α−β+ T cells in the colonic lamina propria of the diseased mice. DNA sequencing revealed the accumulation of the α−β+ T cells with the same CDR3 sequences in the colon. These findings suggest that the pathogenic CD4+ α−β+ T cells expressing a homodimeric form of the TCRβ-chains can be clonally expanded upon the stimulation with gut-derived Ags.
Cutting Edge: Primary Structure of the Light Chain of Fusion Regulatory Protein-1/CD98/4F2 Predicts a Protein with Multiple Transmembrane Domains That Is Almost Identical to the Amino Acid Transporter E16
Authors: Tsurudome Masato , Ito Morihiro , Takebayashi Shin-Ichiro , Okumura Katsuzumi , Nishio Machiko , Kawano Mitsuo , Kusagawa Shigeru , Komada Hiroshi , Ito Yasuhiko
Published: December 2022

The CD98 light chain (CD98LC) was copurified from HeLa S3 cells by an affinity chromatography using a mAb specific for the fusion regulatory protein-1 (FRP-1) which is identical to the CD98 heavy chain. On the basis of the N-terminal sequence (63 amino acids) of purified CD98LC polypeptide, we have cloned a PCR fragment (155 bp) from a HeLa S3 cDNA library and finally obtained a full cDNA clone encoding the CD98LC. Fluorescence in situ hybridization analysis using the cDNA assigned the CD98LC gene to the long arm of human chromosome 16 (16q24). The predicted amino acid sequence suggested that CD98LC is a protein with multiple transmembrane domains and is almost identical to the amino acid transporter E16. Resting monocytes and lymphocytes expressed CD98LC as analyzed by a newly isolated anti-CD98LC mAb, which showed cross-reactivity with insect Sf9 cells as well as with various mammalian cell lines.
Mucosal Immunity to Influenza Without IgA: An IgA Knockout Mouse Model
Authors: Mbawuike Innocent N. , Pacheco Susan , Acuna Catherine L. , Switzer Kirsten C. , Zhang Yongxin , Harriman Gregory R.
Published: December 2022

IgA knockout mice (IgA−/−) were generated by gene targeting and were used to determine the role of IgA in protection against mucosal infection by influenza and the value of immunization for preferential induction of secretory IgA. Aerosol challenge of naive IgA−/− mice and their wild-type IgA+/+ littermates with sublethal and lethal doses of influenza virus resulted in similar levels of pulmonary virus infection and mortality. Intranasal and i.p. immunization with influenza vaccine plus cholera toxin/cholera toxin B induced significant mucosal and serum influenza hemagglutinin-specific IgA Abs in IgA+/+ (but not IgA−/−) mice as well as IgG and IgM Abs in both IgA−/− and IgA+/+ mice; both exhibited similar levels of pulmonary and nasal virus replication and mortality following a lethal influenza virus challenge. Monoclonal anti-hemagglutinin IgG1, IgG2a, IgM, and polymeric IgA Abs were equally effective in preventing influenza virus infection in IgA−/− mice. These results indicate that IgA is not required for prevention of influenza virus infection and disease. Indeed, while mucosal immunization for selective induction of IgA against influenza may constitute a useful approach for control of influenza and other respiratory viral infections, strategies that stimulate other Igs in addition may be more desirable.
Cellular and Molecular Characterization of thescurfyMouse Mutant
Authors: Clark Lisa B. , Appleby Mark W. , Brunkow Mary E. , Wilkinson J. Erby , Ziegler Steven F. , Ramsdell Fred
Published: December 2022

Mice hemizygous (Xsf/Y) for the X-linked mutation scurfy (sf) develop a severe and rapidly fatal lymphoproliferative disease mediated by CD4+CD8− T lymphocytes. We have undertaken phenotypic and functional studies to more accurately identify the immunologic pathway(s) affected by this important mutation. Flow cytometric analyses of lymphoid cell populations reveal that scurfy syndrome is characterized by changes in several phenotypic parameters, including an increase in Mac-1+ cells and a decrease in B220+ cells, changes that may result from the production of extremely high levels of the cytokine granulocyte-macrophage CSF by scurfy T cells. Scurfy T cells also exhibit strong up-regulation of cell surface Ags indicative of in vivo activation, including CD69, CD25, CD80, and CD86. Both scurfy and normal T cells are responsive to two distinct signals provided by the TCR and by ligation of CD28; scurfy cells, however, are hyperresponsive to TCR ligation and exhibit a decreased requirement for costimulation through CD28 relative to normal controls. This hypersensitivity may result, in part, from increased costimulation through B7-1 and B7-2, whose expression is up-regulated on scurfy T cells. Although the specific defect leading to this hyperactivation has not been identified, we also demonstrate that scurfy T cells are less sensitive than normal controls to inhibitors of tyrosine kinases such as genistein and herbimycin A, and the immunosuppressant cyclosporin A. One interpretation of our data would suggest that the scurfy mutation results in a defect, which interferes with the normal down-regulation of T cell activation.
The EBV Transforming Protein, Latent Membrane Protein 1, Mimics and Cooperates with CD40 Signaling in B Lymphocytes
Authors: Busch Lisa K. , Bishop Gail A.
Published: December 2022

