bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Distinct Phospholipases C ofListeria monocytogenesInduce Ceramide Generation, Nuclear Factor-κB Activation, and E-Selectin Expression in Human Endothelial Cells
Authors: Schwarzer Nicole , Nöst Ralph , Seybold Joachim , Parida Shreemanta K. , Fuhrmann Oliver , Krüll Matthias , Schmidt Reinhold , Newton Robert , Hippenstiel Stefan , Domann Eugen , Chakraborty Trinad , Suttorp Norbert
Published: December 2022

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGDΔplcA and EGDΔplcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGDΔplcA or EGDΔplcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-κB activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-κB activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.
High Resolution Mapping of the B Cell Epitopes of Staphylokinase in Humans Using Negative Selection of a Phage-Displayed Antigen Library
Authors: Jenné Stéphane , Brepoels Katleen , Collen Désiré , Jespers Laurent
Published: December 2022

Staphylokinase (Sak), a 16-kDa protein secreted by Staphylococcus aureus, induces fibrin-specific thrombolysis in patients with thrombotic disorders. However, Sak also elicits high titers of neutralizing Abs that persist for several months and preclude its repeated use in humans. To identify the antigenic determinants of Sak recognized by humans, a phage-displayed library of Sak variants was selected for mutants that escape binding to an affinity matrix derivatized with patient-specific polyclonal anti-Sak Abs. Fifty-six escape Sak variants were identified after three selection cycles using human polyclonal anti-Sak IgGs obtained from four different patients. DNA sequencing revealed 213 amino acid substitutions, of which 73% were found at 25 positions clustered in eight discontinuous Sak antigenic segments. Although each antigenic segment was recognized to a variable extent by each patient antiserum, the main epitopes of Sak in all patients were roughly targeted to two large discontinuous areas covering 35% of the solvent-accessible surface of Sak. The antigenic area I comprises three segments centered on residues 66, 73, and 135, while the antigenic area II consists of four segments centered on positions 20, 95, 102, and 121. These results suggest that a secondary immune response against Sak can occur in patients, and confirm an initial site-directed mutagenesis study wherein amino acid Lys74 was shown to play a prominent antigenic role. Comprehensive mapping of the most relevant sites of Sak that are antigenic for humans will guide efforts to modulate the immunogenicity of this therapeutically important molecule.
Cyclosporin A Enhances IL-12 Production by CpG Motifs in Bacterial DNA and Synthetic Oligodeoxynucleotides
Authors: Redford Thomas W. , Yi Ae-Kyung , Ward Courtney T. , Krieg Arthur M.
Published: December 2022

Certain sequences of nucleotides (CpG motifs) in bacterial DNA or synthetic oligonucleotides (CpG DNA) promote the production of proinflammatory cytokines, including TNF-α, IFN-γ, IL-6, and IL-12. Here we demonstrate that the immunosuppressant cyclosporin A (CsA) unexpectedly enhanced CpG DNA-induced IL-12 production in murine splenocytes. CsA did not inhibit CpG DNA-induced TNF-α or IL-6 production, but decreased the production of IFN-γ by CpG DNA. Upon examining mechanisms by which CsA increases IL-12 production, we found that CpG DNA can also induce IL-10 production in B cells and that this production was sensitive to CsA. IL-10 has anti-inflammatory effects and can reduce the production of IL-12. To determine the possible role of CsA-modulated IL-10 production in mediating the increased IL-12 levels, splenocytes from IL-10 gene-disrupted mice (IL-10 −/−) and splenocytes cultured in anti-IL-10 Ab were studied. CpG DNA-stimulated IL-10 (−/−) splenocytes demonstrated no increase in IL-12 levels in the presence of CsA. Anti-IL-10 Ab treatment of normal splenocytes increased the magnitude of CpG DNA-induced IL-12 production to that seen with CsA. These results suggest that CpG DNA induces CsA-sensitive IL-10 production in B cells and that IL-10 acts as a negative feedback regulator of CpG DNA-induced IL-12 production.
Factor Xa Induces Cytokine Production and Expression of Adhesion Molecules by Human Umbilical Vein Endothelial Cells
Authors: Senden Nicole H. M. , Jeunhomme Trudi M. A. A. , Heemskerk Johan W. M. , Wagenvoord Rob , van’t Veer Cornelis , Hemker H. Coenraad , Buurman Wim A.
Published: December 2022

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5–80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1α and TNF-α were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from γ-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and γ-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.
Endothelial Cell Activation by Leukocyte Microparticles
Authors: Mesri Mehdi , Altieri Dario C.
Published: December 2022

