bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: A Dominant Negative Form of TNF-α Converting Enzyme Inhibits ProTNF and TNFRII Secretion
Authors: Solomon Kimberly A. , Pesti Nancy , Wu Guoxin , Newton Robert C.
Published: December 2022

TNF-α converting enzyme (TACE) is the protease responsible for processing proTNF from the 26-kDa membrane-anchored precursor to the secreted 17-kDa TNF-α. We show here that a deletion mutant of TACE (dTACE), lacking the pro and catalytic domains of the protease, acts as a dominant negative for proTNF processing in transfected HEK293 cells. We used the same system to test the effect of dTACE on TNFRII processing. Overexpression of dTACE with TNFRII resulted in >80% inhibition of TNFRII shedding. Although significant inhibition of TNF-α and TNFRII shedding was achieved with dTACE, we could not detect a cell surface accumulation of the noncleaved substrates above that observed in the absence of dTACE. Our results suggest that TNFRII is a substrate for TACE, and that dTACE is capable of interfering with the function of endogenous TACE, either by binding and sequestering TACE substrates via the disintegrin domain, transmembrane domain, or cytoplasmic tail, or by some other mechanism that has yet to be determined.
A Caspase-Independent Pathway of MHC Class II Antigen-Mediated Apoptosis of Human B Lymphocytes
Authors: Drénou Bernard , Blancheteau Vincent , Burgess David H. , Fauchet Renée , Charron Dominique J. , Mooney Nuala A.
Published: December 2022

MHC class II molecules have a crucial role in thymic selection and in generating Ag-specific T cell responses. There is extensive evidence for second messenger generation via MHC class II molecules, which can lead to apoptosis of B lymphocytes. We have examined HLA class II-mediated apoptosis in both normal and tumoral human B lymphocytes. Phosphatidylserine exposure and DNA fragmentation were observed in B cells within 24 h of stimulation via HLA class II. In marked comparison with Fas, the cell-permeable and irreversible caspase inhibitors zVAD-fmk and DEVD-fmk failed to inhibit HLA-DR-mediated apoptosis. No direct activation of caspase 3 was detected, and cleavage of pro-caspase 3 was not observed. Cleavage of poly(ADP-ribose) polymerase was detected via Fas but not via HLA class II. Although phosphatidylinositol-3-kinase has been implicated in HLA class I-mediated apoptosis, neither wortmannin nor LY294002 affected HLA class II-mediated apoptosis. CD95-sensitive cells were used to reveal that death occurred independently of CD95-CD95 ligand interactions. Overall, these data reveal a pathway of HLA-DR-mediated apoptosis that neither requires nor involves caspases. Moreover, it is phosphatidylinositol-3-kinase independent and Fas/CD95 independent. This pathway of HLA class II-mediated apoptosis could have an important role in the regulation of APC populations or in the control of malignant B lymphocyte proliferations.
Integrating Signals from IFN-γ and IL-4 by B Cells: Positive and Negative Effects on CD40 Ligand-Induced Proliferation, Survival, and Division-Linked Isotype Switching to IgG1, IgE, and IgG2a
Authors: Hasbold Jhagvaral , Hong Jonathan Sui-Yin , Kehry Marilyn R. , Hodgkin Philip D.
Published: December 2022

IL-4 and IFN-γ each have potent effects on B cell responses as well as strong mutual antagonism. Here we have examined the quantitative effects of these cytokines on CD40 ligand-induced B cell proliferation, cell survival, and division-linked isotype switching. Both IL-4 (strongly) and IFN-γ (weakly) enhanced the number of B cells found in culture by reducing the average time cells take to enter the first division cycle and by promoting B cell survival. When added in combination, the net effect of IL-4 and IFN-γ on time to division and survival was a response intermediate between that of the two cytokines alone, indicating a partial antagonism of IL-4 by IFN-γ. By modulating both time to division and cell survival, these small effects of IFN-γ are amplified and give rise to large reductions in cell number in the presence of IL-4. At higher concentrations, IFN-γ had minor inhibitory effects on IL-4-induced isotype switching to IgG1 and greater effects on IgE. A reciprocal relation was observed between the ability to inhibit IgE at late cell divisions vs induction of IgG2a. In contrast, IL-4 did not prevent switching to IgG2a induced by IFN-γ alone. Therefore, antagonism between IFN-γ and IL-4 is observed at multiple levels and over different concentration ranges, resulting in complex net outcomes. The evolutionary significance of this complexity is discussed.
Role of IL-12 in the Induction and Potentiation of IFN-γ in Response to Bacillus Calmette-Guérin
Authors: O’Donnell Michael A. , Luo Yi , Chen Xiaohong , Szilvasi Akos , Hunter Sharon E. , Clinton Steven K.
Published: December 2022

Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been accepted as the most effective agent in clinical use against superficial bladder cancer, its mechanism of action remains incompletely understood. A kinetic analysis in assessing the potential role of cytokines from BCG-stimulated murine splenocytes showed that IL-12 expression preceded that of other cytokines. Experiments subtracting endogenous BCG-driven IL-12 using neutralizing Ab or augmenting its activity with supplemental rIL-12 revealed not only that IL-12 plays a dominant role in IFN-γ induction but also that it is normally dose limiting. A striking increase in IFN-γ production could be generated in both mouse and human immunocompetent cell culture by the addition of even a small amount of rIL-12. Moreover, this same synergistic effect could be replicated during in vivo administration of BCG plus rIL-12 into the mouse bladder and was observed in a patient receiving intravesical combination therapy. In costimulation cultures, this synergy appeared to partially rely on IL-18 and IL-2 and could be down-regulated by IL-10. This suggests that a dynamic interplay between Th1 and Th2 cytokines is responsible for net IFN-γ production. The ability of supplemental exogenous IL-12 to strongly shift this balance toward Th1 provides an immunological basis for using it in conjunction with intravesical BCG for bladder cancer immunotherapy.
LFA-1 to LFA-1 Signals Involve ζ-Associated Protein-70 (ZAP-70) Tyrosine Kinase: Relevance for Invasion and Migration of a T Cell Hybridoma
Authors: Soede Ron D. M. , Driessens Mariëtte H. E. , Ruuls-Van Stalle Lisette , Van Hulten Paula E. M. , Brink Arjen , Roos Ed
Published: December 2022

We previously showed that LFA-1-dependent in vitro invasion and in vivo migration of a T cell hybridoma was blocked in cells overexpressing a truncated dominant-negative ζ-associated protein (ZAP)-70. The truncated ZAP-70 also blocked LFA-1-dependent chemotaxis through ICAM-1-coated filters induced by 1 ng/ml stromal cell-derived factor-1, but not LFA-1-independent chemotaxis induced by 100 ng/ml stromal cell-derived factor-1. This suggested that LFA-1 engagement triggers a signal that amplifies a weak chemokine signal and that dominant-negative ZAP-70 blocks this LFA-1 signal. Here we show that cross-linking of part of the LFA-1 molecules with Abs causes activation of free LFA-1 molecules (not occupied by the Ab) on the same cell, which then bind to ICAM-2 on other cells. This causes cell aggregation that was also blocked by dominant-negative ZAP-70. Thus, an LFA-1 signal involving ZAP-70 activates other LFA-1 molecules, suggesting that the chemokine signal can be amplified by multiple cycles of LFA-1 activation. The chemokine and the LFA-1 signal were both blocked by a phospholipase C inhibitor and a calpain inhibitor, suggesting that one of the amplified signals is the phospholipase C-dependent activation of calpain. Finally, we show that both Src-homology 2 domains are required for inhibition of invasion, chemotaxis, and aggregation by the truncated ZAP-70, suggesting that ZAP-70 interacts with a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequence. Remarkably, this is not an ITAM in the TCR/CD3 complex because this is not expressed by this T cell hybridoma.
A Soluble Factor Produced by Lamina Propria Mononuclear Cells Is Required for TNF-α Enhancement of IFN-γ Production by T Cells
Authors: Prehn John L. , Landers Carol J. , Targan Stephan R.
Published: December 2022

The role of TNF-α in the mucosal inflammation of Crohn’s disease has been demonstrated by the prolonged clinical responses and/or remissions among patients receiving i.v. infusion of anti-TNF-α. A correlation between TNF-α and elevated IFN-γ production is suggested by the reduction in the number of IFN-γ producing lamina propria mononuclear cells (LPMC) found in colonic biopsies from anti-TNF-α-treated patients. The aim of this study was to define the mechanism of TNF-α-augmented mucosal T cell IFN-γ production. In this paper we present evidence that cultured LPMC secrete a factor which acts on preactivated T cells in concert with TNF-α to augment IFN-γ production. This activity is independent of IL-12 and IL-18, the well-documented potentiators of IFN-γ expression, and is not produced by PBMC. Peripheral blood PHA-activated T cells incubated in supernatants from LPMC became responsive to TNF-α by increasing IFN-γ output upon stimulation. These results are consistent with a model in which LPMC, but not PBMC, release an unidentified substance when cultured in vitro with low dose IL-2. This substance can act on preactivated peripheral T cells, as well as on lamina propria T cells, conditioning them to respond to TNF-α by increased IFN-γ secretion upon stimulation. Expression of this factor in the gut mucosa could contribute to up-regulation of the Th1 response in the presence of TNF-α, and could be important for mucosal immunoregulation.
Inhibition of CD28 Expression by Oligonucleotide Decoys to the Regulatory Element in Exon 1 of the CD28 Gene
Authors: Tam Robert C. , Lin Catherine J. , Lim Charmaine , Pai Bharati , Stoisavljevic Vesna
Published: December 2022

