bluuebunny/crossref_metadata_2025 huggingface dataset:
SB 203580 Inhibits p38 Mitogen-Activated Protein Kinase, Nitric Oxide Production, and Inducible Nitric Oxide Synthase in Bovine Cartilage-Derived Chondrocytes
Authors: Badger Alison M. , Cook Michael N. , Lark Michael W. , Newman-Tarr Tonie M. , Swift Barbara A. , Nelson Allen H. , Barone Frank C. , Kumar Sanjay
Published: January 2023

Nitric oxide (NO) is implicated in a number of inflammatory processes and is an important mediator in animal models of rheumatoid arthritis and in in vitro models of cartilage degradation. The pyridinyl imidazole SB 203580 inhibits p38 mitogen-activated protein (MAP) kinase in vitro, blocks proinflammatory cytokine production in vitro and in vivo, and is effective in animal models of arthritis. The purpose of this study was to determine whether SB 203580 could inhibit p38 MAP kinase activity, NO production, and inducible NO synthase (iNOS) in IL-1 stimulated bovine articular cartilage/chondrocyte cultures. The results indicated that SB 203580 inhibited both IL-1 stimulated p38 MAP kinase activity in isolated chondrocytes and NO production in bovine chondrocytes and cartilage explants with an IC50 value of approximately 1 μM. To inhibit NO production, SB 203580 had to be present in cartilage explant cultures during the first 8 h of IL-1 stimulation, and activity was lost when it was added 24 h following IL-1. SB 203580 did not inhibit iNOS activity, as measured by the conversion of arginine to citrulline, when added directly to cultures where the enzyme had already been induced, but had to be present during the induction period. Using a 372-bp probe for bovine iNOS we demonstrated inhibition of IL-1-induced mRNA by SB 203580 at both 4 and 24 h following IL-1 treatment. The iNOS mRNA levels were consistent with NO levels in 24-h cell culture supernatants of the IL-1-stimulated bovine chondrocytes used to obtain the RNA.
Requirements for B7-CD28 Costimulation in Mucosal IgA Responses: Paradoxes Observed in CTLA4-Hγ1 Transgenic Mice
Authors: Gärdby Eva , Lane Peter , Lycke Nils Y.
Published: January 2023

The block in the CD80/CD86-CD28/CTLA-4 pathway in CTLA4-Hγ1 transgenic (Tg) mice results in strongly impaired systemic IgG immunity and failure to develop germinal center reactions. By contrast, here we report that mucosal immunity and IgA B cell differentiation are not affected by this block. We found abundant germinal centers and evidence of IgA switch differentiation in Peyer’s patches, normal total IgA levels, and normal numbers of IgA-labeling cells in the gut mucosa. The distribution of B-1 and B-2 cells and the relative contribution of B-1 cells to the total IgA B cells were similar in Tg and wild-type mice. Despite this, oral immunizations with keyhole limpet hemocyanin plus cholera toxin adjuvant failed to stimulate Ag-specific mucosal IgA responses in CTLA4-Hγ1 Tg mice. This was not due to a lack of adjuvant activity of cholera toxin in Tg mice, nor was this secondary to an inability to take up Ag from the gut lumen. Rather, CD4+ T cells stimulated by oral immunization in Tg mice appeared to be inappropriately primed, as evidenced by a significantly reduced level of CD40 ligand and CD44 expression and an increased expression of CD95 compared to those in wild-type mice. This study reveals a paradox in the regulation of mucosal IgA responses.
Cutting Edge: Linked Suppression of Skin Graft Rejection Can Operate Through Indirect Recognition
Authors: Wise Matt P. , Bemelman Frederike , Cobbold Steve P. , Waldmann Herman
Published: January 2023

Adult mice can be rendered immunologically tolerant of allogeneic tissues if transplanted under cover of mAbs to CD4 and CD8. Tolerance generated in this manner is characterized by the presence of regulatory CD4+ T cells that can recruit naive T cells to become tolerant also through “infectious tolerance.” Regulatory CD4+ T cells can also suppress rejection of third party transplant Ags provided they are expressed on the same graft as the tolerated Ags. This process of linked suppression can act across whole MHC barriers and represents a powerful mechanism with therapeutic potential. Tolerance can also be induced to reprocessed minor transplantation Ags presented through host APCs (indirect recognition). We here demonstrate that linked suppression can also be induced through the indirect pathway. This finding may be important in the development of transplantation tolerance in the clinic.
The Role of Host (Endogenous) T Cells in Chronic Graft-Versus-Host Autoimmune Disease
Authors: Chen Fangqi , Maldonado Michael A. , Madaio Michael , Eisenberg Robert A.
Published: January 2023

