bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary Human CD4+ T Cells Contain Heterogeneous IκB Kinase Complexes: Role in Activation of the IL-2 Promoter
Authors: Khoshnan Ali , Kempiak Stephan J. , Bennett Brydon L. , Bae David , Xu Weiming , Manning Anthony M. , June Carl H. , Nel Andre E.
Published: December 2022

NF-κB transcription factors play an important role in the activation of the IL-2 gene in response to TCR ligation. The release of NF-κB factors to the nucleus requires phosphorylation and degradation of the inhibitory κ-B proteins (IκBs). IκBα and IκBβ phosphorylation is dependent on dual signaling by the TCR and the CD28 accessory receptor. This pathway involves a multisubunit IκB kinase (IKK) complex, which includes the IKKα (IKK-1) and IKKβ (IKK-2) kinases. We demonstrate that stimulation of primary human CD4+ T cells by CD3/CD28 activates two distinct endogenous IKK complexes, a heterodimeric IKKα/β and a homodimeric IKKβ complex. IKKβ overexpression in a Jurkat cell line resulted in the formation of a constitutively active IKK complex, which was CD3/CD28 inducible. In contrast, ectopic expression of IKKα assembled into a complex with negligible IκB kinase activity. Moreover, IKKβ, but not IKKα, overexpression enhanced transcriptional activation of the CD28 response element in the IL-2 promoter. Conversely, only kinase-inactive IKKβ interfered in the activation of the IL-2 promoter. Sodium salicylate, an inhibitor of IKKβ, but not IKKα, activity, inhibited IL-2 promoter activation as well as IL-2 secretion and interfered in activation of both the heterodimeric as well as the homodimeric IKK complexes in primary CD4+ T cells. Taken together, these data demonstrate the presence of an IKKβ-mediated signaling pathway that is activated by TCR and CD28 coligation and regulates IL-2 promoter activity.
Inducible Nitric Oxide Synthase Protection Against Coxsackievirus Pancreatitis
Authors: Zaragoza Carlos , Ocampo Christopher J. , Saura Marta , Bao Clare , Leppo Michelle , Lafond-Walker Anne , Thiemann David R. , Hruban Ralph , Lowenstein Charles J.
Published: December 2022

Coxsackievirus infection causes myocarditis and pancreatitis in humans. In certain strains of mice, Coxsackievirus causes a severe pancreatitis. We explored the role of NO in the host immune response to viral pancreatitis. Coxsackievirus replicates to higher titers in mice lacking NO synthase 2 (NOS2) than in wild-type mice, with particularly high viral titers and viral RNA levels in the pancreas. Mice lacking NOS have a severe, necrotizing pancreatitis, with elevated pancreatic enzymes in the blood and necrotic acinar cells. Lack of NOS2 leads to a rapid increase in the mortality of infected mice. Thus, NOS2 is a critical component in the immune response to Coxsackievirus infection.
CD8+ T Cells Modulate Late Allergic Airway Responses in Brown Norway Rats
Authors: Suzuki Masaru , Taha Rame , Ihaku Daizo , Hamid Qutayba , Martin James G.
Published: December 2022

To test the hypothesis that CD8+ T cells may suppress the allergen-induced late airway response (LAR) and airway eosinophilia, we examined the effect of administration of Ag-primed CD8+ T cells on allergic airway responses, bronchoalveolar lavage (BAL) leukocytes, and mRNA expression for cytokines (IL-4, IL-5, and IFN-γ) in OVA-sensitized Brown Norway rats. On day 12 postsensitization to OVA, test rats were administered 2 million CD8+ T cells i.p. isolated from either the cervical lymph nodes (LN group; n = 8) or the spleen (Spl group; n = 6) of sensitized donors. On day 14, test rats were challenged with aerosolized OVA. Control rats were administered PBS i.p. on day 12, and challenged with OVA (n = 10) or BSA (n = 6) on day 14. The lung resistance was measured for 8 h after challenge. BAL was performed at 8 h. Cytospin slides of BAL were analyzed for major basic protein by immunostaining and for cytokine mRNA by in situ hybridization. The LAR was significantly less in the LN group (1.8 ± 0.5 U; p < 0.01) and BSA controls (1.4 ± 0.7; p < 0.01), but not in the Spl group (6.7 ± 2.2), compared with that in OVA controls (8.1 ± 1.8). In BAL, the number of major basic protein-positive cells was lower in the LN and Spl groups compared with OVA controls (p < 0.05 and p < 0.01). IL-4- and IL-5-positive cells were decreased in the LN group compared with the OVA controls (p < 0.01). INF-γ-positive cells were increased in the LN and Spl groups compared with the OVA controls (p < 0.01). Serum OVA-specific IgE levels were unaffected by CD8+ T cell transfers. These results indicate that Ag-primed CD8+ T cells have a potent suppressive effect on LAR.
Alloantibody-Mediated Class I Signal Transduction in Endothelial Cells and Smooth Muscle Cells: Enhancement by IFN-γ and TNF-α
Authors: Bian Hong , Reed Elaine F.
Published: December 2022

