bluuebunny/crossref_metadata_2025 huggingface dataset:
Different Doses of Adenoviral Vector Expressing IL-12 Enhance or Depress the Immune Response to a Coadministered Antigen: the Role of Nitric Oxide
Authors: Lasarte Juan José , Corrales Fernando José , Casares Noelia , López-Díaz de Cerio Ascensión , Qian Cheng , Xie Xiaoming , Borrás-Cuesta Francisco , Prieto Jesús
Published: December 2022

Joint immunization with two recombinant adenoviruses, one expressing hepatitis C virus (HCV) core and E1 proteins and another expressing IL-12 (RAdIL-12), strongly potentiates cellular immune response against HCV Ags in BALB/c mice when RAdIL-12 was used at doses of 1 × 105–1 × 107 plaque-forming units. However, cellular immunity against HCV Ags was abolished when higher doses (1 × 108 plaque-forming units) of RAdIL-12 were used. This immunosuppressive effect was associated with marked elevation of IFN-γ and nitric oxide in the serum and increased cell apoptosis in the spleen. Administration of N-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase, to mice that received high doses of RAdIL-12 was lethal, whereas no apparent systemic toxicity by l-NAME was observed in those immunized with lower doses of the adenovirus. Interestingly, in mice immunized with recombinant adenovirus expressing core and E1 proteins of HCV in combination with RAdIL-12 at low doses (1 × 107 plaque-forming units), l-NAME inhibited T cell proliferation and CTL activity in response to HCV Ags and also production of Abs against adenoviral proteins. In conclusion, gene transfer of IL-12 can increase or abolish cell immunity against an Ag depending of the dose of the vector expressing the cytokine. IL-12 stimulates the synthesis of NO which is needed for the immunostimulating effects of IL-12, but apoptosis of T cells and immunosuppression ensues when IFN-γ and NO are generated at very high concentrations.
The Evolutionarily Conserved Sequence Upstream of the Human Ig Heavy Chain Sγ3 Region Is an Inducible Promoter: Synergistic Activation by CD40 Ligand and IL-4 Via Cooperative NF-κB and STAT-6 Binding Sites
Authors: Schaffer András , Cerutti Andrea , Shah Shefali , Zan Hong , Casali Paolo
Published: December 2022

Germline Cγ gene transcription is a crucial event in the process that leads to switch DNA recombination to IgG, but its regulation in the human is poorly understood. We took advantage of our monoclonal model of germinal center B cell differentiation, IgM+ IgD+ CL-01 cells, to define the role of the Iγ3 evolutionarily conserved sequence (ECS) in the germline transcriptional activation of the human Cγ3 gene. The Iγ3 ECS lies upstream of the major Iγ3 transcription initiation site and displays more than 90% identity with the corresponding human Iγ1, Iγ2, and Iγ4 regions. Reporter luciferase gene vectors containing the human γ3 ECS were used to transfect CL-01 cells, which have been shown to undergo Sμ→Sγ3 DNA recombination, upon engagement of CD40 by CD40 ligand (CD40L) and exposure to IL-4. In these transfected CL-01 cells, CD40:CD40L engagement and exposure to IL-4 synergistically induced γ3 ECS-dependent luciferase reporter gene activation. Targeted mutational analysis demonstrated that a tandem NF-κB/Rel binding motif is critical for the γ3 ECS responsiveness to both CD40L and IL-4, while a STAT-6-binding site is additionally required for IL-4 inducibility. Electrophoretic mobility shift assays showed that p50/p65/c-Rel and STAT-6 are effectively induced by CD40L and IL-4, respectively, and bind to specific DNA motifs within the ECS. These partially overlapping CD40L and IL-4 responsive elements are functionally cooperative as the disruption of one of them prevents synergistic promoter activation. Thus, the γ3 ECS is an inducible promoter containing cis elements that critically mediate CD40L and IL-4-triggered transcriptional activation of the human Cγ3 gene.
Apoptosis Induced by Granzyme B-Glycosaminoglycan Complexes: Implications for Granule-Mediated Apoptosis In Vivo
Authors: Galvin John P. , Spaeny-Dekking Liesbeth H. A. , Wang Baikun , Seth Prem , Hack C. Erik , Froelich Christopher J.
Published: December 2022

Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro.
Response toListeria monocytogenesin Mice Lacking MHC Class Ia Molecules
Authors: Seaman Michael S. , Pérarnau Béatrice , Lindahl Kirsten Fischer , Lemonnier Francois A. , Forman James
Published: December 2022