Ligation of CD28 provides a costimulatory signal essential for Ag-mediated T cell activation via the TCR. Previously we demonstrated that inhibition of human and murine CD28 expression by a guanosine (G)-rich oligonucleotide (ODN), GR1, led to immunosuppression in vitro and in vivo. The bioactivity of GR1 was dependent on a G-rich DNA sequence motif consisting of two G tetrads separated by four nucleotides, (G4N4G4). We have shown recently that a G-rich region, designated CD28GR, in exon 1 of the CD28 gene is such a motif and is a positive regulatory element that binds the transcription factors Sp1 and EGR-1. Here we showed that the bioactivity of GR1 and the related GR2 correlated with the sequence-specific formation of distinct nuclear protein complexes and a high degree of ODN secondary structure. In addition, these ODN blocked transcription factor binding to CD28GR (also in a sequence-specific manner) and prevented CD28GR from driving transcription of a reporter gene. Interestingly, GR1 potently inhibited CD28, but not the expression of other Sp1- and EGR-1-regulated genes, an effect associated with lower Sp1 protein binding affinity of GR1 and GR2 compared with that of canonical Sp1 sites. These data show that DNA sequences that contain the G-rich sequence motif, G4N4G4, such as GR1 and GR2, can functionally mimic the regulatory protein binding ability of CD28GR. Thus, GR1 and GR2 act as molecular decoys to selectively interfere with transcriptional regulation of the CD28 gene.
Cerebral Endothelial Cells Release TNF-α After Stimulation with Cell Walls of Streptococcus pneumoniae and Regulate Inducible Nitric Oxide Synthase and ICAM-1 Expression Via Autocrine Loops
Authors: Freyer Dorette , Manz Rahel , Ziegenhorn Andreas , Weih Markus , Angstwurm Klemens , Döcke Wolf-Dietrich , Meisel Andreas , Schumann Ralf R. , Schönfelder Gilbert , Dirnagl Ulrich , Weber Joerg R.
Published: December 2022

TNF-α, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-α, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF-α mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF-α protein peaked at 4 h (9.4 ± 3.6 vs 0.1 ± 0.1 pg/μg protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 ± 4 vs 5.8 ± 1.8 at 12 h; 18.1 ± 4 vs 5.8 ± 1.8 pmol/μg protein at 72 h). The addition of TNF-α Ab significantly reduced nitrite production to 62.2 ± 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF-α Ab (142 ± 18.6 vs 97.5 ± 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF-α mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-α, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.
Specificity of CTL Interactions with Peptide-MHC Class I Tetrameric Complexes Is Temperature Dependent
Authors: Whelan Joseph A. , Dunbar P. Rod , Price David A. , Purbhoo Marco A. , Lechner Franziska , Ogg Graham S. , Griffiths Gillian , Phillips Rodney E. , Cerundolo Vincenzo , Sewell Andrew K.
Published: December 2022

Tetrameric peptide-MHC class I complexes (“tetramers”) are proving invaluable as reagents for characterizing immune responses involving CTLs. However, because the TCR can exhibit a degree of promiscuity for binding peptide-MHC class I ligands, there is potential for cross-reactivity. Recent reports showing that the TCR/peptide-MHC interaction is dramatically dependent upon temperature led us to investigate the effects of incubation temperature on tetramer staining. We find that tetramers rapidly stain CTLs with high intensity at 37°C. We examine the fine specificity of tetramer staining using a well-characterized set of natural epitope variants. Peptide variants that elicit little or no functional cellular response from CTLs can stain these cells at 4°C but not at 37°C when incorporated into tetramers. These results suggest that some studies reporting tetramer incubations at 4°C could detect cross-reactive populations of CTLs with minimal avidity for the tetramer peptide, especially in the tetramer-low population. For identifying specific CTLs among polyclonal cell populations such as PBLs, incubation with tetramers at 37°C improves the staining intensity of specific CTLs, resulting in improved separation of tetramer-high CD8+ cells. Confocal microscopy reveals that tetramers incubated at 37°C can be rapidly internalized by specific CTLs into vesicles that overlap with the early endocytic compartment. This TCR-specific internalization suggests that coupling of tetramers or analogues with toxins, which are activated only after receptor internalization, may create immunotoxins capable of killing CTLs of single specificities.
The Epithelial Cell Response to Rotavirus Infection
Authors: Rollo Ellen E. , Kumar K. Prasanna , Reich Nancy C. , Cohen Jean , Angel Juana , Greenberg Harry B. , Sheth Riten , Anderson Joseph , Oh Brian , Hempson Scott J. , Mackow Erich R. , Shaw Robert D.
Published: December 2022