Chronic rejection is the major limiting factor to long term survival of solid organ allografts. The hallmark of chronic rejection is transplant atherosclerosis, which is characterized by the intimal proliferation of smooth muscle cells, endothelial cells, and fibroblasts, leading to vessel obstruction, fibrosis, and eventual graft loss. The mechanism of chronic rejection is poorly understood, but it is suspected that the associated vascular changes are a result of anti-HLA Ab-mediated injury to the endothelium and smooth muscle of the graft. In this study we have investigated whether anti-HLA Abs, developed by transplant recipients following transplantation, are capable of transducing signals via HLA class I molecules, which stimulate cell proliferation. In this report we show that ligation of class I molecules with Abs to distinct HLA-A locus and HLA-B locus molecules results in increased tyrosine phosphorylation of intracellular proteins and induction of fibroblast growth factor receptor expression on endothelial and smooth muscle cells. Treatment of cells with IFN-γ and TNF-α up-regulated MHC class I expression and potentiated anti-HLA Ab-induced fibroblast growth factor receptor expression. Engagement of class I molecules also stimulated enhanced proliferative responses to basic fibroblast growth factor, which augmented endothelial cell proliferation. These findings support a role for anti-HLA Abs and cytokines in the transduction of proliferative signals, which stimulate the development of myointimal hyperplasia associated with chronic rejection of human allografts.
Highly Th2-Skewed Cytokine Profile of β-Lactam-Specific T Cells from Nonatopic Subjects with Adverse Drug Reactions
Authors: Brugnolo Francesca , Annunziato Francesco , Sampognaro Salvatore , Campi Paolo , Manfredi Mariangela , Matucci Andrea , Blanca Miguel , Romagnani Sergio , Maggi Enrico , Parronchi Paola
Published: December 2022

A positive lymphocyte transformation test to β-lactams (β-L) was found in 12 of 29 subjects with adverse drug reaction (ADR) to β-L, irrespective of either the type of clinical manifestation or the presence of specific serum IgE. Short-term T cell lines specific for penicillin G, amoxicillin, and ampicillin could be generated only from subjects with ADR (eight with positive and one with negative lymphocyte transformation test), while streptokinase and Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells were obtained from all these subjects, from 7 atopic Der p-sensitive donors without history of ADR and 17 healthy nonatopic donors. Streptokinase-specific T cells from all subjects showed intracellular expression of IFN-γ with poor or no IL-4, whereas Der p 1-specific T cells exhibited IFN-γ but low or no IL-4 expression in nonatopics, and remarkable IL-4 expression in atopic donors. By contrast, all penicillin G-, ampicillin-, and amoxicillin-specific short-term T cell lines showed high intracellular expression of IL-4, IL-5, and IL-13, but poor or no expression of IFN-γ, thus exhibiting a clear-cut Th2 profile. Accordingly, most penicillin G-specific T cell clones derived from two subjects with ADR released high concentrations of IL-4 alone or IL-4 and IFN-γ. These data suggest that cytokines produced by Th2 cells play an important role in all β-L-induced ADR, even when late clinical manifestations occur and an IgE-mediated mechanism is apparently indemonstrable.
Cutting Edge: Combined Treatment of TNF-α and IFN-γ Causes Redistribution of Junctional Adhesion Molecule in Human Endothelial Cells
Authors: Ozaki Harunobu , Ishii Kenji , Horiuchi Hisanori , Arai Hidenori , Kawamoto Takahiro , Okawa Katsuya , Iwamatsu Akihiro , Kita Toru
Published: December 2022

Proinflammatory cytokines such as TNF-α and IFN-γ induce cell adhesion molecules in endothelial cells and promote transmigration of leukocytes across endothelial cells. However, when those two were administered together, leukocyte transmigration paradoxically decreased. We cloned a human and bovine homologue of the junctional adhesion molecule (JAM), a novel molecule at the tight junction, and examined the effects of proinflammatory cytokines on JAM in HUVECs. The combined treatment of TNF-α plus IFN-γ caused a disappearance of JAM from intercellular junctions. However, flow cytometry, cell ELISA, and subcellular fractionation analysis demonstrated that the amount of JAM was not reduced. This suggested that JAM changed its distribution in response to proinflammatory cytokines. This redistribution of JAM might be involved in a decrease in transendothelial migration of leukocytes at inflammatory sites.
Cutting Edge: Purinergic Signaling Regulates Radical-Mediated Bacterial Killing Mechanisms in Macrophages Through a P2X7-Independent Mechanism
Authors: Sikora Andrew , Liu Judy , Brosnan Celia , Buell Gary , Chessel Iain , Bloom Barry R.
Published: December 2022

Signaling by extracellular nucleotides through P2 purinergic receptors affects diverse macrophage functions; however, its role in regulating antimicrobial radicals during bacterial infection has not been investigated. Mycobacterium tuberculosis-infected macrophages released ATP in a dose-dependent manner, which correlated with nitrite accumulation. P2 receptor inhibitors, including oxidized ATP, blocked NO synthase (NOSII) up-regulation and NO production induced by infection with M. tuberculosis or bacille Calmette-Guérin, or treatment with LPS or TNF-α. Oxidized ATP also inhibited oxygen radical production and activation of NF-κB and AP-1 in response to infection and inhibited NO-dependent killing of bacille Calmette-Guérin by macrophages. Experiments using macrophages derived from P2X7 gene-disrupted mice ruled out an essential role for P2X7 in NOSII regulation. These data demonstrate that P2 receptors regulate macrophage activation in response to bacteria and proinflammatory stimuli, and suggest that extracellular nucleotides released from infected macrophages may enhance production of oxygen radicals and NO at sites of infection.
Cas-L Is Required for β1 Integrin-Mediated Costimulation in Human T Cells
Authors: Kamiguchi Kenjiro , Tachibana Kouichi , Iwata Satoshi , Ohashi Yoshiyuki , Morimoto Chikao
Published: December 2022