MHC class Ia-deficient mice (H2 Kb−/− Db−/−) inoculated with the intracellular pathogen Listeria monocytogenes (LM) displayed a three- to fourfold expansion of splenic CD8+ T cells 6 days following infection. Culture of these spleen cells in vitro gave rise to CTL that recognized LM-infected target cells and were restricted by the class Ib molecules, Qa1b and M3. Exposure of target cells to heat-killed LM (HKLM) rather than live bacteria did not result in CTL-mediated lysis. Target cells pulsed with three LM peptides known to bind M3, f-MIGWII, f-MIVTLF, and f-MIVIL, were recognized by effector cells from both B6 and Kb−/− Db−/− animals. In vivo analysis showed that B6 and Kb−/− Db−/− mice clear LM from the spleen and liver rapidly with similar kinetics, whereas TAP.1−/− mice, which are deficient in class Ia and Ib molecules, clear LM slowly upon infection. To establish the in vivo role of CD8+ T cells in Kb−/− Db−/− animals, we showed that depletion of such cells from the spleens of immune mice prevented the adoptive transfer of protective immunity to syngeneic recipients. Spleen cells from Kb−/− Db−/− mice were also capable of generating responses directed against syngeneic as well as allogeneic class Ia molecules in vitro. Thus, class Ia-deficient animals have a CD8+ T cell repertoire capable of recognizing both class Ia and class Ib molecules and can generate protective immunity to LM.
IgE Enhances Fcε Receptor I Expression and IgE-Dependent Release of Histamine and Lipid Mediators from Human Umbilical Cord Blood-Derived Mast Cells: Synergistic Effect of IL-4 and IgE on Human Mast Cell Fcε Receptor I Expression and Mediator Release
Authors: Yamaguchi Masao , Sayama Koichi , Yano Koji , Lantz Chris S. , Noben-Trauth Nancy , Ra Chisei , Costa John J. , Galli Stephen J.
Published: December 2022

We investigated the effects of IgE versus IL-4 on FcεRI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 μg/ml) much more strikingly enhanced surface expression of FcεRI than did IL-4 (at 0.1–100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance FcεRI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4−/− or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of FcεRI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on FcεRI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.
Neutralization of the CXC Chemokine, Macrophage Inflammatory Protein-2, Attenuates Bleomycin-Induced Pulmonary Fibrosis
Authors: Keane Michael P. , Belperio John A. , Moore Thomas A. , Moore Bethany B. , Arenberg Douglas A. , Smith Robert E. , Burdick Marie D. , Kunkel Steven L. , Strieter Robert M.
Published: December 2022

Few studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis. For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during bleomycin-induced pulmonary fibrosis are dependent in part upon an overexpression of the angiogenic CXC chemokine, macrophage inflammatory protein-2 (MIP-2). To test this hypothesis, we measured MIP-2 by specific ELISA in whole lung homogenates in either bleomycin-treated or control CBA/J mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a significant increase in the presence of MIP-2 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Neutralizing anti-MIP-2 Abs inhibited the angiogenic activity of day 16 bleomycin-treated lung specimens using an in vivo angiogenesis bioassay. Furthermore, when MIP-2 was depleted in vivo by passive immunization, bleomycin-induced pulmonary fibrosis was significantly reduced without a change in the presence of pulmonary neutrophils, fibroblast proliferation, or collagen gene expression. This was also paralleled by a reduction in angiogenesis. These results demonstrate that the angiogenic CXC chemokine, MIP-2, is an important factor that regulates angiogenesis/fibrosis in pulmonary fibrosis.
Differential Requirements for NF-κB and AP-1trans-Activation in Response to Minimal TCR Engagement by a Partial Agonist in Naive CD8 T Cells
Authors: Auphan Nathalie , Ghosh Sankar , Flavell Richard A. , Schmitt-Verhulst Anne-Marie
Published: December 2022

We investigated the basis for partial reactivity of naive CD8 T cells expressing an alloreactive transgenic TCR in response to a mutant alloantigen. When unstimulated APCs were used, IFN-γ as well as IL-2 and cell proliferation were observed in response to wild-type Ag, whereas mutant Ag induced only IFN-γ. DNA binding and reporter gene assays showed that the response to mutant Ag involved NF-κB, but not AP-1 activation, whereas wild-type Ag activated both transcription factors. Increasing the contribution of costimulatory signals by using LPS-activated APCs partially corrected the activation by mutant Ag, because proliferation and weak IL-2 production could be measured. This also led to AP-1 activation, albeit with delayed kinetics, in response to mutant Ag. To explain how engagement of the same TCR by distinct ligands results in different T cell responses, it may be proposed, in line with models stressing the importance of the kinetics of Ag/TCR interaction, that two types of signals be distinguished: a “fast” short-lived signal is sufficient to activate NF-κB; whereas a “slow” signal obtained after prolonged TCR engagement is required for AP-1 activation. Failure to activate AP-1 in limiting conditions (unstimulated mutant APC) was partially corrected by increasing costimulation.
Ly-49CB6 NK Inhibitory Receptor Recognizes Peptide-Receptive H-2Kb 1
Authors: Su Ruey-Chyi , Kung Sam Kam-Pun , Silver Elizabeth T. , Lemieux Suzanne , Kane Kevin P. , Miller Richard G.
Published: December 2022