Rotavirus is the most important worldwide cause of severe gastroenteritis in infants and young children. Intestinal epithelial cells are the principal targets of rotavirus infection, but the response of enterocytes to rotavirus infection is largely unknown. We determined that rotavirus infection of HT-29 intestinal epithelial cells results in prompt activation of NF-κB (<2 h), STAT1, and ISG F3 (3 h). Genetically inactivated rotavirus and virus-like particles assembled from baculovirus-expressed viral proteins also activated NF-κB. Rotavirus infection of HT-29 cells induced mRNA for several C-C and C-X-C chemokines as well as IFNs and GM-CSF. Mice infected with simian rotavirus or murine rotavirus responded similarly with the enhanced expression of a profile of C-C and C-X-C chemokines. The rotavirus-stimulated increase in chemokine mRNA was undiminished in mice lacking mast cells or lymphocytes. Rotavirus induced chemokines only in mice <15 days of age despite documented infection in older mice. Macrophage inflammatory protein-1β and IFN-stimulated protein 10 mRNA responses occurred, but were reduced in p50−/− mice. Macrophage inflammatory protein-1β expression during rotavirus infection localized to the intestinal epithelial cell in murine intestine. These results show that the intestinal epithelial cell is an active component of the host response to rotavirus infection.
The NF-κB Family Member RelB Is Required for Innate and Adaptive Immunity toToxoplasma gondii
Authors: Caamaño Jorge , Alexander James , Craig Linden , Bravo Rodrigo , Hunter Christopher A.
Published: December 2022

The NF-κB family of transcription factors are associated with the regulation of innate and adaptive immunity to infection. Infection of C57BL/6 mice with Toxoplasma gondii resulted in up-regulation of NF-κB activity that included the NF-κB family member RelB. To assess the role of RelB in the regulation of the immune response to this infection, we challenged RelB-deficient mice (RelB−/−) and wild-type (WT) littermate controls with T. gondii. Although WT controls were resistant to T. gondii, RelB−/− mice succumbed 10–15 days after infection. Examination of accessory cell functions associated with resistance to T. gondii revealed that RelB−/− macrophages stimulated with IFN-γ plus LPS or TNF-α produced IL-12 as well as reactive nitrogen intermediates and inhibited parasite replication similar to WT macrophages. Analysis of the systemic responses of RelB−/− and WT mice revealed that infected mice had similar serum levels of IL-12. However, RelB−/− mice challenged with T. gondii produced negligible levels of IFN-γ and had reduced NK cell activity compared with WT mice. Similarly, splenocytes from uninfected RelB−/− mice stimulated with polyclonal stimuli were deficient in their ability to produce IFN-γ. Together, our results demonstrate that RelB is essential for the development of innate NK and adaptive T cell responses that lead to the production of IFN-γ and resistance to T. gondii.
Nitric Oxide Participates in the Recovery of Normal Jejunal Epithelial Ion Transport Following Exposure to the Superantigen,Staphylococcus aureusEnterotoxin B
Authors: McKay Derek M. , Lu Jun , Jedrzkiewicz Sean , Ho Winnie , Sharkey Keith A.
Published: December 2022

Bacterial superantigens (SAgs) are potent T cell activators. Mice treated 4 h previously with the SAg, Staphylococcus aureus enterotoxin B (SEB), display reduced ion transport (assessed by short circuit current) responses to prosecretory stimuli, which normalize 24 h posttreatment. Here, mice were treated with SEB alone or in combination with an inhibitor of the inducible form of NO synthase (iNOS), l-NIL. Subsequently, jejunal iNOS expression was detected by immunohistochemistry, ion transport was evaluated in Ussing chambers, and serum levels of TNF-α and IFN-γ were measured by ELISA. SEB-treated mice had increased epithelial iNOS immunoreactivity, and numerous iNOS-positive CD3+ T cells occurred in their mucosa and submucosa. Concomitant treatment with l-NIL did not affect the reduced short circuit current responsiveness to electrical nerve stimulation or the prosecretory agents, carbachol and forskolin, that occurred 4 h post-SEB (5 μg) treatment. However, Isc responses in l-NIL- plus SEB-treated mice were still significantly reduced 24 h posttreatment, indicating a role for NO in the restoration of normal ion transport following exposure to SAgs. The prolongation of epithelial ion transport abnormalities correlated with elevated serum levels of TNF-α and IFN-γ in mice treated 24 h previously with l-NIL plus SEB compared with those in controls and SEB-only-treated mice. Additionally, mice treated with l-NIL plus SEB and TNF-α- or IFN-γ-neutralizing Abs displayed normal jejunal ion transport characteristics 24 h posttreatment. We conclude that NO mobilization is important in the homeostatic recovery response following immune stimulation by SAgs and that the beneficial effect of NO in this model system is probably via regulation of TNF-α and IFN-γ production.
Activation of p90RSK and cAMP Response Element Binding Protein in Stimulated Neutrophils: Novel Effects of the Pyridinyl Imidazole SB 203580 on Activation of the Extracellular Signal-Regulated Kinase Cascade
Authors: Lian Jian P. , Huang RiYun , Robinson Dwight , Badwey John A.
Published: December 2022