Latent membrane protein 1 (LMP1) is required for EBV-induced immortalization of human B cells, and expression of the protein in the absence of other viral proteins leads to an activated phenotype in B cells. It has been well documented that LMP1 causes B cells to up-regulate adhesion molecules, such as LFA-1 and ICAM-1, and coactivation molecules, such as B7-1 and CD23, as well as to activate NF-κB. Ligation of the endogenous B cell CD40 molecule also induces these and other activated phenotypic changes. Here, we report that expression of LMP1 also activates B cells to secrete Ig and IL-6 and rescues them from B cell receptor-mediated growth arrest analogous to CD40 signaling. Furthermore, an HLA-A2LMP1 chimeric construct demonstrates that the oligomerization of the carboxyl-terminal 200 amino acids of LMP1 is sufficient for B cell signaling. Finally, we demonstrate that LMP1 and CD40 signaling pathways interact cooperatively in inducing B cell effector functions.
TRANCE, a TNF Family Member, Is Differentially Expressed on T Cell Subsets and Induces Cytokine Production in Dendritic Cells
Authors: Josien Régis , Wong Brian R. , Li Hong-Li , Steinman Ralph M. , Choi Yongwon
Published: December 2022

TNF-related activation-induced cytokine (TRANCE) is a member of the TNF family recently identified in activated T cells. We report here that TRANCE mRNA is constitutively expressed in memory, but not naive, T cells and in single-positive thymocytes. Upon TCR/CD3 stimulation, TRANCE mRNA and surface protein expression are rapidly up-regulated in CD4+ and CD8+ T cells, which can be further enhanced on CD4+ T cells by CD28-mediated costimulation. However, TRANCE induction is significantly suppressed when cells are stimulated in the presence of IL-4, but is not modified in the presence of IFN-α, IFN-γ, TGF-β, TNF-α, or IL-2. High levels of TRANCE receptor expression are found on mature dendritic cells (DCs). In this study we show that activated T and B cells also express TRANCE receptor, but only at low levels. TRANCE, however, does not exert any significant effect on the proliferation, activation, or survival of those cells. In DCs, TRANCE induces the expression of proinflammatory cytokines (IL-6, IL-1) and T cell growth and differentiation factors (IL-12, IL-15) in addition to enhancing DC survival. Moreover, TRANCE cooperates with CD40 ligand or TNF-α to further increase the viability of DCs, suggesting that several TNF-related molecules on activated T cells may cooperatively regulate the function and survival of DCs to enhance T cell-mediated immune responses.
Pregnancy Ameliorates Induction and Expression of Experimental Autoimmune Uveitis
Authors: Agarwal Rajeev K. , Chan Chi-Chao , Wiggert Barbara , Caspi Rachel R.
Published: December 2022

Female patients suffering from autoimmune uveitis are reported to experience a temporary remission during pregnancy. Experimental autoimmune uveitis (EAU) is a model for human uveitis. Here we examine the effect of pregnancy on the development of EAU and its associated immunological responses. Susceptible C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP). EAU scores and Ag-specific responses were evaluated 21 days later. Mice immunized during pregnancy developed significantly less EAU than nonpregnant controls. Their lymph node cells and splenocytes produced a distinct pattern of cytokines in response to IRBP: reduced IFN-γ and IL-12 p40, but unchanged levels of TNF-α, IL-4, IL-5, and IL-10. Anti-IRBP Ab isotypes revealed an up-regulation of IgG1, indicating a possible Th2 bias at the humoral level. Ag-specific proliferation and delayed hypersensitivity, as well as mitogen-induced IFN-γ production, remained undiminished, arguing against an overall immune deficit. Interestingly, pregnant mice that received an infusion of IRBP-primed lymphoid cells from nonpregnant donors also developed reduced EAU, suggesting that pregnancy suppresses not only the generation, but also the function of mature uveitogenic effector T cells. Pregnant mice at the time of immunization exhibited elevated levels of TGF-β, but not of IL-10, in the serum. We suggest that protection from EAU during pregnancy is due primarily to a selective reduction of Ag-specific Th1 responses with only marginal enhancement of Th2 function, and that these effects may in part be secondary to elevated systemic levels of TGF-β.