NK-mediated cytotoxicity involves two families of receptors: activating receptors that trigger lysis of the target cells being recognized and inhibitory receptors specific primarily for MHC I on the target cell surface that can override the activating signal. MHC I molecules on the cell surface can be classified into molecules made stable by the binding of peptide with high affinity or unstable molecules potentially capable of binding high affinity peptide (hence, peptide receptive) and being converted into stable molecules. It has been previously shown that the Ly-49A inhibitory receptor recognizes stable Dd molecules. We show in this study that the inhibitory receptor Ly-49CB6 recognizes peptide-receptive Kb molecules, but does not recognize Kb molecules once they have bound high affinity peptide.
Role of Macrophage Lysosomal Enzymes in the Degradation of Nucleosomes of Apoptotic Cells
Authors: Odaka Chikako , Mizuochi Toshiaki
Published: December 2022

Although apoptotic cells are recognized and engulfed by macrophages via a number of membrane receptors, little is known about the fate of apoptotic cells after the engulfment. We observed in this study that nucleosomal DNA fragments of apoptotic cells disappeared when they were engulfed by the macrophage cell line J774.1 at 37oC. Pretreatment of J774.1 cells with chloroquine inhibited intensive DNA degradation, indicating that the cleavage of nucleosomal DNA fragments of apoptotic cells may take place in the lysosomes of J774.1. When apoptotic cells were exposed to a lysosome-rich fraction derived from J774.1 cells under an acidic condition, nucleosomal DNA fragments of apoptotic cells were no longer detectable by agarose gel electrophoresis. Additionally, we found that the lysosome-rich fraction of J774.1 cells contained an acid DNase that is similar to DNase II with respect to its m.w., optimal pH, and sensitivity to the inhibitors of DNase II. By exposure of apoptotic cells to the lysosomal-rich fraction, nucleosomal core histones of apoptotic cells were hydrolyzed along with degradation of nucleosomal DNA fragments. Addition of pepstatin A to the reaction buffer resulted in accumulation of ∼180-bp DNA fragments and inhibition of hydrolysis of nucleosomal core histones. Leupeptin or CA-074 partially inhibited the degradation of nucleosomal DNA fragments and core histones. These findings suggest that lysosomal enzymes of macrophages, e.g., DNase II-like acid DNase and cathepsins, are responsible for the degradation of nucleosomes of apoptotic cells.
CD4+ CD45RB Low-Density Cells from Untreated Mice Prevent Acute Allograft Rejection
Authors: Davies Joanna D. , O’Connor Eric , Hall DeShon , Krahl Troy , Trotter Joseph , Sarvetnick Nora
Published: December 2022

In the absence of therapy that suppresses the action of the immune system, the immune response to transplantation Ags results in rapid rejection of the transplant. The most successful mechanism so far described that achieves organ-specific immunological tolerance is that which controls peripheral tolerance to self-tissue. Until now, no similarities have been documented between the peripheral response to self-Ags and the response to transplantation Ags. CD4+ cells that express a high density of CD45RB (in the mouse) and CD45RC (in the rat) on their surface have been shown to cause a number of autoimmune disorders. In contrast, autoimmunity caused by the CD45RB high-density cells is inhibited by CD4+ CD45RB cells that express a low density of CD45RB (CD45RC in the rat). In this paper we show that CD4+ CD45RB high-density cells are sufficient to cause rejection of a MHC-mismatched pancreas allograft, whereas CD4+ CD45RB low-density cells are not. Unexpectedly, the CD45RB low-density cells prevent the CD45RBhigh expressing cells from causing rejection. These data suggest that the response to foreign tissue can be controlled in the same way as the response to self-tissue.
Role of Protein Kinase C-α in the Control of Infection by Intracellular Pathogens in Macrophages
Authors: St-Denis Anik , Caouras Vassiliki , Gervais Francine , Descoteaux Albert
Published: December 2022

The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In this study, we investigated the role of macrophage PKC-α in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections. To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-α. While phagocytosis of L. donovani promastigotes was not affected by DN PKC-α overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection. Intracellular survival of a L. donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection. However, IFN-γ-induced leishmanicidal activity was not affected by DN PKC-α overexpression. Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila. In contrast, DN PKC-α-overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L. pneumophila. Permissiveness to L. pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC-α-overexpressing RAW 264.7 clones. Collectively, these results support a role for PKC-α in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.
Impaired Anti-Viral T Cell Responses Due to Expression of the LY49A Inhibitory Receptor
Authors: Zajac Allan J. , Vance Russell E. , Held Werner , Sourdive David J. D. , Altman John D. , Raulet David H. , Ahmed Rafi
Published: December 2022