Neutrophils stimulated with the chemoattractant FMLP or the phorbol ester PMA are known to exhibit activation of a 90-kDa renaturable protein kinase. Activation of this kinase was maximal at ∼1–3 min after cell stimulation and the time course for activation was similar to that of the extracellular-regulated kinases (ERKs) and p38-mitogen activated protein kinase (p38MAPK). Compounds that block activation of ERK-1/2 (PD 98059) or that inhibit the activity of p38MAPK (SB 203580) blocked activation of this 90-kDa kinase. SB 203580 is a highly selective inhibitor of p38MAPK in vitro and is under intense study as a lead compound for developing novel anti-inflammatory agents. However, we demonstrate that SB 203580 at concentrations ≥10 μM can also inhibit activation of ERK-1/2 in neutrophils. An Ab to the protein kinase p90RSK2 (also referred to as MAPKAP-K1b, or p90rsk) immunoprecipitated the active 90-kDa kinase from lysates of stimulated neutrophils. No activity was observed for this enzyme in immunoprecipitates obtained from unstimulated cells, and the amounts of activity were markedly reduced if the cells were treated with PD 98059 or SB 203580 before stimulation. Neutrophils stimulated with FMLP exhibited phosphorylation of the cAMP response element binding protein (CREB), and this reaction was inhibited by SB 203580 and PD 98059. These data establish that the renaturable 90-kDa protein kinase is p90RSK2 and that CREB may be a substrate for this enzyme in these cells. Novel effects of compound SB 203580 on stimulated neutrophils are also described.
Posttranscriptional Regulation of Acute Phase Serum Amyloid A2 Expression by the 5′- and 3′-Untranslated Regions of Its mRNA
Authors: Longley Daniel B. , Steel Diana M. , Whitehead Alexander S.
Published: December 2022

Human acute-phase serum amyloid A protein (A-SAA) is a major acute phase reactant, the concentration of which increases dramatically as part of the body’s early response to inflammation. A-SAA is the product of two almost identical genes, SAA1 and SAA2, which are induced by the pro-inflammatory cytokines, IL-1 and IL-6. In this study, we examine the roles played by the 5′- and 3′-untranslated regions (UTRs) of the SAA2 mRNA in regulating A-SAA2 expression. SAA2 promoter-driven luciferase reporter gene constructs carrying the SAA2 5′-UTR and/or 3′-UTR were transiently transfected into the HepG2 human hepatoma cell line. After induction of chimeric mRNA with IL-1β and IL-6, the SAA2 5′- and 3′-UTRs were both able to posttranscriptionally modify the expression of the luciferase reporter. The SAA2 5′-UTR promotes efficient translation of the chimeric luciferase transcripts, whereas the SAA2 3′-UTR shares this property and also significantly accelerates the rate of reporter mRNA degradation. Our data strongly suggest that the SAA2 5′- and 3′-UTRs each play significant independent roles in the posttranscriptional regulation of A-SAA2 protein synthesis.
Adenoviral Vector-Mediated Overexpression of IL-4 in the Knee Joint of Mice with Collagen-Induced Arthritis Prevents Cartilage Destruction
Authors: Lubberts Erik , Joosten Leo A. B. , van den Bersselaar Liduine , Helsen Monique M. A. , Bakker Andrew C. , van Meurs Joyce B. J. , Graham Frank L. , Richards Carl D. , van den Berg Wim B.
Published: December 2022

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-α and IL-1β were suppressed as well, and IL-1β and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.
Novel Path to Activation of Vascular Smooth Muscle Cells: Up-Regulation of gp130 Creates an Autocrine Activation Loop by IL-6 and Its Soluble Receptor
Authors: Klouche Mariam , Bhakdi Sucharit , Hemmes Monika , Rose-John Stefan
Published: December 2022

This study describes a novel path to the activation of smooth muscle cells (SMC) by the IL-6/soluble IL-6 receptor (sIL-6R) system. Human vascular SMC constitutively express only scant amounts of IL-6R and so do not respond to stimulation with this cytokine. We show that SMC also do not constitutively express appreciable levels of gp130, which would render them sensitive to transsignaling by the IL-6/sIL-6R complex. Because gp130 is generally believed not to be subject to regulation, SMC would thus appear not to qualify as targets for the IL-6/sIL-6R system. However, we report that treatment of SMC with IL-6/sIL-6R provokes marked up-regulation of gp130 mRNA and surface protein expression. This is accompanied by secretion of IL-6 by the cells, so that an autocrine stimulation loop is created. In the wake of this self-sustaining system, there is a selective induction and secretion of MCP-1, up-regulation of ICAM-1, and marked cell proliferation. The study identifies SMC as the first example of cells in which gp130 expression is subject to substantive up-regulation, and discovers a novel amplification loop involving IL-6 and its soluble receptor that drives SMC into a proinflammatory state.
Nephritogenic λ Light Chain Dimer: A Unique Human Miniautoantibody Against Complement Factor H
Authors: Jokiranta T. Sakari , Solomon Alan , Pangburn Michael K. , Zipfel Peter F. , Meri Seppo
Published: December 2022