The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 ± 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 ± 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-α, IL-1β, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or β2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.
Uncoupling Activation-Dependent HS1 Phosphorylation from Nuclear Factor of Activated T Cells Transcriptional Activation in Jurkat T Cells: Differential Signaling Through CD3 and the Costimulatory Receptors CD2 and CD28
Authors: Hutchcroft Jill E. , Slavik Jacqueline M. , Lin Huamao , Watanabe Takeshi , Bierer Barbara E.
Published: December 2022

CD3, CD2, and CD28 are functionally distinct receptors on T lymphocytes. Engagement of any of these receptors induces the rapid tyrosine phosphorylation of a shared group of intracellular signaling proteins, including Vav, Cbl, p85 phosphoinositide 3-kinase, and the Src family kinases Lck and Fyn. Ligation of CD3 also induces the tyrosine phosphorylation of HS1, a 75-kDa hematopoietic cell-specific intracellular signaling protein of unknown function. We have examined changes in HS1 phosphorylation after differential stimulation of CD3, CD2, and CD28 to elucidate its role in T cells and to further delineate the signaling pathways recruited by these receptors. Unlike ligation of CD3, stimulation with anti-CD28 mAb or CHO cells expressing the CD28 ligands CD80 or CD86 did not lead to tyrosine phosphorylation of HS1 in Jurkat T cells. Additionally, no tyrosine phosphorylation of HS1 was induced by mitogenic pairs of anti-CD2 mAbs capable of activating the transcription factor NFAT (nuclear factor of activated T cells). Costimulation through CD28 and/or CD2 did not modulate the CD3-dependent phosphorylation of HS1. In vivo studies indicated that CD3-induced HS1 phosphorylation was dependent upon both the Src family tyrosine kinase Lck and the tyrosine phosphatase CD45, did not require MEK1 kinase activity, and was regulated by protein kinase C activation. Thus, although CD3, CD28, and CD2 activate many of the same signaling molecules, they differed in their capacity to induce the tyrosine phosphorylation of HS1. Furthermore, activation-dependent tyrosine phosphorylation of HS1 was not required for NFAT transcriptional activation.
Self-Restricted Dual Receptor Memory T Cells
Authors: Lee William T. , Shiledar-Baxi Vaibhavi , Winslow Gary M. , Mix Denise , Murphy Donal B.
Published: December 2022

Enhanced immune responses during secondary exposure to Ag result from the development of memory cells. In the present report we show that stimulation through one receptor on dual receptor CD4 cells can promote the generation of T cells capable of giving a memory response through the second receptor, even though the cells had not been previously exposed to the Ag recognized by the second receptor. Cloned cells generated from dual receptor memory T cells proliferated and secreted the same lymphokines after stimulation with either Ag. Independent recognition of both Ags by distinct TCRs was shown by production of variants that had lost either Ag specificity along with the corresponding TCR. Recognition of both Ags is MHC restricted, since the cells recognize Ag presented by self, but not non-self, MHC class II molecules. These results raise the possibility that one potential mechanism of maintaining specific memory to a given Ag is through stimulation by an unrelated Ag via the second TCR.
Evidence for an Important Interaction Between a Complement-Derived CD21 Ligand on Follicular Dendritic Cells and CD21 on B Cells in the Initiation of IgG Responses
Authors: Qin Dahui , Wu Jiuhua , Carroll Michael C. , Burton Gregory F. , Szakal Andras K. , Tew John G.
Published: December 2022

The addition of Ags to mononuclear leukocyte cultures typically elicits modest Ab responses, implying that cosignals beyond those provided by T cells and macrophages may be needed. Recently, we reported that Ab responses could be dramatically enhanced (10–1000-fold) by the addition of follicular dendritic cells (FDC), suggesting that FDC may provide an important costimulatory signal. This result prompted a study of molecules involved in FDC-mediated enhancement of Ab responses stimulated by specific Ag with memory T and B cells or nonspecifically by the addition of LPS. In this study, we report evidence supporting the concept that FDC bear a ligand that engages complement receptor II (CR2 or CD21) on B cells and provides a critical cosignal for both Ag-specific and polyclonal responses. A blockade of the CR2 ligand on FDC by the use of soluble CR2 or a blockade of CR2 on B cells by use of CR2 knockout mice (or B cells with CR2 blocked) reduced Ab responses from the μg/ml to the ng/ml range (10–1000-fold reductions). FDC from C3 knockout mice, which cannot generate the CR2-binding fragments (iC3b, C3d, and C3dg), were unable to provide costimulatory activity, suggesting the CR2 ligand on FDC consists of C3 fragments. FDC trap complement-activating Ag-Ab complexes, and it appears that FDC present B cells with both specific Ag to engage B cell receptors and a CR2 ligand to engage B cell-CR2. In short, optimal induction of specific Ab responses appears to require the combination of specific Ag and costimulatory molecules from both T cells and FDC.
CD99 Engagement on Human Peripheral Blood T Cells Results in TCR/CD3-Dependent Cellular Activation and Allows for Th1-Restricted Cytokine Production
Authors: Waclavicek Martina , Majdic Otto , Stulnig Thomas , Berger Markus , Sunder-Plassmann Raute , Zlabinger Gerhard J. , Baumruker Thomas , Stöckl Johannes , Ebner Christof , Knapp Walter , Pickl Winfried F.
Published: December 2022