β1 integrins provide a costimulus for TCR/CD3-driven T cell activation and IL-2 production in human peripheral T cells. However, this β1 integrin-mediated costimulation is impaired in a human T lymphoblastic line, Jurkat. We studied the molecular basis of this impaired costimulation and found that Cas-L, a 105-kDa docking protein, is marginally expressed in Jurkat T cells, whereas Cas-L is well expressed in peripheral T cells. Cas-L is a binding protein and a substrate for focal adhesion kinase and is tyrosine phosphorylated by β1 integrin stimulation. We here show that the transfection of wild-type Cas-L in Jurkat T cells restores β1 integrin-mediated costimulation. However, Cas-L transfection had no effect on CD28-mediated costimulation, indicating that Cas-L is specifically involved in the β1 integrin-mediated signaling pathway. Furthermore, transfection of the Cas-LΔSH3 mutant failed to restore β1 integrin-mediated costimulation in Jurkat cells. Cas-LΔSH3 mutant lacks the binding site for focal adhesion kinase and is not tyrosine phosphorylated after β1 integrin stimulation. These findings strongly suggest that the tyrosine phosphorylation of Cas-L plays a key role in the signal transduction in the β1 integrin-mediated T cell costimulation.
How Specific Should Immunological Memory Be?
Authors: Borghans José A. M. , Noest André J. , De Boer Rob J.
Published: December 2022

Protection against infection hinges on a close interplay between the innate immune system and the adaptive immune system. Depending on the type and context of a pathogen, the innate system instructs the adaptive immune system to induce an appropriate immune response. Here, we hypothesize that the adaptive immune system stores these instructions by changing from a naive to an appropriate memory phenotype. In a secondary immune reaction, memory lymphocytes adhere to their instructed phenotype. Because cross-reactions with unrelated Ags can be detrimental, such a qualitative form of memory requires a sufficient degree of specificity of the adaptive immune system. For example, lymphocytes instructed to clear a particular pathogen may cause autoimmunity when cross-reacting with ignored self molecules. Alternatively, memory cells may induce an immune response of the wrong mode when cross-reacting with subsequent pathogens. To maximize the likelihood of responding to a wide variety of pathogens, it is also required that the immune system be sufficiently cross-reactive. By means of a probabilistic model, we show that these conflicting requirements are met optimally by a highly specific memory lymphocyte repertoire. This explains why the lymphocyte system that was built on a preserved functional innate immune system has such a high degree of specificity. Our analysis suggests that 1) memory lymphocytes should be more specific than naive lymphocytes and 2) species with small lymphocyte repertoires should be more vulnerable to both infection and autoimmune diseases.
Immune System Development and Function in Prolactin Receptor-Deficient Mice
Authors: Bouchard Brigitte , Ormandy Christopher J. , Di Santo James P. , Kelly Paul A.
Published: December 2022

Prolactin (PRL) is the primary lactogenic pituitary hormone that plays an essential role in many aspects of reproduction, from fertilization to mammary gland development and maternal behavior. PRL has also been reported to play a role in immunoregulation. Because initial observations indicated that hypophysectomized rats present abnormalities of the immune system, including increased thymic atrophy and lymphopenia, a number of studies have focused on the potential immunomodulatory roles of PRL. This hormone exerts its biological activities following binding to specific cell surface PRL receptors (PRLRs). In this report, we have characterized the development and function of the immune system in PRLR-deficient mice. Compared with wild-type control mice, PRLR−/− mice demonstrate no alterations in thymic or splenic cellularity or in the composition of the lymphocyte subsets present in primary (bone marrow and thymus) or secondary (spleen and lymph nodes) lymphoid organs. Lymphocytes from PRLR−/− mice are functional in vitro, as they can proliferate normally to mitogens, cytokines, and allogeneic cells. PRLR−/− splenocytes display normal NK-mediated cytotoxicity to YAC-1 target cells. In vivo studies have revealed that PRLR−/− mice are able to 1) generate normal steady-state Ig levels, 2) mount a normal specific Ig response following immunization with a T-dependent Ag, 3) eliminate injected allogeneic tumor cells, and 4) effectively control Listeria monocytogenes infection. Taken together, these results show that immune system development and function proceed normally in the absence of PRL-mediated signaling and suggest that PRLR pathways are not essential for immunomodulation in vivo.
The Transcription Factors Elf-1 and GATA-1 Bind to Cell-Specific Enhancer Elements of Human High-Affinity IgE Receptor α-Chain Gene
Authors: Nishiyama Chiharu , Yokota Toyokazu , Okumura Ko , Ra1 Chisei
Published: December 2022