A unique monoclonal Ig λ light chain dimer (protein LOI) was isolated from the serum and urine of a patient with hypocomplementemic membranoproliferative glomerulonephritis. In vitro the λ light chain dimer efficiently activated the alternative pathway of complement (AP). When added to normal human serum, LOI temporarily enhanced AP hemolytic activity, but during a prolonged incubation the hemolytic activity was depleted. Protein LOI was found to bind to factor H, the main regulator molecule of AP. By binding to the short consensus repeat domain 3 of factor H, the dimer LOI blocked one of three interaction sites between H and C3b and thus inhibited the activity of H and induced an uncontrolled activation of the AP. Structural analysis showed that LOI belonged to the Vλ3a subgroup of λ light chains. The variable (V) region of LOI was most closely related to the predicted product of the Vλ3 germline gene Iglv3s2, although it contained several unique residues that in a tertiary homology model structure form an unusual ring of charged residues around a hydrophobic groove in the putative Ag binding site. This site fitted considerably well with a putative binding site in the molecular model of domain 3 of factor H containing a reciprocal ring of charged amino acids around a hydrophobic area. Apparently, functional blocking of factor H by the Ab fragment-like λ light chain dimer had initiated the development of a severe form of membranoproliferative glomerulonephritis. Thus, the λ light chain dimer LOI represents the first described pathogenic miniautoantibody in human disease.
Switching to IgG3, IgG2b, and IgA Is Division Linked and Independent, Revealing a Stochastic Framework for Describing Differentiation
Authors: Deenick Elissa K. , Hasbold Jhagvaral , Hodgkin Philip D.
Published: December 2022

LPS was used to induce switching of B cells to IgG3 and, in the presence of TGF-β, to IgG2b and IgA. Switching to all three isotypes increased with division number according to a consistent relationship that was independent of time in culture. The mode of activation altered the relationship with division, as CD40 ligand increased switching to IgA and decreased switching to IgG2b and IgG3 when measured per division. This division-linked switching behavior could be described by Gaussian probability distributions centered around a mean division number. The divisions at which switching to IgG3 and IgG2b occurred overlapped, raising the possibility that the two switching mechanisms were linked. However, when IgG3+ and IgG3− B cells were sorted and placed back in culture, they switched to IgG2b at an equivalent rate, indicating that alternative switching decisions were made independently within a single cell. As a consequence, isotype switching could be predicted at the population level by standard probability laws. Therefore, division number provides a framework for a stochastic description of differentiation that may be widely applicable.
Protein Kinase C Inhibits CD95 (Fas/APO-1)-Mediated Apoptosis by at Least Two Different Mechanisms in Jurkat T Cells
Authors: Ruiz-Ruiz Carmen , Robledo Gema , Font Jovita , Izquierdo Manuel , López-Rivas Abelardo
Published: December 2022

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we demonstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane.
Visualization, Fate, and Pathogenicity of Antigen-Specific CD8+ T Cells in the Graft-Versus-Host Reaction
Authors: Yu Xue-Zhong , Bidwell Sasha , Martin Paul J. , Anasetti Claudio
Published: December 2022

To follow the fate of alloreactive T cell effectors in graft-vs-host disease, Ld-specific CD8+ T cells from C57BL/6 2C TCR-transgenic donors were transplanted into sublethally irradiated (750 cGy) Ld+ or Ld− recipients. In Ld− C57BL/6 or (BALB/c-dm2 × C57BL/6)F1 recipients, naive 2C T cells engrafted and survived long term, but did not acquire effector function. In Ld+ (BALB/c × C57BL/6)F1 recipients, 2C T cells engrafted, expanded, became cytolytic, destroyed host B cells and double-positive thymocytes, and later disappeared. Despite marked damage to lymphoid and hemopoietic cells by 2C T cells, no significant pathology was detected in other organs, and recipients survived. Ld+ (BALB/c × C57BL/6)F1 recipients died when LPS/endotoxin was administered on day 7 after cell transfer, while Ld− (BALB/c-dm2 × C57BL/6)F1 recipients survived. Our findings show that under certain conditions, a CD8+ T cell population recognizing an extremely limited repertoire of Ags can initiate graft-vs-host disease.
Peripheral CD4+ T Cell Maturation Recognized by Increased Expression of Thy-1/CD90 Bearing the 6C10 Carbohydrate Epitope
Authors: Gui Ming , Wiest David L. , Li Jin , Kappes Dietmar , Hardy Richard R. , Hayakawa Kyoko
Published: December 2022