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.
Activation of T Cells Recognizing an Epitope of Heat-Shock Protein 70 Can Protect Against Rat Adjuvant Arthritis
Authors: Tanaka Shigehisa , Kimura Yuki , Mitani Akio , Yamamoto Genta , Nishimura Hitoshi , Spallek Ralf , Singh Mahavir , Noguchi Toshihide , Yoshikai Yasunobu
Published: December 2022

We have previously reported that CD4+ T cells recognizing a peptide comprising residues 234–252 of the heat shock protein (HSP)70 of Mycobacterium tuberculosis (M.tb) in the context of RT1.B MHC class II molecule emerged in the peritoneal cavity during the course of Listeria monocytogenes infection in rats and suppressed the inflammatory responses against listerial infection via IL-10 production. We report in this work that pretreatment with peptide 234–252 of HSP70 derived from M.tb suppressed the development of adjuvant arthritis (AA) in Lewis rats induced using heat-killed M.tb. T cells from rats pretreated with peptide 234–252 produced a significant amount of IL-10 in response to the epitope. T cells from rats pretreated with the peptide and immunized with M.tb produced the larger amount of IL-10 in response to the peptide, but only a marginal level of IFN-γ in response to purified protein derivative of M.tb. Administration of anti-IL-10 Ab partly inhibited the suppressive effect of pretreatment with peptide 234–252 on the development of AA. Furthermore, transfer of a T cell line specific for the epitope at the time of AA induction markedly suppressed AA. These findings suggested that T cells recognizing peptide 234–252 may play a regulatory role in inflammation during AA via the production of suppressive cytokines including IL-10.
Chemicals in Diesel Exhaust Particles Generate Reactive Oxygen Radicals and Induce Apoptosis in Macrophages
Authors: Hiura Timothy S. , Kaszubowski Martin P. , Li Ning , Nel Andre E.
Published: December 2022

There is increasing evidence that particulate air pollutants, such as diesel exhaust particles (DEP), potentiate chronic inflammatory processes as well as acute symptomatic responses in the respiratory tract. The mechanisms of action as well as the cellular targets for DEP remain to be elucidated. We show in this paper that the phagocytosis of DEP by primary alveolar macrophages or macrophage cell lines, RAW 264.7 and THP-1, leads to the induction of apoptosis through generation of reactive oxygen radicals (ROR). This oxidative stress initiates two caspase cascades and a series of cellular events, including loss of surface membrane asymmetry and DNA damage. The apoptotic effect on macrophages is cell specific, because DEP did not induce similar effects in nonphagocytic cells. DEP that had their organic constituents extracted were no longer able to induce apoptosis or generate ROR. The organic extracts were, however, able to induce apoptosis. DEP chemicals also induced the activation of stress-activated protein kinases, which play a role in cellular apoptotic pathways. The injurious effects of native particles or DEP extracts on macrophages could be reversed by the antioxidant, N-acetyl-cysteine. Taken together, these data suggest that organic compounds contained in DEP may exert acute toxic effects via the generation of ROR in macrophages.
Identification of CD66a and CD66b as the Major Galectin-3 Receptor Candidates in Human Neutrophils
Authors: Feuk-Lagerstedt Elisabeth , Jordan Elizabeth T. , Leffler Hakon , Dahlgren Claes , Karlsson Anna
Published: December 2022

The mammalian lectin galectin-3 is a potent stimulus of human neutrophils, provided that the receptor(s) for the lectin has been mobilized to the cell surface before activation. We have recently shown that the receptors for galectin-3 are stored in intracellular mobilizable granules. Here we show supportive evidence for this in that DMSO-differentiated (neutrophil-like) HL-60 cells, which lack gelatinase and specific granules, are nonresponsive when exposed to galectin-3. Neutrophil granules were subsequently used for isolation of galectin-3 receptors by affinity chromatography. Proteins eluted from a galectin-3-Sepharose column by lactose were analyzed on SDS-polyacrylamide gels and showed two major bands of 100 and 160 kDa and a minor band of 120 kDa. By immunoblotting, these proteins were shown to correspond to CD66a (160 kDa), CD66b (100 kDa), and lysosome-associated membrane glycoprotein-1 and -2 (Lamp-1 and -2; 120 kDa). The unresponsive HL-60 cells lacked the CD66 Ags but contained the Lamps, implying that neutrophil CD66a and/or CD66b may be the functional galectin-3 receptors. This conclusion was supported by the subcellular localization of the CD66 proteins to the gelatinase and specific granules in resting neutrophils.
The Antiinflammatory Sesquiterpene Lactone Parthenolide Inhibits NF-κB by Targeting the IκB Kinase Complex
Authors: Hehner Steffen P. , Hofmann Thomas G. , Dröge Wulf , Schmitz M. Lienhard
Published: December 2022