Key regulatory regions necessary for the expression of the gene encoding FcεRI α-chain, a component of the high-affinity IgE receptor primarily responsible for IgE-dependent allergic response, were investigated. Two regions, −74/−69 and −55/−47, which contained binding motifs for proteins belonging to the Ets family and the GATA family, respectively, were shown to be necessary for the activation of the α-chain promoter. Both the regulatory elements enhanced the promoter activity only in α-chain-producing cells PT18 and RBL-2H3 (mast cell lines), indicating that the elements required specific trans-acting proteins present in the α-chain-producing cells. EMSA using nuclear extracts and in vitro-translated proteins revealed that Elf-1 and GATA-1 bound to the enhancer elements. This is the first report describing the regulation in the expression of the FcεRI α-chain.
ICAM-1-Coupled Signaling Pathways in Astrocytes Converge to Cyclic AMP Response Element-Binding Protein Phosphorylation and TNF-α Secretion
Authors: Etienne-Manneville Sandrine , Chaverot Nathalie , Strosberg A. Donny , Couraud Pierre-Olivier
Published: December 2022

In the CNS, astrocytes play a key role in immunological and inflammatory responses through ICAM-1 expression, cytokine secretion (including TNF-α), and regulation of blood-brain barrier permeability. Because ICAM-1 transduces intracellular signals in lymphocytes and endothelial cells, we investigated in the present study ICAM-1-coupled signaling pathways in astrocytes. Using rat astrocytes in culture, we report that ICAM-1 binding by specific Abs induces TNF-α secretion together with phosphorylation of the transcription factor cAMP response element-binding protein. We show that ICAM-1 binding induces cAMP accumulation and activation of the mitogen-activated protein kinase extracellular signal-regulated kinase. Both pathways are responsible for cAMP response element-binding protein phosphorylation and TNF-α secretion. Moreover, these responses are partially dependent protein kinase C, which acts indirectly, as a common activator of cAMP/protein kinase A and extracellular signal-regulated kinase pathways. These results constitute the first evidence of ICAM-1 coupling to intracellular signaling pathways in glial cells and demonstrate the convergence of these pathways onto transcription factor regulation and TNF-α secretion. They strongly suggest that ICAM-1-dependent cellular adhesion to astrocytes could contribute to the inflammatory processes observed during leukocyte infiltration in the CNS.
Phenotypic Identification of Antigen-Dependent and Antigen-Independent CD8 CTL Precursors in the Draining Lymph Node During Acute Cutaneous Herpes Simplex Virus Type 1 Infection
Authors: McNally James M. , Dempsey Deborah , Wolcott R. Michael , Chervenak Robert , Jennings Stephen R.
Published: December 2022

Optimal immunological control of cutaneous herpes simplex virus type 1 (HSV-1) infections initiated in the hind footpad of C57BL/6 (B6, H-2b) mice is dependent upon the presence of functional HSV-1-specific T lymphocytes. The class I MHC-restricted, CD8+ T cell subpopulation is involved in the clearance of infectious HSV-1 from the skin and limiting HSV-1 replication and spread within the peripheral nervous system. However, the frequency of HSV-1-specific CTL precursors (CTLp), as a measure of potential anti-viral CD8+ T cell function, is relatively low compared with other acute viral infections. To gain insight into the basis for this low functional frequency, changes in the CD8+ T cell subpopulation phenotype associated with activation and differentiation were investigated. Analysis of the phenotypic changes showed that HSV-1-specific CTLp were found predominantly within a subpopulation of CD8+ T cells expressing high levels of CD44 (CD44high) and high levels of the IL-2 receptor α-chain (CD25high). A second activated subpopulation of CD8+ T cells expressing the CD44high CD25low phenotype did not contain detectable HSV-1-specific CTLp, even after the addition of HSV-1-infected stimulator cells as a source of an exogenous Ag. These data suggested that HSV-1-specific CD8+ T cells must increase expression of CD25 before attaining the potential to become CTL effector cells. These findings also indicated that the up-regulation of CD44 alone is not sufficient to identify precisely HSV-1-specific CD8+ T cells.
I-Ag7-Mediated Antigen Presentation by B Lymphocytes Is Critical in Overcoming a Checkpoint in T Cell Tolerance to Islet β Cells of Nonobese Diabetic Mice
Authors: Noorchashm Hooman , Lieu Yen K. , Noorchashm Negin , Rostami Susan Y. , Greeley Siri Atma S. , Schlachterman Alexander , Song Howard K. , Noto Lauren E. , Jevnikar Anthony M. , Barker Clyde F. , Naji Ali
Published: December 2022

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet β cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet β cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.
Conformational Epitope of the Type III Group B Streptococcus Capsular Polysaccharide
Authors: Zou Wei , Mackenzie Roger , Thérien Lina , Hirama Tomoko , Yang Qingling , Gidney Margaret A. , Jennings Harold J.
Published: December 2022