We have assessed the functional effect of CD99 engagement on resting human peripheral blood (PB) T cells. CD99, as detected by the mAb 3B2/TA8, is constitutively expressed on all PB T cells and becomes further up-regulated upon cellular activation. In this study we demonstrate that cross-linking of the CD99 molecule with the agonistic mAb 3B2/TA8 cooperates with suboptimal TCR/CD3 signals, but not with phorbol ester, ionomycin, or CD28 mAb stimulation, to induce proliferation of resting PB T cells. Comparable stimulatory effects were observed with the CD99 mAb 12E7. Characterization of the signaling pathways involved revealed that CD99 engagement leads to the elevation of intracellular Ca2+, which is dependent on the cell surface expression of the TCR/CD3 complex. No CD99 mAb-induced calcium mobilization was observed on TCR/CD3-modulated or TCR/CD3-negative T cells. To examine the impact of CD99 stimulation on subsequent cytokine production by T cells, we cross-linked CD99 molecules in the presence of a suboptimal TCR/CD3 trigger followed by determination of intracellular cytokine levels. Significantly, T cell lines as well as Th1 and Th0 clones synthesized TNF-α and IFN-γ after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-γ when stimulated in a similar fashion. We conclude that CD99 is a receptor that mediates TCR/CD3-dependent activation of resting PB T cells and specifically induces Th1-type cytokine production in polyclonally activated T cell lines, Th1 and Th0 clones.
Soluble VCAM-1 Induces Chemotaxis of Jurkat and Synovial Fluid T Cells Bearing High Affinity Very Late Antigen-4
Authors: Kitani Atsushi , Nakashima Noriko , Izumihara Tomomaro , Inagaki Masaki , Baoui Xu , Yu Su , Matsuda Takemasa , Matsuyama Takami
Published: December 2022

It has been shown that cells with high affinity very late Ag (VLA)-integrins have up-regulated expression of a β1-subunit epitope, which is detected by 15/7 mAb. In this study, we demonstrate that soluble VCAM-1 (sVCAM-1) exhibits chemotactic activity of T cells with high affinity VLA-4 against VCAM-1, such as Jurkat T cells and IL-2-dependent T cells. Moreover, we found that T cells in the synovial fluid show high basal migration in the absence of sVCAM-1, compared with peripheral blood T cells in patients with rheumatoid arthritis. Among T cells in the synovial fluid, CD45RO+ memory T cells, in response to sVCAM-1, showed a much higher than basal migratory response when compared with CD45RA+ naive cells, while no significant difference was observed between CD4+ and CD8+ T cells. The chemotactic activity of sVCAM-1 is inhibited in the presence of anti-VCAM-1 and anti-VLA-4, which interfered with the binding between VCAM-1 and VLA-4. Inhibition studies using various kinase inhibitors (C3 exoenzyme, KN62, and H7) show that Rho, Ca2+/calmodulin-dependent kinase II, and protein kinase C are involved in signal transduction in sVCAM-1-induced chemotaxis, respectively, whereas tyrosine kinase seems to play a lesser role, since genistein showed only partial inhibition of T cell chemotaxis. Western blot analysis using an anti-phospho-serine mAb (MO82) reveals that Ser82 in the vimentin is phosphorylated specifically by Ca2+/calmodulin-dependent kinase II through sVCAM-1 activation in the IL-2 dependent T cells. Collectively, by inducing migration and recruitment of T cells through several kinase activations, sVCAM-1 contributes to the development of the inflammation of synovial lesion.
Contributions of Direct and Indirect T Cell Alloreactivity During Allograft Rejection in Mice
Authors: Benichou Gilles , Valujskikh Anna , Heeger Peter S.
Published: December 2022