The transcription factor NF-κB is a key regulator of the cellular inflammatory and immune response. Therefore, components of the NF-κB-activating signaling pathways are frequent targets for antiinflammatory agents. This study shows that the sesquiterpene lactone parthenolide inhibits a common step in NF-κB activation by preventing the TNF-α-induced induction of IκB kinase (IKK) and IKKβ, without affecting the activation of p38 and c-Jun N-terminal kinase. Parthenolide impairs NF-κB-dependent transcription triggered by expression of TNFR-associated factor-2, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK1), and NF-κB-inducing kinase. This compound also prevents activation of both IKKs and DNA binding of NF-κB induced by MEKK and NF-κB-inducing kinase. Parthenolide targets a component of the IκB kinase complex without directly inhibiting IKKα, IKKβ, or MEKK1. Therefore, this sesquiterpene lactone could serve as a lead compound for the development of antiinflammatory remedies and is suitable as a molecular tool, allowing the dissection of TNF-α-derived signaling pathways leading to the activation of NF-κB, c-Jun N-terminal kinase, and p38.
Production of Metalloproteinase-7 (Matrilysin) by Human Myeloma Cells and Its Potential Involvement in Metalloproteinase-2 Activation
Authors: Barillé Sophie , Bataille Régis , Rapp Marie-José , Harousseau Jean-Luc , Amiot Martine
Published: December 2022

Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor spreading. Multiple myeloma (MM) is a plasma cell malignancy primarily localized within the bone marrow and characterized by its capacity to destroy bone matrix and to disseminate. We have reported recently that human myeloma cells were able to induce the conversion of pro-MMP-2 produced by the tumoral environment in its activated form. In the current study, we have investigated the mechanism involved in this process. We demonstrate that a soluble MMP constitutively produced by myeloma cells was responsible for pro-MMP-2 activation. Furthermore, we show that the soluble MMP, MMP-7, also known as matrilysin, was able to activate the MMP-2 produced in its latent form by bone marrow stromal cells. Finally, we demonstrate that myeloma cells constitutively produce MMP-7 with expected proteolytic activity. Our results suggest that MMP-7 produced by myeloma cells could participate in bone destruction and tumor spreading in MM, on one hand by its own proteolytic activity and on the other hand by its capacity to activate pro-MMP-2. These findings strengthen the idea that inhibition of MMP activity could represent an interesting therapeutic approach in MM.
A New Role for Platelet-Endothelial Cell Adhesion Molecule-1 (CD31): Inhibition of TCR-Mediated Signal Transduction
Authors: Newton-Nash Debra K. , Newman Peter J.
Published: December 2022

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils, and certain T cell subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3ε mAbs, anti-PECAM-1 mAbs, or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction.
Diacylglycerol Kinase Inhibition Prevents IL-2-Induced G1 to S Transition Through a Phosphatidylinositol-3 Kinase-Independent Mechanism
Authors: Flores Ignacio , Jones David R. , Ciprés Angel , Díaz-Flores Ernesto , Sanjuan Miguel Angel , Mérida Isabel
Published: December 2022

Stimulation via IL-2R ligation causes T lymphocytes to transit through the cell cycle. Previous experiments by our group have demonstrated that, in human T cells, IL-2 binding induces phosphatidic acid production through activation of the α isoform of diacylglycerol kinase. In this study, using the IL-2-dependent mouse T cell line CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced diacylglycerol kinase activation is found to block IL-2-induced late G1 to S transition without affecting cell viability. Herein, we demonstrate that diacylglycerol kinase inhibition has a profound effect on the induction of the protooncogenes c-myc, c-fos, and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL are not affected. When the IL-2-regulated cell cycle control checkpoints are examined in detail, we demonstrate that inhibition of diacylglycerol kinase activation prevents IL-2 induction of cyclin D3 without affecting p27 down-regulation. The strict control of cell proliferation exerted by phosphatidic acid through activation of diacylglycerol kinase is independent of other well-characterized IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt pathway, indicating the existence of a different and important mechanism to control cell division.
Antigen Concentration and Precursor Frequency Determine the Rate of CD8+ T Cell Tolerance to Peripherally Expressed Antigens
Authors: Morgan David J. , Kreuwel Huub T. C. , Sherman Linda A.
Published: December 2022