The protective epitope of the type III group B streptococcal polysaccharide (GBSPIII) is length dependent and conformational. To obtain a more accurate characterization of the conformational epitope, ELISA inhibition and surface plasmon resonance studies were conducted on two GBSPIII-specific mAbs using a large panel of oligosaccharide probes. The results of the studies confirmed that 2 repeating units (RU) is the minimum binding unit and that, while increases in chain length from 2 RU to 7 RU caused further optimization of the epitope, it remained monovalent. A 3-fold increase in affinity was observed between 7 RU and 20 RU, which, by surface plasmon resonance studies on a Fab, was shown to be due to both further optimization of the individual epitope and the occurrence of multivalency of epitope. The data support our hypothesis that the conformational epitope is an extended helical segment of the GBSPIII. GBSPIII exists mainly in the random coil form, which structurally mimics short oligosaccharide self Ags, but it can infrequently and spontaneously form extended helices. Although not prevalent in GBSPIII, the immune system preferentially selects these helical epitopes because they are unique to the polysaccharide. Contrary to a previously proposed model of GBSPIII binding in which the binding of the first Ab propagates a continuum of helical epitopes, our binding kinetics are consistent only with the helical epitope’s being discontinuous and infrequent.
Studies with Double Cytokine-Deficient Mice Reveal That Highly Polarized Th1- and Th2-Type Cytokine and Antibody Responses Contribute Equally to Vaccine-Induced Immunity toSchistosoma mansoni
Authors: Hoffmann Karl F. , James Stephanie L. , Cheever Allen W. , Wynn Thomas A.
Published: December 2022

A fundamental obstacle to vaccine development in schistosomiasis mansoni is a lack of understanding of what type of an immune response should be invoked. We have addressed this central issue by using the radiation-attenuated cercariae vaccine in mice genetically engineered to exhibit highly polarized type 1 (IL-10/IL-4-deficient) or type 2 (IL-10/IL-12-deficient) cytokine and Ab phenotypes. Our data show that while significant differences in immunity exist after a single vaccination with irradiated cercariae in double cytokine-deficient vs wild-type mice, these differences disappear after two vaccinations. The most important finding of these studies, however, was revealed in vaccinated IL-10-deficient mice. These mice developed a mixed and elevated type 1- and type 2-associated immune response and developed anti-schistosome immunity at levels equal to or better than those in wild-type mice. This immunity in IL-10-deficient mice correlated with higher parasite-specific Ab titers, greater proliferative capacity of lymphocytes, increased frequency of IFN-γ- and IL-4-secreting cells, elevated perivascular/peribronchial inflammatory responses in the lung, and greater in vitro schistosomulacidal capacity of parasite Ag-elicited cells. These results suggest that optimal vaccine-induced immunity against schistosomes is linked not to the development of a highly polarized response, but, rather, to the induction of both type 1- and type 2-associated immune responses.
Lipopolysaccharide Modulates Cyclooxygenase-2 Transcriptionally and Posttranscriptionally in Human Macrophages Independently from Endogenous IL-1β and TNF-α
Authors: Barrios-Rodiles Miriam , Tiraloche Gabrielle , Chadee Kris
Published: December 2022

The pathogenesis of septicemia can be triggered by LPS, a potent stimulus for PG synthesis. The enzyme cyclooxygenase (COX) is a rate-limiting step in PG production. COX exists as two isoforms: COX-1, which is constitutively expressed in most cell types, and COX-2, which is inducible by LPS and cytokines in a variety of cells. In this study we determined the role of the proinflammatory cytokines IL-1β and TNF-α released by LPS-stimulated U937 human macrophages in the regulation of COX-2. Macrophages exposed to LPS showed a rapid and sustained expression of COX-2 mRNA and protein for up to 48 h, whereas PGE2 production was notably enhanced only after 12 h. LPS increased COX-2 gene transcription and activation of the transcription factor NF-κB in a transient manner. LPS-treated macrophages produced high levels of TNF-α and moderate amounts of IL-1β protein. However, neutralizing Abs against these cytokines had no effect on COX-2 mRNA and protein expression, nor did they affect the stability of COX-2 mRNA. Interestingly, in the presence of LPS or exogenous IL-1β, COX-2 transcripts were stabilized, and actinomycin D inhibited their degradation. Only when LPS or IL-1β was removed did COX-2 mRNA decay with a t1/2 of ≥5 h. In contrast, dexamethasone promoted a faster decay of the LPS-induced COX-2 transcripts (t1/2 = 2.5 h). These results clearly demonstrate that LPS can regulate COX-2 at both transcriptional and posttranscriptional levels independently from endogenous IL-1β and TNF-α in human macrophages.
Impaired Cytokine Signaling in Mice Lacking the IL-1 Receptor-Associated Kinase
Authors: Thomas James A. , Allen Jerry L. , Tsen May , Dubnicoff Todd , Danao Jay , Liao X. Charlene , Cao Zhaodan , Wasserman Steven A.
Published: December 2022

Stimulation of the type 1 IL-1R (IL-1R1) and the IL-18R by their cognate ligands induces recruitment of the IL-1R-associated kinase (IRAK). Activation of IRAK leads in turn to nuclear translocation of NF-κB, which directs expression of innate and adaptive immune response genes. To study IRAK function in cytokine signaling, we generated cells and mice lacking the IRAK protein. IRAK-deficient fibroblasts show diminished activation of NF-κB when stimulated with IL-1. Immune effector cells without IRAK exhibit a defective IFN-γ response to costimulation with IL-18. Furthermore, mice lacking the Irak gene demonstrate an attenuated response to injected IL-1. Deletion of Irak, however, does not affect the ability of mice to develop delayed-type hypersensitivity or clear infection with the intracellular parasite, Listeria monocytogenes. These results demonstrate that although IRAK participates in IL-1 and IL-18 signal transduction, residual cytokine responsiveness operates through an IRAK-independent pathway.
Selection of a C5a Receptor Antagonist from Phage Libraries Attenuating the Inflammatory Response in Immune Complex Disease and Ischemia/Reperfusion Injury
Authors: Heller Tanja , Hennecke Meike , Baumann Ulrich , Gessner J. Engelbert , Meyer zu Vilsendorf Andreas , Baensch Melanie , Boulay Francois , Kola Axel , Klos Andreas , Bautsch Wilfried , Köhl Jörg
Published: December 2022