The immune response to transplanted allogeneic tissues is mediated by T cells that recognize donor histocompatibility Ags either via direct (donor MHC and peptides) or indirect (recipient MHC and donor-derived peptides) allorecognition pathways. The relative contribution of each of these pathways to allograft rejection remains largely unknown. To address this, we used an enzyme-linked immunospot assay to define the frequency and cytokine phenotype of T cells responding via direct and indirect pathways to alloantigens at various time points following placement of allogeneic B10.A skin grafts on BALB/c recipient mice. During acute graft rejection >90% of the anti-B10.A T cell repertoire was directed toward intact donor MHC molecules, while T cells recognizing indirectly presented, donor-derived peptides accounted for <10%. This indirect response was comprised of reactivity toward both MHC-derived and, to a lesser extent, minor Ag-derived determinants. The direct and indirect alloresponses were predominantly detected in recipient lymph nodes and were mediated by T cells displaying a mixed type 1/type 2 cytokine phenotype. Six weeks following rejection, however, the memory allospecific T cell response became predominant in the recipient spleen, with only minimal activity detectable in the draining lymph nodes. This work provides a new approach for analysis of the immunophysiology of allograft rejection and should be useful for monitoring immune responses to graft Ags in human transplant recipients.
CD1-Restricted Microbial Lipid Antigen-Specific Recognition Found in the CD8+ αβ T Cell Pool
Authors: Rosat Jean-Pierre , Grant Ethan P. , Beckman Evan M. , Dascher Christopher C. , Sieling Peter A. , Frederique Daphney , Modlin Robert L. , Porcelli Steven A. , Furlong Stephen T. , Brenner Michael B.
Published: December 2022

It is generally accepted that TCR αβ+ CD8+ T cells recognize immunogenic peptides bound to MHC-encoded class I molecules. This recognition is a major component of the cellular response mediating immune protection and recovery from viral infections and from certain intracellular bacterial infections. Here, we report two human CD8+ TCR αβ+ T cell lines specific for Mycobacterium tuberculosis Ags presented in the context of CD1a or CD1c Ag-presenting molecules. These T cells recognize lipid Ags and display cytotoxicity as well as strong Th cell type I cytokine responses. By extending presentation by the CD1 system to the major TCR αβ+ CD8+ T cell pool, this system gains wider applicability beyond the double negative subset of T cells previously shown to have this reactivity. This implies that previous assumptions about the role of CD8+ T cells in microbial immunity may require revision as the relative proportions of CD1-restricted and MHC class I-restricted CD8+ T cells are further defined.
Phagocytosis Triggers Macrophage Release of Fas Ligand and Induces Apoptosis of Bystander Leukocytes
Authors: Brown Simon B. , Savill John
Published: December 2022

Human monocyte/macrophages (Mφ) exposed to nonparticulate stimuli can express cell surface Fas ligand (FasL) and release active soluble FasL (sFasL). We now report that monocyte/Mφ-ingesting opsonized zymosan released sFasL and conditioned supernatants so that these triggered Fas-mediated apoptosis of “bystander” monocytes and FasL-negative neutrophils. Furthermore, identical results were seen with Mφ taking up apoptotic neutrophils, whereas medium conditioned by Mφ phagocytizing latex beads had no proapoptotic effects upon neutrophils despite the presence of sFasL. These data suggest the hitherto unrecognized existence of a feedback loop requiring soluble factors in addition to sFasL that may promote resolution of inflammation-phagocytic clearance of apoptotic cells leading to Fas-mediated killing of bystander leukocytes by phagocytizing macrophages.
Accelerated Rejection of FAS Ligand-Expressing Heart Grafts
Authors: Takeuchi Takumi , Ueki Tetsuo , Nishimatsu Hiroaki , Kajiwara Takahiro , Ishida Toshimitsu , Jishage Kou-ichi , Ueda Otoya , Suzuki Hiroshi , Li Baoxing , Moriyama Nobuo , Kitamura Tadaichi
Published: December 2022

The Fas/Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Recently, there has been renewed interest in immune privilege, as it was shown that two privileged sites (the eye and testes) constitutively express FasL, which kills lymphoid cells that invade these areas. We have established murine FasL-transgenic mice (B6) under the control of the cardiac α-myosin heavy chain promotor, and transplanted FasL-expressing F1(B6 × C3H/HeJ) heart grafts into syngeneic (F1) and allogeneic (C3H/HeJ) recipients. FasL-expressing F1 heart allografts placed in C3H/HeJ recipients as well as FasL-expressing F1 isografts placed in nontransgenic and FasL-transgenic F1 were more rapidly rejected, and their survival was much shorter than that of nontransgenic control F1 allografts placed in C3H/HeJ. Native control and FasL-expressing hearts looked normal in mice up to 8 wk of age on hematoxylin-eosin staining. Control heart allografts undergoing ordinally acute rejection showed moderate focal lymphocyte infiltrates, while FasL-expressing F1 allografts and isografts showed massive hemorrhage, edema, and massive neutrophil infiltration as early as 1 day after transplantation. In conclusion, FasL expression and surgical procedure (ischemia/reperfusion) were synergistic in the induction of accelerated heart graft rejection, while allogenicity was not necessary. It may be necessary to find ways of controlling neutrophilic reaction/apoptosis in infiltrating lymphocytes to use FasL in clinical organ transplantation.
In the Normal Repertoire of CD4+ T Cells, a Single Class II MHC/Peptide Complex Positively Selects TCRs with Various Antigen Specificities
Authors: Chmielowski Bartosz , Muranski Pawel , Ignatowicz Leszek
Published: December 2022