Expression of transgene-encoded proteins in the pancreatic islets can cause peripheral deletion of T cells. However, tolerance has not been observed in all transgenic models. It has been proposed that the determining factor for successful peripheral tolerance is the amount of Ag cross-presented by quiescent APCs. Using InsHA mice, which demonstrate peripheral tolerance to the influenza virus hemagglutinin (HA) expressed in the pancreatic islet β cells, we have investigated the consequences when different amounts of HA are expressed. As compared with InsHA mice that are heterozygous for the InsHA transgene, homozygous InsHA mice demonstrated enhanced activation and proliferation of Kd-restricted HA-specific CD8+ T cells in the pancreatic lymph nodes. However, despite such activation, insulitis was not observed, and the T cells were gradually functionally deleted. Deletion of these activated cells occurred much more rapidly in homozygous than in heterozygous InsHA mice. These data demonstrate that there is a direct correlation between the amount of HA expressed in the periphery, and both the degree of T cell proliferation in the pancreatic lymph nodes and the rate of tolerance of HA-specific CD8+ T cells. This strongly supports the hypothesis that activation of T cells through cross-presentation of peripheral Ags in a noninflammatory environment is an important part of the normal mechanism of tolerance to Ags expressed in the pancreatic islets.
TCR and IL-12 Receptor Signals Cooperate to Activate an Individual Response Element in the IFN-γ Promoter in Effector Th Cells
Authors: Zhang Feng , Nakamura Tetsuo , Aune Thomas M.
Published: December 2022

IFN-γ is a key regulatory cytokine of the immune system. Reporter transgenic mice expressing the luciferase gene under the control of separate TCR-response elements (TCR-RE) from the IFN-γ promoter or expressing the green fluorescent protein gene under the control of an IFN-γ “minigene” were employed to explore the basis for IL-12 regulation of IFN-γ gene transcription. In the absence of TCR stimulation, IL-12 did not activate the TCR-REs but did induce green fluorescent protein expression. TCR plus IL-12R stimulation of effector Th cells resulted in: 1) enhanced activation of the proximal, but not the distal, TCR-RE, and 2) increased induction of cJun-proximal TCR-RE complexes and c-Jun protein expression. Overexpression of cJun, but not cFos, increased activity of the proximal TCR-RE in T cells. These results suggest that IL-12R signaling affects IFN-γ gene transcription by at least two separate mechanisms; IL-12R signaling without TCR signaling targets promoter regions outside of the ∼100-bp IFN-γ TCR-RE, and IL-12R signaling also stimulates TCR-induced activity of the proximal TCR-RE.
M1204, a Novel 2′,5′ Oligoadenylate Synthetase with a Ubiquitin-Like Extension, Is Induced During Maturation of Murine Dendritic Cells
Authors: Tiefenthaler Martin , Marksteiner Rainer , Neyer Susanne , Koch Franz , Hofer Susanne , Schuler Gerold , Nussenzweig Michel , Schneider Rainer , Heufler Christine
Published: December 2022

A novel molecule expressed by spleen dendritic cells (DC) was isolated using a subtractive hybridization approach. The full-length M1204 clone has 3063 bp, with 1415 bp spanning a single open reading frame, coding for a protein of a predicted size of about 50 kDa. This sequence has strong homology to 2′, 5′ oligoadenylate synthetase and contains a ubiquitin-like domain. In Northern blot analyses the mRNA is strongly expressed in spleen DC, whereas, in bone marrow-derived DC, the amount of mRNA increases during the maturation process. None of the other leukocytes nor several hemopoietic cell lines tested express this mRNA, but clear expression occurs in many organs, the highest levels being in thymus, lung, and bone marrow. In situ hybridization, combined with immunocytochemical staining of tissue sections of lung and spleen, shows colocalization of M1204 with the 2A1 and NLDC DC markers. In Western blot experiments, an antiserum raised against the recombinant M1204 recognizes a single band in bone marrow-derived DC and in the lung. The expressed oligoadenylate synthetase domain is active in synthesizing 2′,5′ diadenylate, which by itself may inhibit viral protein synthesis and may also function as a substrate for 2′,5′ oligoadenylate synthetase. Since the oligoadenylate/RNase L system provides early protection against virus infection, we hypothesize that M1204 prevents virus-induced cell death in DC.
A Novel Lyn-Binding Peptide Inhibitor Blocks Eosinophil Differentiation, Survival, and Airway Eosinophilic Inflammation1, 2
Authors: Adachi Tetsuya , Stafford Susan , Sur Sanjiv , Alam Rafeul
Published: December 2022