A C5a-receptor antagonist was selected from human C5a phage display libraries in which the C terminus of des-Arg74-hC5a was mutated. The selected molecule is a competitive C5a receptor antagonist in vitro and in vivo. Signal transduction is interrupted at the level of G-protein activation. In addition, the antagonist does not cause any C5a receptor phosphorylation. Proinflammatory properties such as chemotaxis or lysosomal enzyme release of differentiated U937 cells, as well as C5a-induced changes in intracellular Ca2+ concentration of murine peritoneal macrophages, are inhibited. The in vivo efficacy was evaluated in three different animal models of immune complex diseases in mice, i.e., the reverse passive Arthus reaction in the peritoneum, skin, and lung. The i.v. application of the C5a receptor antagonist abrogated polymorphonuclear neutrophil accumulation in peritoneum and markedly attenuated polymorphonuclear neutrophil migration into the skin and the lung. In a model of intestinal ischemia/reperfusion injury, i.v. administration of the C5a receptor antagonist decreased local and remote tissue injury: bowel wall edema and hemorrhage as well as pulmonary microvascular dysfunction. These data give evidence that C5a is an important mediator triggering the inflammatory sequelae seen in immune complex diseases and ischemia/reperfusion injury. The selected C5a receptor antagonist may prove useful to attenuate the inflammatory response in these disorders.
Activated Human T Cells Release Bioactive Fas Ligand and APO2 Ligand in Microvesicles
Authors: Martínez-Lorenzo María José , Anel Alberto , Gamen Susana , Monleón Inmaculada , Lasierra Pilar , Larrad Luis , Piñeiro Andrés , Alava María A. , Naval Javier
Published: December 2022

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100–200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (≤1 h) after PHA stimulation, well before the cell enters apoptosis. FasL- and APO2L-containing vesicles are also present in supernatants from PHA-activated fresh human PBMC. These observations provide the basis for a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation.
Critical Role of Costimulation in the Activation of Naive Antigen-Specific TCR Transgenic CD8+ T Cells In Vitro
Authors: Chai Jian-Guo , Vendetti Silvia , Bartok Istvan , Schoendorf Diana , Takacs Katalin , Elliott James , Lechler Robert , Dyson Julian
Published: December 2022

The influence of costimulation on the activation of naive CD8+ T cells and thymocytes was studied in vitro using H-Y-specific TCR-transgenic mice and H-Y antigenic peptide. Using a variety of physiological APC types, the activation of naive CD8+ T cells depended strictly on costimulation, which could not be substituted by high epitope density. T cell activation is known to be regulated by the interactions between CD86/CD80 and CD28/CD152, although it remains unclear whether the B7 isoforms have distinct roles. Addition of soluble anti-CD86 Ab led to profound inhibition of T cell reactivity, further confirming the importance of costimulation in naive CD8+ T cell activation. Finally, TCR engagement in the absence of costimulation had no effect on the subsequent reactivity of peripheral naive transgenic CD8+ T cells, but induced nonresponsiveness in mature CD8+ transgenic thymocytes. Collectively, these results demonstrate the importance of costimulation for naive CD8+ T cell activation, suggest that CD80 and CD86 can mediate opposing effects, possibly due to differential interaction with CD152 and CD28, and indicate differences in the sensitivity of immature vs mature CD8+ T cells to the induction of nonresponsiveness following costimulation-deficient Ag presentation.
TCRαβ-Independent CD28 Signaling and Costimulation Require Non-CD4-Associated Lck
Authors: Leung Bernadine L. , Haughn Loralee , Veillette André , Hawley Robert G. , Rottapel Robert , Julius Michael
Published: December 2022

Whether the sequelae of signals generated through CD28 either directly or in circumstances of costimulation require proximal events mediated by p56lck remains contentious. We demonstrate that CD4−, but not CD4+ clonal variants respond to CD28-specific mAb with both early and late indicators of activation. Forced expression of A418/A420-mutated CD4 or wild-type CD4 in the CD4− variant recapitulated the CD28-mediated responses of the CD4− and CD4+ variants, respectively. The implicated involvement of non-CD4-associated Lck is formally demonstrated by overexpressing S20/S23 Lck or wild-type Lck in CD4+ variants. The former, but not latter, rescues direct CD28 signaling, and supports costimulation. The results demonstrate that constitutive levels of non-CD4-associated Lck functionally limit CD28-mediated signaling.
Nasal-Associated Lymphoid Tissue: Phenotypic and Functional Evidence for the Primary Role of Peripheral Node Addressin in Naive Lymphocyte Adhesion to High Endothelial Venules in a Mucosal Site
Authors: Csencsits Keri L. , Jutila Mark A. , Pascual David W.
Published: December 2022