In the thymus, immature T cells are positively and negatively selected by multiple interactions between their Ag receptors (TCRs) and self MHC/peptide complexes expressed on thymic stromal cells. Here we show that in the milieu of negative selection on physiological self class II MHC/peptide complexes (Abwt), a single class II/peptide complex AbEp52–68 positively selects a number of TCRs with various Ag specificities. This TCR repertoire is semidiverse and not biased toward Ep-like Ags. Our finding implies that the degeneracy of positive selection for peptide ligands exceeds peptide-specific negative selection and is essential to increase the efficiency and diversity of the repertoire so that T cells with the same Ag specificity can be selected by different self MHC/peptide complexes.
A Single Intramuscular Injection with an Adenovirus- Expressing IL-12 Protects BALB/c Mice AgainstLeishmania majorInfection, While Treatment with an IL-4-Expressing Vector Increases Disease Susceptibility in B10.D2 Mice
Authors: Gabaglia Claudia Raja , Pedersen Brian , Hitt Mary , Burdin Nicolas , Sercarz Eli E. , Graham Frank L. , Gauldie Jack , Braciak Todd A.
Published: December 2022

Experimental infection of the susceptible BALB/c (H-2d) mouse with the intracellular parasite Leishmania major induces a predominant Th2-type T cell response that eventually leads to death. In contrast, the resistant B10.D2 (H-2d) strain develops Th1 cells that control parasite replication and disease. In this study, we tested the ability of a recombinant adenovirus vector-expressing IL-12 to skew the immune response in a Th1 direction and prevent leishmaniasis in susceptible mice. We report that BALB/c mice treated with the Ad5IL-12 vector on the same day as parasitic challenge are significantly protected against leishmaniasis and acquired long-lasting immunity, because upon rechallenge with L. major parasites they were resistant to disease. The vector-derived IL-12 expression was transient and highly localized to the tissue after i.m. injection; it caused an increase in the number of Ag-specific IFN-γ-secreting lymphocytes and enhanced NK cell activity in the draining popliteal node. In contrast, resistant B10.D2 mice given i.m. injections with a recombinant adenovirus-expressing IL-4 displayed greater susceptibility to disease, and severe lesions were produced in some of the infected animals. These results suggest the potential use of recombinant adenoviruses expressing cytokines as potent immunomodulatory agents for the generation of protective immune responses against intracellular pathogens.
Multiple Signal Transduction Pathways Regulate TNF-Induced Actin Reorganization in Macrophages: Inhibition of Cdc42-Mediated Filopodium Formation by TNF
Authors: Peppelenbosch Maikel , Boone Elke , Jones Gareth E. , Deventer S.J.H. van , Haegeman Guy , Fiers Walter , Grooten Johan , Ridley Anne J.
Published: December 2022

TNF is known to regulate macrophage (Mφ) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent filopodium formation, and stimulated macropinocytosis. Using a panel of TNFR-1 mutants, the regions of the receptor required for each of these responses were mapped. The decrease in F-actin required both the death domain and the membrane proximal part of the receptor, whereas inhibition of filopodium formation and increased pinocytosis were only dependent upon a functional death domain. When the TNF-induced decrease in F-actin was inhibited using either receptor mutants or the compound D609, TNF-stimulated actin reorganization at the cell cortex became apparent. This activity was dependent upon the FAN-binding region of TNFR-1. We conclude that different domains of TNFR-1 mediate distinct changes in the Mφ cytoskeleton, and that the ability of TNF to inhibit Mφ chemotaxis may be due to decreased filopodium formation downstream of Cdc42.
Endotoxin Down-Regulates T Cell Activation by Antigen-Presenting Liver Sinusoidal Endothelial Cells
Authors: Knolle Percy A. , Germann Tieno , Treichel Ulrich , Uhrig Anja , Schmitt Edgar , Hegenbarth Silke , Lohse Ansgar W. , Gerken Guido
Published: December 2022