The SM6C10 IgM autoantibody recognizes a surface determinant, 6C10, that is highly expressed on all immature thymocytes. In contrast, its expression on peripheral T cells appears developmentally regulated, i.e., absent from most naive T cells in spleen of neonatal mice, but expressed on 40–80% of naive CD4+ T cells in adult. In this paper, we demonstrate that SM6C10 recognizes a carbohydrate epitope on the Thy-1 glycoprotein using immunoprecipitation analysis, by binding to affinity-purified Thy-1 in an ELISA, and by sensitivity to N-glycosidase-F treatment. Retroviral Thy-1 gene transduction experiments into Thy-1− variant T cell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but depends on the recipient cell. Therefore, differences in 6C10 levels among Thy-1+ T cells in mice likely reflect developmental regulation of posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD4+ T cells to respond to anti-Thy-1 stimulation increases from neonate to adult, and 6C10− naive cells from adult mice respond poorly compared with 6C10+ cells, similar to the cells in neonatal mice. These results suggest that there is functional maturation by peripheral CD4+ T cells that coincides with 6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope.
Distinct Mechanisms Target Stress and Extracellular Signal-Activated Kinase 1 and Jun N-Terminal Kinase During Infection of Macrophages with Salmonella
Authors: Procyk Katarzyna J. , Rippo Maria Rita , Testi Roberto , Hoffmann Fred , Parker Peter J. , Baccarini Manuela
Published: December 2022

The interaction between bacteria and macrophages is central to the outcome of Salmonella infections. Salmonella can escape killing by these phagocytes and survive and multiply within them, giving rise to chronic infections. Cytokines produced by infected macrophages are involved in the early gastrointestinal pathology of the infection as well as in the induction and maintenance of the immune response against the invaders. Jun N-terminal kinases (JNK) are activated by inflammatory stimuli and play a role in cytokine production. We have investigated the signaling routes leading to JNK activation in Salmonella-infected macrophages and have discovered that they differ radically from the mechanisms operating in epithelial cells. In particular, activation of the JNK kinase stress and extracellular-activated kinase 1 (SEK1) and of JNK in macrophages occurs independently of actin rearrangements and of the GTPases Cdc42 and Rac, essential mediators in other cells. Activation of JNK is effected by a novel pathway comprising tyrosine kinase(s), phosphoinositide 3-kinase and, likely, atypical protein kinase C ζ. SEK1 is stimulated by a distinct mechanism involving phosphatidylcholine-phospholipase C and acidic sphingomyelinase. Dominant-negative SEK1 can block JNK activation by LPS, but not by Salmonella. These data demonstrate that SEK1 and JNK are activated independently in Salmonella-infected macrophages and offer experimental support for the concept that incoming signals can direct the selective coupling of downstream pathways to elicit highly specific responses. Inhibitors of stress kinase pathways are receiving increasing attention as potential anti-inflammatory drugs. The precise reconstruction of stimulus-specific pathways will be instrumental in predicting/evaluating the effects of the inhibitors on a given pathological condition.
Immune-Associated Nucleotide-1 (IAN-1) Is a Thymic Selection Marker and Defines a Novel Gene Family Conserved in Plants
Authors: Poirier Ghislaine M. C. , Anderson Graham , Huvar Arne , Wagaman Pamela C. , Shuttleworth John , Jenkinson Eric , Jackson Michael R. , Peterson Per A. , Erlander Mark G.
Published: December 2022

Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.
A Cyclophilin B Gene Encodes Antigenic Epitopes Recognized by HLA-A24-Restricted and Tumor-Specific CTLs
Authors: Gomi Shinya , Nakao Masanobu , Niiya Fumihiko , Imamura Yutaka , Kawano Kouichiro , Nishizaka Shinya , Hayashi Akihiko , Sobao Yuji , Oizumi Kotaro , Itoh Kyogo
Published: December 2022

We have studied Ags recognized by HLA class I-restricted CTLs established from tumor site to better understand the molecular basis of tumor immunology. HLA-A24-restricted and tumor-specific CTLs established from T cells infiltrating into lung adenocarcinoma recognized the two antigenic peptides encoded by a cyclophilin B gene, a family of genes for cyclophilins involved in T cell activation. These two cyclophilin B peptides at positions 84–92 and 91–99 induced HLA-A24-restricted CTL activity against tumor cells in PBMCs of leukemia patients, but not in epithelial cancer patients or in healthy donors. In contrast, the modified peptides at position 2 from phenylalanine to tyrosine, which had more than 10 times higher binding affinities to HLA-A24 molecules, could induce HLA-A24-restricted CTL activity against tumor cells in PBMCs from leukemia patients, epithelial cancer patients, or healthy donors. PHA-activated normal T cells were resistant to lysis by the CTL line or by these peptide-induced CTLs. These results indicate that a cyclophilin B gene encodes antigenic epitopes recognized by CTLs at the tumor site, although T cells in peripheral blood (except for those from leukemia patients) are immunologically tolerant to the cyclophilin B. These peptides might be applicable for use in specific immunotherapy of leukemia patients or that of epithelial cancer patients.
Nematode Infection Enhances Survival of Activated T Cells by Modulating Accessory Cell Function
Authors: Liwski Robert S. , Lee Timothy D. G.
Published: December 2022