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafficking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunofluorescent and immunoperoxidase staining of frozen NALT sections were performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression profiles were distinct from those of the gut mucosal inductive site, Peyer’s patches (PP). The functionality of NALT HEV was determined using a Stamper-Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV. NALT lymphocytes exhibited a unique L-selectin expression profile, differing from both PP and peripheral lymph nodes. Finally, NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than α4β7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention.
Heat Shock Protein 70 Induced During Tumor Cell Killing Induces Th1 Cytokines and Targets Immature Dendritic Cell Precursors to Enhance Antigen Uptake
Authors: Todryk Stephen , Melcher Alan A. , Hardwick Nicola , Linardakis Emmanouela , Bateman Andrew , Colombo Mario P. , Stoppacciaro Antonella , Vile Richard G.
Published: December 2022

Previously, we reported that killing tumor cells in vivo with the HSV thymidine kinase/ganciclovir system generates potent antitumor immunity, determined in part by the mechanism by which the cells die and by the levels of inducible heat shock protein (hsp) expression induced during the process of cell death. Here, we show that induction of hsp70 expression induces an infiltrate of T cells, macrophages, and predominantly dendritic cells (DCs) into the tumors as well as an intratumoral profile of Th1 cytokine expression (IFN-γ, TNF-α, and IL-12) and enhances immunogenicity via a T cell-mediated mechanism. In addition, the protection conferred by hsp70 is both tumor and cell specific. We also demonstrate that hsp70 targets immature APC to make them significantly more able to capture Ags. This is likely to optimize cross-priming of the infiltrating APC with tumor Ags, which are simultaneously being released by the dying cells. In addition, using an Myc epitope-tagged hsp70 expression vector, we present evidence that hsp70 released from dying tumor cells is taken up directly into DCs and may, therefore, be involved in direct chaperoning of Ags into DCs. Taken together, our data suggest that hsp70 induction serves to signal the immune system of the presence of an immunologically relevant (dangerous) situation against which an immune reaction should be raised.
Th1 T Cell Responses to HIV-1 Gag Protein Delivered by aListeria monocytogenesVaccine Are Similar to Those Induced by Endogenous Listerial Antigens
Authors: Mata Marielena , Paterson Yvonne
Published: December 2022

Listeria monocytogenes is a facultative intracellular bacterium that lives and grows in the cytoplasm of the host cell. The hallmark of a listerial infection is a cell-mediated immune response to its own secreted virulence factors. Thus, L. monocytogenes vaccines engineered to secrete HIV proteins may be ideal vectors for boosting cellular immune responses against HIV. Using strains of L. monocytogenes that stably express and secrete HIV Gag (Lm-Gag) to deliver this Ag to the immune system, we have previously shown strong MHC class I-restricted cytotoxic T cell responses to this protein. In this study, we examine MHC class II-restricted T cell responses to HIV-Gag delivered by Lm-Gag. We demonstrate the induction of CD4+ T cells that are HIV-Gag specific and identify three epitopes in two strains of mice, BALB/c (H-2d) and C57BL/6 (H-2b), two of which are both H-2d and H-2b restricted, but are not immunodominant for both haplotypes. In addition, we show that the CD4+ T cells induced are of the Th1 phenotype that produce IFN-γ at levels similar to CD4+ T cells induced to endogenous listerial Ags. These studies suggest that chromosomally modified strains of L. monocytogenes may be useful as vaccine vectors for the induction of Th1 T cell responses against HIV.
Human NK Cells Express Endothelial Nitric Oxide Synthase, and Nitric Oxide Protects Them from Activation-Induced Cell Death by Regulating Expression of TNF-α
Authors: Furuke Keizo , Burd Parris R. , Horvath-Arcidiacono Judith A. , Hori Kotaro , Mostowski Howard , Bloom Eda T.
Published: December 2022

Although NO appears important in rodent immune responses, its involvement in the human immune system is unclear. We report that human NK cells express constitutive endothelial NO synthase mRNA and protein, but not detectable levels of inducible NO synthase. They produce NO following activation by coculture with target cells or cross-linking with anti-CD16 mAb, and production is increased in the presence of IL-2. N-monomethyl-l-arginine (l-NMA), a NOS inhibitor, partially inhibited NK cell lysis of four different target cells (<40% inhibition at 500 μM l-NMA), but not granule release following coculture with target cells, or Fas ligand induction following cross-linking with anti-CD16 mAb. However, l-NMA augmented apoptosis of NK cells induced by activation through CD16 ligation or coculture with K562. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), suppressed apoptosis of NK cells induced by CD16 cross-linking or coculture with target cells, suggesting that endogenous NO production is involved in protection of NK cells from activation-induced apoptosis, thereby maintaining NK activity. SNAP also suppressed, and l-NMA enhanced, expression of TNF-α, reported to be involved in activation-induced NK cell death, in response to CD16 cross-linking. Suppression of anti-CD16-induced apoptosis by SNAP was reversed by the addition of rTNF-α. DNA-binding activity of the transcription factor, NF-AT, which is involved in TNF-α induction upon ligation of CD16, was inhibited by SNAP and enhanced by l-NMA. Our results suggest that down-regulation of TNF-α expression, possibly due to suppression of NF-AT activation, is a mechanism by which endogenous NO protects NK cells from activation-induced apoptosis, and maintains lytic capacity.
Infection of HIV-1 Transgenic Mice withMycobacterium aviumInduces the Expression of Infectious Virus Selectively from a Mac-1-Positive Host Cell Population
Authors: Doherty T. Mark , Chougnet Claire , Schito Marco , Patterson Bruce K. , Fox Cecil , Shearer Gene M. , Englund George , Sher Alan
Published: December 2022