Endotoxin is physiologically present in portal venous blood at concentrations of 100 pg/ml to 1 ng/ml. Clearance of endotoxin from portal blood occurs through sinusoidal lining cells, i.e., Kupffer cells, and liver sinusoidal endothelial cells (LSEC). We have recently shown that LSEC are fully efficient APCs. Here, we studied the influence of endotoxin on the accessory function of LSEC. Incubation of Ag-presenting LSEC with physiological concentrations of endotoxin lead to ≥80% reduction of the accessory function, measured by release of IFN-γ from CD4+ T cells. In contrast, conventional APC populations rather showed an increase of the accessory function after endotoxin treatment. Inhibition of the accessory function in LSEC by endotoxin was not due to lack of soluble costimulatory signals, because neither supplemental IL-1β, IL-2, IFN-γ, or IL-12 could rescue the accessory function. Ag uptake was not influenced by endotoxin in LSEC. However, we found that endotoxin led to alkalinization of the endosomal/lysomal compartment specifically in LSEC but not in bone marrow macrophages, which indicated that Ag processing, i.e., proteolytic cleavage of protein Ags into peptide fragments, was affected by endotoxin. Furthermore, endotoxin treatment down-regulated surface expression of constitutively expressed MHC class II, CD80, and CD86. In conclusion, it is conceivable that endotoxin does not alter the clearance function of LSEC to remove gut-derived Ags from portal blood but specifically affects Ag processing and expression of the accessory molecules in these cells. Consequently, Ag-specific immune responses by CD4+ T cells are efficiently down-regulated in the hepatic microenvironment.
Germline Structure and Differential Utilization ofIghaandIghbVH10 Genes
Authors: Whitcomb Elizabeth A. , Haines Brian B. , Parmelee Allen P. , Pearlman Adam M. , Brodeur Peter H.
Published: December 2022

Ab heavy chains encoded by mouse VH10 genes have been of particular interest due to their frequent association with DNA binding. We reported previously that VH10 sequences are over-represented in the preimmune repertoire considering the apparent number of germline-encoded VH10 gene segments. In this report, we show that the VH10 family consists of three and two germline genes in the Igha and Ighb haplotypes, respectively. The complete nucleotide sequences of these five genes, including promoters and recombination signal sequences, were determined and allow unambiguous assignment of allelic relationships. The usage of individual VH10 genes varied significantly and ranged from 0.2% to an extraordinary 7.2% of the VH genes expressed by splenic B cells. Since the promoter and recombination signal sequence elements of all five VH10 genes are identical, we suggest that the few amino acid differences encoded by these five germline VH10 genes determine their representation in the preimmune repertoire. Rearrangements of the most frequently used VH10 gene have an apparent bias for histidine at position 95 of complementarity-determining region-3 (CDR3). These CDR3s are also biased for asparagine, an amino acid associated with the CDRs of DNA binding Abs. Together, these results suggest that high VH10 gene use is the result of B cell receptor-mediated selection and may involve DNA and/or ligands that share antigenic features with DNA.
Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Enhance IL-10 Production by Murine Macrophages: In Vitro and In Vivo Studies
Authors: Delgado Mario , Munoz-Elias Ernesto J. , Gomariz Rosa P. , Ganea Doina
Published: December 2022

Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, and the structurally related pituitary adenylate cyclase-activating polypeptide (PACAP) act as potent anti-inflammatory agents that inhibit the function of activated macrophages and TH cells. Previous reports showed that VIP/PACAP inhibit IL-6 and TNF-α production in LPS-stimulated macrophages. The present study reports on the effect of VIP/PACAP on IL-10 production. Although VIP/PACAP do not induce IL-10 by themselves, they enhance IL-10 production in LPS-stimulated macrophages. The specific VPAC1 receptor mediates the stimulatory effect of VIP/PACAP, and cAMP is the major second messenger involved. VIP/PACAP increase IL-10 mRNA in LPS-stimulated cells, and the effect of transcriptional and protein synthesis inhibitors indicates de novo IL-10 production. Electromobility shift assays show that VIP/PACAP induce an increase in nuclear cAMP response element (CRE)-binding complexes, with CRE binding protein as the major active component. Treatments with either a VPAC1 antagonist or a protein kinase A inhibitor abolish IL-10 stimulation and, concomitantly, the increase in CRE binding. Effects similar to the in vitro stimulation of IL-10 were obtained in vivo in mice treated with LPS and VIP or PACAP. The neuropeptides induce increased levels of IL-10 in both serum and peritoneal fluid, and increased expression of the IL-10 mRNA in peritoneal exudate cells. The stimulation of IL-10 production in activated macrophages represents a novel anti-inflammatory activity of VIP and PACAP, which presumably acts in vivo in conjunction with the inhibition of proinflammatory cytokines such as IL-6 and TNF-α to reduce the magnitude of the immune response.
Fas Ligand-Mediated Exocrinopathy Resembling Sjögren’s Syndrome in Mice Transgenic for IL-10
Authors: Saito Ichiro , Haruta Kumiko , Shimuta Misa , Inoue Hiroko , Sakurai Hiroshi , Yamada Koichi , Ishimaru Naozumi , Higashiyama Hiroyuki , Sumida Takayuki , Ishida Hiroshi , Suda Takashi , Noda Tetsuo , Hayashi Yoshio , Tsubota Kazuo
Published: December 2022