The type of immune response generated following exposure to Ag depends on a variety of factors, including the nature of the Ag, the type of adjuvant used, the site of antigenic entry, and the immune status of the host. We have previously shown that infection of rodents with Nippostrongylus brasiliensis (Nb) shifts the development of type 1 allo-specific responses toward type 2 immunity, suggesting nematode modulation of T cell activation. In this report we explore the immunomodulatory effects of Nb on T cell activation. We found that spleen cells from Nb-infected mice exhibited dramatically increased proliferation in response to Con A and anti-CD3. This hyperproliferation could be transferred in vitro to naive splenocytes by coculture with mitomycin C-treated cells from Nb-infected animals. The transfer was mediated by non-T accessory cells and supernatants derived from Con A-activated non-T cells, suggesting the involvement of a soluble factor secreted by accessory cells. The accessory cells secreted high levels of IL-6, and anti-IL-6 treatment abrogated the supernatant-induced hyperproliferation, thus confirming that IL-6 was mediating the effect. Further, spleen cells from Nb-infected mice were more resistant to activation-induced cell death (AICD) following mitogenic stimulation. Reduced AICD was also transferable and IL-6 dependent. Thus, the hyperproliferation was in part due to enhanced activated T cell survival. These phenomena mediated by accessory cells may contribute to the powerful polyclonal activation of type 2 immunity caused by nematode infection.
Relative Contribution of LFA-1 and Mac-1 to Neutrophil Adhesion and Migration
Authors: Ding Zhi-Ming , Babensee Julia E. , Simon Scott I. , Lu Huifang , Perrard Jerry L. , Bullard Daniel C. , Dai Xiao Y. , Bromley Shannon K. , Dustin Michael L. , Entman Mark L. , Smith C. Wayne , Ballantyne Christie M.
Published: December 2022

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-α was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-α in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.
A Novel LPS-Inducible C-Type Lectin Is a Transcriptional Target of NF-IL6 in Macrophages
Authors: Matsumoto Makoto , Tanaka Takashi , Kaisho Tsuneyasu , Sanjo Hideki , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Akira Shizuo
Published: December 2022

C-type lectins serve multiple functions through recognizing carbohydrate chains. Here we report a novel C-type lectin, macrophage-inducible C-type lectin (Mincle), as a downstream target of NF-IL6 in macrophages. NF-IL6 belongs to the CCAAT/enhancer binding protein (C/EBP) of transcription factors and plays a crucial role in activated macrophages. However, what particular genes are regulated by NF-IL6 has been poorly defined in macrophages. Identification of downstream targets is required to elucidate the function of NF-IL6 in more detail. To identify downstream genes of NF-IL6, we screened a subtraction library constructed from wild-type and NF-IL6-deficient peritoneal macrophages and isolated Mincle that exhibits the highest homology to the members of group II C-type lectins. Mincle mRNA expression was strongly induced in response to several inflammatory stimuli, such as LPS, TNF-α, IL-6, and IFN-γ in wild-type macrophages. In contrast, NF-IL6-deficient macrophages displayed a much lower level of Mincle mRNA induction following treatment with these inflammatory reagents. The mouse Mincle proximal promoter region contains an indispensable NF-IL6 binding element, demonstrating that Mincle is a direct target of NF-IL6. The Mincle gene locus was mapped at 0.6 centiMorgans proximal to CD4 on mouse chromosome 6.
Src-Regulated Extracellular Signal-Related Kinase and Syk-Regulated c-Jun N-Terminal Kinase Pathways Act in Conjunction to Induce IL-1 Synthesis in Response to Microtubule Disruption in HL60 Cells
Authors: Cambien Béatrice , Millet Marie-Ange , Schmid-Antomarchi Heidy , Brossette Nicole , Rossi Bernard , Schmid-Alliana Annie
Published: December 2022

A microtubule reorganization is often observed during cellular contacts that are associated to IL-1 production. Here, we show that in HL60 cells, vincristine, a microtubule-disrupting agent that induces a strong production of IL-1, triggers the activation of both extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK-1). While ERK activation is rapid and transient, peaking at 10 min, the JNK1 activation is delayed and more sustained reaching a maximum at 2 h. ERK activation was blocked by CP 118556, indicating it is regulated by a Src-like kinase, while JNK1 was inhibited by piceatannol, revealing an upstream regulation by Syk. Each kind of the nonreceptor tyrosine kinase blockers efficiently inhibits the vincristine-induced IL-1 production and diminishes the level of IL-1 transcripts, indicating that the ERK and JNK pathways act coordinately to elicit the transcription of the IL-1 gene. Furthermore, we found that pertussis toxin, a blocker of Go/Gi proteins, abrogated the vincristine-induced activation of both Src and Syk. Our data support a model where the status of microtubule polymerization influences the activity of Go or Gi proteins that control, in turn, two independent Src/ERK and Syk/JNK1 cascades that are both necessary to sustain IL-1 synthesis.