Receptor antagonists block all receptor-coupled signaling pathways indiscriminately. We introduce a novel class of peptide inhibitors that is designed to block a specific signal from a receptor while keeping other signals intact. This concept was tested in the model of IL-5 signaling via Lyn kinase. We have previously mapped the Lyn-binding site of the IL-5/GM-CSF receptor common β (βc) subunit. In the present study, we designed a peptide inhibitor using the Lyn-binding sequence. The peptide was N-stearated to enable cellular internalization. The stearated peptide blocked the binding of Lyn to the βc receptor and the activation of Lyn. The lipopeptide did not affect the activation of Janus kinase 2 or its association with βc. The inhibitor blocked the Lyn-dependent functions of IL-5 in vitro (e.g., eosinophil differentiation from stem cells and eosinophil survival). It did not affect eosinophil degranulation. When applied in vivo, the Lyn-binding peptide significantly inhibited airway eosinophil influx in a mouse model of asthma. The lipopeptide had no effect on basophil histamine release or on the proliferation of B cells and T cells. To our knowledge, this is the first report on an inhibitor of IL-5 that blocks eosinophil differentiation, survival, and airway eosinophilic inflammation. This novel strategy to develop peptide inhibitors can be applied to other receptors.
Cutting Edge: RANTES Regulates Fas Ligand Expression and Killing by HIV-Specific CD8 Cytotoxic T Cells
Authors: Hadida Fabienne , Vieillard Vincent , Mollet Lucile , Clark-Lewis Ian , Baggiolini Marco , Debré Patrice
Published: December 2022

Based on the previous observation that RANTES mediates the cytotoxic activity of human HIV-specific CD8+ T cells via the chemokine receptor CCR3, we studied the effect of this chemokine on different effector CD8+ cytolytic cells requiring Fas/Fas ligand (FasL) or perforin-dependent pathway. In CTLs derived from PBMCs of HIV-infected patients, both the spontaneous and the RANTES-induced cytotoxicity were inhibited by anti-FasL neutralizing Abs. In contrast, allogeneic CTLs or NK cells killing through perforin were not affected by RANTES and anti-FasL Ab. Accordingly, RANTES enhanced the expression of FasL in a concentration- and time-dependent manner in HIV-specific CTLs, whereas anti-RANTES Ab decreased markedly FasL expression. Finally, cell surface expression of FasL protein in HIV-specific CTLs was also up-regulated by eotaxin, a selective ligand for CCR3. Our observations show that the action of RANTES via CCR3 is necessary to regulate FasL expression on HIV-specific CD8+ T cells that kill through the Fas/FasL pathway.
IL-18 Inhibits Diabetes Development in Nonobese Diabetic Mice by Counterregulation of Th1-Dependent Destructive Insulitis
Authors: Rothe Helga , Hausmann Andreas , Casteels Kristina , Okamura Hakuri , Kurimoto Masashi , Burkart Volker , Mathieu Chantal , Kolb Hubert
Published: December 2022

The development of type 1 diabetes in animal models is T cell and macrophage dependent. Islet inflammation begins as peripheral benign Th2 type insulitis and progresses to destructive Th1 type insulitis, which is driven by the innate immune system via secretion of IL-12 and IL-18. We now report that daily application of IL-18 to diabetes-prone female nonobese diabetic mice, starting at 10 wk of age, suppresses diabetes development (p < 0.001, 65% in sham-treated animals vs 33% in IL-18-treated animals by 140 days of age). In IL-18-treated animals, we detected significantly lower intraislet infiltration (p < 0.05) and concomitantly an impaired progression from Th2 insulitis to Th1-dependent insulitis, as evidenced from IFN-γ and IL-10 mRNA levels in tissue. The deficient progression was probably due to lesser mRNA expression of the Th1 driving cytokines IL-12 and IL-18 by the innate immune system (p < 0.05). Furthermore, the mRNA expression of inducible NO synthase, a marker of destructive insulitis, was also not up-regulated in the IL-18-treated group. IL-18 did not exert its effect at the levels of islet cells. Cultivation of islets with IL-18 affected NO production or mitochondrial activity and did not protect from the toxicity mediated by IL-1β, TNF-α, and IFN-γ. In conclusion, we show for the first time that administration of IL-18, a mediator of the innate immune system, suppresses autoimmune diabetes in nonobese diabetic mice by targeting the Th1/Th2 balance of inflammatory immune reactivity in the pancreas.
Distinct In Vivo and In Vitro Cytokine Profiles of Draining Lymph Node Cells in Acute and Chronic Phases of Contact Hypersensitivity: Importance of a Type 2 Cytokine-Rich Cutaneous Milieu for the Development of an Early-Type Response in the Chronic Phase
Authors: Kitagaki Hideki , Kimishima Momoko , Teraki Yuichi , Hayakawa Jun , Hayakawa Kazuhito , Fujisawa Shigeki , Shiohara Tetsuo
Published: December 2022