Although IL-10 has been implicated in the pathogenesis of several autoimmune diseases, the mechanisms by which this cytokine mediates inflammatory lesions remain to be elucidated. Exocrine gland destruction is an important early step in the development of Sjögren’s syndrome. To better understand the role of IL-10 in Sjögren’s syndrome, we made transgenic mice in which the mouse IL-10 gene was regulated by the human salivary amylase promoter. Transgenic expression of IL-10 induced apoptosis of glandular tissue destruction and lymphocyte infiltration consisting primarily of Fas-ligand (FasL)+ CD4+ T cells, as well as in vitro up-regulation of FasL expression on T cells. These data suggest that overexpression of IL-10 in the glands and their subsequent Fas/FasL-mediated bystander tissue destruction is a causal factor in the development of this disease.
The Porcine 2A10 Antigen Is Homologous to Human CD163 and Related to Macrophage Differentiation
Authors: Sánchez Carmen , Doménech Nieves , Vázquez Jesús , Alonso Fernando , Ezquerra Angel , Domínguez Javier
Published: December 2022

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5–50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10− monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10− monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10− cells. LPS-stimulated 2A10+ and 2A10− monocytes both produce proinflammatory cytokines (TNF-α and IL-1α), but antiinflammatory IL-10 is only detected on the latter population. When 2A10− monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10− circulating monocytes.
Augmentation of the CD8+ T Cell Response by IFN-γ in IL-12-Deficient Mice DuringToxoplasma gondiiInfection
Authors: Ely Kenneth H. , Kasper Lloyd H. , Khan Imtiaz A.
Published: December 2022

The importance of IFN-γ in regulating the host CD8+ T cell response during microbial infection has not been delineated. Mice deficient for the p40 chain of the IL-12 heterodimer have impaired IFN-γ production and are susceptible to infection with the intracellular parasite Toxoplasma gondii. The administration of exogenous IFN-γ to parasite-infected p40−/− mice increases survival and up-regulates the depressed CD8+ T cell response following infection. CD8+ T cells isolated from cytokine-treated p40−/− mice exhibit an increase in both precursor CTL frequency and IFN-γ production compared with untreated controls. The enhancement of the CD8+ T cell response is independent of CD4+ T cell help. These CD8+ T cells induce protective immunity against a lethal challenge when adoptively transferred into naive p40−/− and IFN-γ−/− mice. These observations indicate that IFN-γ can regulate the CD8+ T cell response during T. gondii infection.
Autocrine Regulation of IL-12 Receptor Expression Is Independent of Secondary IFN-γ Secretion and not Restricted to T and NK Cells
Authors: Thibodeaux Deborah K. , Hunter Sharon E. , Waldburger Kristine E. , Bliss Judy L. , Trepicchio William L. , Sypek Joseph P. , Dunussi-Joannopoulos Kyriaki , Goldman Samuel J. , Leonard John P.
Published: December 2022

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rβ1 and IL-12Rβ2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rβ2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rβ2 expression can be prevented by treatment with IFN-γ, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rβ1 and β2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-γ secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-γ secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-γ production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-γ secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.
Disruption of the STAT4 Signaling Pathway Protects from Autoimmune Diabetes While Retaining Antiviral Immune Competence
Authors: Holz Andreas , Bot Adrian , Coon Bryan , Wolfe Tom , Grusby Michael J. , von Herrath Matthias G.
Published: December 2022