Infection of HIV-1-transgenic mice with Mycobacterium avium, a common opportunistic pathogen in AIDS patients, was shown to result in increased tissue expression of viral specific transcripts. Moreover, by coculturing splenocytes from the transgenic animals with human T cells it was possible to demonstrate that the elevation in HIV-1 mRNA triggered by M. avium infection reflects increased production of infectious virions. Viral immune activation was also shown to correlate with a marked elevation of p24 in supernatants of ex vivo-cultured tissues and, more importantly, in systemic increases in the HIV-1 protein in plasma. Interestingly, these tissue and systemic p24 responses were found to be differentially regulated. Thus, while in vitro p24 production by cultured splenocytes increased concurrently with bacterial loads during the first 6 wk of infection, levels of the Ag in plasma actually decreased. In situ localization experiments together with FACS analysis of HIV-1-expressing splenocytes indicated that virus production is restricted largely to cells of the monocyte/macrophage lineage. Indeed, in vitro p24 expression by cells from noninfected transgenic mice was up-regulated by polyclonal stimulation of macrophages but not T cells. Together these results underscore the importance of the macrophage reservoir in persistent virus expression and establish a convenient and relevant animal model for studying the factors responsible for immune activation of HIV-1 induced by mycobacterial as well as other common coinfections encountered by AIDS patients.
Adenovirus-Induced Liver Pathology Is Mediated Through TNF Receptors I and II but Is Independent of TNF or Lymphotoxin
Authors: Hayder Hikmat , Blanden Robert V. , Körner Heinrich , Riminton D. Sean , Sedgwick Jonathon D. , Müllbacher Arno
Published: December 2022

Mice infected with an adenovirus mutant in which the E3 region is deleted, including TNF-resistance genes, develop fatal liver pathology within 3–4 days after infection. At least 10-fold more wild-type virus was needed to cause comparable pathology. These results indicate that the E3 region is critically involved in modulating the pathogenesis of adenovirus infection and that TNF may play a role in liver damage. To explore the latter possibility, the course of disease was examined in infected mice lacking TNFR-I and/or TNFRII, TNF only, or both TNF and lymphotoxin-α. Only mice lacking both TNFRI and TNFRII were protected from the lethal affects of the mutant adenovirus. Mice deficient in TNF or TNF and lymphotoxin-α displayed the fatal pathology. This outcome is consistent with the existence of another related ligand that binds TNFRI/II to mediate liver damage during infection with this mutant.
Evidence That β Cell Death in the Nonobese Diabetic Mouse Is Fas Independent
Authors: Thomas Helen E. , Darwiche Rima , Corbett John A. , Kay Thomas W. H.
Published: December 2022

Recent studies suggest that Fas expression on pancreatic β cells may be important in the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse. To address this, pancreatic islets from NOD mice were analyzed by flow cytometry to directly identify which cells express Fas and Fas ligand (FasL) ex vivo and after in vitro culture with cytokines. Fas expression was not detected on β cells isolated from young (35 days) NOD mice. In vitro, incubation of NOD mouse islets with both IL-1 and IFN-γ was required to achieve sufficient Fas expression and sensitivity for islets to be susceptible to lysis by soluble FasL. In islets isolated from older (≥125 days) NOD mice, Fas expression was detected on a limited number of β cells (1–5%). FasL was not detected on β cells from either NOD or Fas-deficient MRLlpr/lpr islets. Also, both NOD and MRLlpr/lpr islets were equally susceptible to cytokine-induced cell death. This eliminates the possibility that cytokine-treated murine islet cells commit “suicide” due to simultaneous expression of Fas and FasL. Last, we show that NO is not required for cytokine-induced Fas expression and Fas-mediated apoptosis of islet cells. These findings indicate that β cells can be killed by Fas-dependent cytotoxicity; however, our results raise further doubts about the clinical significance of Fas-mediated β cell destruction because few Fas-positive cells were isolated immediately before the development of diabetes.
Involvement of Distinct Cellular Compartments in the Abnormal Lymphoid Organogenesis in Lymphotoxin-α-Deficient Mice and Alymphoplasia (aly) Mice Defined by the Chimeric Analysis
Authors: Matsumoto Mitsuru , Iwamasa Kikue , Rennert Paul D. , Yamada Takuji , Suzuki Rika , Matsushima Akemi , Okabe Masaru , Fujita Shigeru , Yokoyama Minesuke
Published: December 2022

Both lymphotoxin-α (LTα)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer’s patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTα-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTα expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTα. Rather, abnormal development of lymphoid organs in aly mice seems most likely due to the defective development of the incipient stromal cells of the LN and PP. Supporting this hypothesis, up-regulation of VCAM-1 on aly mouse embryonic fibroblasts by signals through LTβR, which is exclusively expressed by nonlymphoid cells, was disturbed. These studies demonstrate that LTα and the putative aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing the distinct cellular compartments, the former acting as a circulating ligand and the latter as a LTβR-signaling molecule expressed by the stroma of the lymphoid organs.