Although regional lymph nodes (LN) have been extensively studied as rich sources of effector T cells in contact hypersensitivity (CH), it remains unknown whether T cell responses in the LN reflect those in effector skin sites. We previously showed that repeated elicitation of CH results in a shift in the time course of Ag-specific CH from a delayed-type hypersensitivity response to an early-type response, a reflection of a shift in cutaneous cytokine expression from a type 1 to a type 2 profile. To investigate whether repeated elicitation of CH could also drive T cell development to the type 2 phenotype in the regional draining LN, sequential cytokine gene expression after hapten application was assessed during both the acute and the chronic phase of CH. In the draining LN the shift to type 2 cytokine production was also observed, but more mixed patterns of responses were induced than in the corresponding skin sites. The chronic LN cells (LNC), when stimulated in vitro, produced markedly lower levels of type 1 cytokines and higher levels of type 2 cytokines than the acute LNC. A successful passive transfer of an early-type response by the LNC was only induced in the recipient mice when the skin sites chronically treated with hapten were elicited. These results indicate that an early-type response by regional LNC would take place only in a milieu with sufficient levels of type 2 cytokines.
B Cell Responses to a Peptide Epitope. VIII. Immune Complex-Mediated Regulation of Memory B Cell Generation Within Germinal Centers
Authors: Nayak Bishnu P. , Agarwal Anshu , Nakra Pooja , Rao Kanury V. S.
Published: December 2022

Using an in vivo reconstitution assay, we examine here the role of immune complexes in both formation of germinal centers (GC) and processes that occur subsequently within. The presence of Ag, as immune complexes, was found not to constitute a limiting requirement for the initiation of GC formation. No detrimental effect either on numbers or sizes of the resulting GC was observed when Ag-containing immune complexes were omitted during reconstitution. Thus, both recruitment and proliferation of Ag-activated B cells within GC appear not to be limited by Ag concentrations. In contrast, the presence of immune complexes was observed to be obligatory for the generation of Ag-specific memory B cells. This optimally required immune complexes to be constituted by IgG-class Abs with epitope specificities that were homologous to those of the GC B cells. The GC reaction was also found to be characterized by an enhancement of Ab specificity for the homologous epitope. Although some improvement in specificity was noted in recall responses from immune complex-deficient GC, the presence of appropriate immune complexes served to further optimize the outcome. Here again, isotype and epitope-specificity of the Ab constituent in immune complexes proved to be important.
TNF-α Suppresses IFN-γ-Induced MHC Class II Expression in HT1080 Cells by Destabilizing Class IItrans-Activator mRNA
Authors: Han Yulong , Zhou Z-H. Lucy , Ransohoff Richard M.
Published: December 2022

Precise regulation of MHC class II gene expression is crucial for development and function of the immune system. Class II trans-activator (CIITA) has been shown to be required for constitutive and IFN-γ-induced MHC class II transcription. TNF-α is commonly coexpressed with IFN-γ during immune-mediated inflammatory responses and modulates IFN-γ-stimulated MHC class II expression. The effect of TNF-α on MHC class II expression depends on cell type and cellular differentiation state. We show here that TNF-α suppresses IFN-γ-induced CIITA mRNA accumulation, resulting in decreased MHC class II expression in human fibrosarcoma HT1080 cells. TNF-α also inhibits CIITA mRNA accumulation and protein expression in a tetracycline-regulated system without affecting promoter activity. CIITA mRNA, regulated by either IFN-γ or tetracycline, was destabilized in the presence of TNF-α, suggesting that TNF-α utilizes a distinct mechanism to suppress MHC class II expression in HT1080 cells. Consistent with this interpretation, TNF-α blocked IFN-γ-induced CIITA and MHC class II expression in mutant cells that are unresponsive to TGF-β or IFN-β. This is the first instance in which MHC class II expression is inhibited by destabilizing CIITA mRNA.
Apparent MHC-Independent Stimulation of CD8+ T Cells In Vivo During Latent Murine Gammaherpesvirus Infection
Authors: Coppola Michael A. , Flaño Emilio , Nguyen Phuong , Hardy Charles L. , Cardin Rhonda D. , Shastri Nilabh , Woodland David L. , Blackman Marcia A.
Published: December 2022

Like EBV-infected humans with infectious mononucleosis, mice infected with the rodent gammaherpesvirus MHV-68 develop a profound increase in the number of CD8+ T cells in the circulation. In the mouse model, this lymphocytosis consists of highly activated CD8+ T cells strikingly biased toward Vβ4 TCR expression. Moreover, this expansion of Vβ4+CD8+ T cells does not depend on the MHC haplotype of the infected animal. Using a panel of lacZ-inducible T cell hybridomas, we have detected Vβ4-specific T cell stimulatory activity in the spleens of MHV-68-infected mice. We show that the appearance and quantity of this activity correlate with the establishment and magnitude of latent viral infection. Furthermore, on the basis of Ab blocking studies as well as experiments with MHC class II, β2-microglobulin (β2m) and TAP1 knockout mice, the Vβ4-specific T cell stimulatory activity does not appear to depend on conventional presentation by classical MHC class I or class II molecules. Taken together, the data indicate that during latent infection, MHV-68 may express a T cell ligand that differs fundamentally from both conventional peptide Ags and classical viral superantigens.