The role of the STAT4 signaling pathway in autoimmune diabetes was investigated using the rat insulin promoter lymphocytic choriomeningitis virus model of virally induced autoimmune diabetes. Abrogation of STAT4 signaling significantly reduced the development of CD4+-T cell-dependent but not CD4+-T cell-independent diabetes, illustrating the fine-tuned kinetics involved in the pathogenesis of autoimmunity. However, the absence of STAT4 did not prevent the generation of autoreactive Th1/Tc1 T cell responses, as well as protective antiviral immunity. Protection from insulin-dependent diabetes mellitus was associated with decreased numbers of autoreactive CTL precursors in the pancreas and the spleen and a general as well as Ag-specific reduction of IFN-γ secretion by T lymphocytes. A shift from Th1 to Th2 T cell immunity was not observed. Hence, our results implicate both CTL and cytokines in β cell destruction. Selective inhibition of the STAT4 signal transduction pathway might constitute a novel and attractive approach to prevent clinical insulin-dependent diabetes mellitus in prediabetic individuals at risk.
The Jak Family Tyrosine Kinase Jak3 Is Required for IL-2 Synthesis by Naive/Resting CD4+ T Cells
Authors: Thomis Daniel C. , Aramburu José , Berg Leslie J.
Published: December 2022

The Jak family tyrosine kinase, Jak3, is involved in signaling through cytokine receptors using the common γ-chain. Mice deficient in Jak3 have mature T cells, all of which have an activated/memory cell phenotype but are unresponsive to in vitro stimulation. Due to this activated phenotype, it has been impossible to determine whether Jak3 plays a role in the responsiveness of naive/resting T cells. To circumvent this difficulty, we generated naive/resting Jak3-negative T cells by two genetic approaches. After stimulation, these cells failed to produce significant amounts of IL-2. Although no signaling defect could be detected, we did find that naive/resting Jak3-negative T cells have substantially reduced levels of the transcription factor NF-AT1 and moderately reduced levels of c-Jun and c-Fos. On the basis of these data, we propose that Jak3-dependent cytokine signals may be required to maintain the normal levels of basal transcription factors required for immediate responsiveness to Ag activation.
Cytosolic Phospholipase A2-Mediated Regulation of Phospholipase D2 in Leukocyte Cell Lines
Authors: Kim Jae Ho , Lee Byoung Dae , Kim Yong , Lee Sang Do , Suh Pann-Ghill , Ryu Sung Ho
Published: December 2022

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2 , and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.
Molecular, Immunological, and Structural Characterization of Phl p 6, a Major Allergen and P-Particle-Associated Protein from Timothy Grass (Phleum pratense) Pollen
Authors: Vrtala Susanne , Fischer Sabine , Grote Monika , Vangelista Luca , Pastore Annalisa , Sperr Wolfgang R. , Valent Peter , Reichelt Rudolf , Kraft Dietrich , Valenta Rudolf
Published: December 2022

Due to the wide distribution and heavy pollen production of grasses, ∼50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive α-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.
Slow Accumulation of Active Mitogen-Activated Protein Kinase During Thymocyte Differentiation Regulates the Temporal Pattern of Transcription Factor Gene Expression
Authors: Shao Hui , Wilkinson Beverley , Lee Brian , Han Pei-Chen , Kaye Jonathan
Published: December 2022

Thymocyte-positive selection is believed to result from low affinity/avidity interactions of TCR and MHC proteins, but how these weak interactions translate into downstream biochemical signals and how such signals modulate gene expression is unknown. Using a culture system where isolated immature thymocytes can be induced to differentiate along the CD4 lineage pathway, we show that sustained low level mitogen-activated protein/extracellular regulated kinase activity is required for cell differentiation and survival. Furthermore, induction of mitogen-activated protein/extracellular regulated kinase activity is surprisingly slow under conditions that induce differentiation. This pattern of kinase activity not only selects which genes are expressed but also regulates the temporal pattern of expression of transcription factor genes that are induced during double-positive thymocyte differentiation.
Negative Selection of T Cells Occurs Throughout Thymic Development
Authors: Baldwin Kristin K. , Trenchak Brian P. , Altman John D. , Davis3 Mark M.
Published: December 2022

Thymic positive and negative selections govern the development of a self-MHC-reactive, yet self-tolerant, T cell repertoire. Whether these processes occur independently or sequentially remains controversial. To investigate these issues, we have employed tetrameric peptide-MHC complexes to fluorescently label and monitor polyclonal populations of thymocytes that are specific for moth cytochrome c (MCC)/I-Ek. In TCR β mice tetramer-positive thymocytes are detectable even in the most immature TCR-expressing cells. In the presence of MCC peptide, thymocytes that bind strongly to MCC/I-Ek tetramers are deleted earlier in development and more extensively than cells that bind weakly. This negative selection of the MCC/I-Ek-specific cells occurs continuously throughout development and before any evidence of positive selection. Thus, positive and negative selections are independent processes that need not occur sequentially.