Chronic graft-vs-host (cGVH) disease induced by the transfer of Ia-incompatible spleen cells from one normal mouse strain (such as B6.C-H2bm12/KhEg (bm12)) to another (such as C57BL/6) causes an autoimmune syndrome resembling systemic lupus erythematosus (SLE). The role of host-derived T cells in this response is not obvious. Previous reports suggested that host T cells might serve to down-regulate the autoimmune syndrome. To address this issue more definitively, we used CD4 knockout (KO) or CD8KO C57BL/6 (B6) mice as recipients in the bm12→C57B6 cGVH model. CD4KO B6 mice injected with allogeneic bm12 spleen cells (bm12→CD4KO group) showed no evidence of cGVH disease. They made no detectable autoantibodies, including anti-chromatin, anti-dsDNA, anti-ssDNA, and rheumatoid factor. They survived at least 20 wks after induction of cGVH disease; and they did not develop nephritis, based on the absence of detectable levels of proteinuria and normal renal histology at the time of sacrifice. By contrast, CD8KO B6 mice (bm12→CD8KO group) and normal B6 mice (bm12→B6 group) injected with bm12 spleen cells generally showed similar levels of mortality, nephritis, and autoantibodies, although the autoantibody titers declined somewhat after week 8 in the bm12→CD8KO group. Control groups of recipients injected with B6 spleen cells showed no induction of autoantibodies. A surprising finding, however, was that the B6→CD8KO group developed severe histologic glomerulonephritis in the absence of autoantibodies and with decreased immune deposits. These results indicate that endogenous (host) CD4+ T cells play an essential role in the cGVH autoimmune syndrome.
Induction of Antibodies Reactive with SSA/Ro-SSB/La and Development of Congenital Heart Block in a Murine Model
Authors: Miranda-Carús M. Eugenia , Boutjdir Mohamed , Tseng Chung-E , DiDonato Francis , Chan Edward K. L. , Buyon Jill P.
Published: January 2023

To correlate the arrhythmogenic effects of maternal autoantibodies with the genesis of congenital heart block, female BALB/c mice were immunized with human recombinant 48-kDa SSB/La, 60-kDa SSA/Ro, 52-kDa SSA/Ro (52α), and 52β (amino acids 169–245 deleted) as well as with murine recombinant 52-kDa SSA/Ro. Control animals received β-galactosidase or a polypeptide encoded by pET-28 alone. Following primary immunization and two boosters, high titer responses to the respective Ags were established by ELISA, immunoblotting, and immunoprecipitation. Sera from mice immunized with either human 52α or 52β immunoprecipitated murine 52Ro. mRNA and protein expression of 52Ro was demonstrated in the newborn murine heart. A spectrum of atrioventricular nodal conduction abnormalities was identified by electrocardiogram. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48La, 20% of 54 pups born to 60Ro-immunized mothers, 6% of 56 pups born to 52α-immunized mothers, 7% of 86 pups born to 52β-immunized mothers, and 9% of 22 pups born to mothers immunized with murine 52Ro. Advanced conduction abnormalities were only identified in offspring of 52α- or 52β-immunized mice. In the 52α group, one pup had complete block and another had second-degree block (Wenckebach type); in the 52β group, five pups had complete block. Maternal Abs to the primary immunogens were detected in the pups. No control had any conduction abnormalities. This Ab-specific animal model provides strong evidence for a pathogenic role of anti-SSA/Ro-SSB/La Abs, particularly 52Ro, in the development of congenital heart block. The range and frequency of conduction defects suggest that additional factors promote disease expression.
Requirement for Distinct Janus Kinases and STAT Proteins in T Cell Proliferation Versus IFN-γ Production Following IL-12 Stimulation
Authors: Ahn Hyun-Jong , Tomura Michio , Yu Wen-Gong , Iwasaki Masayuki , Park Woong-Ryeon , Hamaoka Toshiyuki , Fujiwara Hiromi
Published: January 2023

While IL-12 is known to activate JAK2 and TYK2 and induce the phosphorylation of STAT4 and STAT3, little is known regarding how the activation of these signaling molecules is related to the biologic effects of IL-12. Using an IL-12-responsive T cell clone (2D6), we investigated their requirements for proliferation and IFN-γ production of 2D6 cells. 2D6 cells could be maintained with either IL-12 or IL-2. 2D6 lines maintained with IL-12 (2D6IL-12) or IL-2 (2D6IL-2) exhibited comparable levels of proliferation, but produced large or only small amounts of IFN-γ, respectively, when restimulated with IL-12 after starvation of either cytokine. 2D6IL-12 induced TYK2 and STAT4 phosphorylation. In contrast, their phosphorylation was marginally induced in 2D6IL-2. The reduced STAT4 phosphorylation was due to a progressive decrease in the amount of STAT4 protein along with the passages in IL-2-containing medium. 2D6IL-12 and 2D6IL-2 similarly proliferating in response to IL-12 induced comparable levels of JAK2 activation and STAT5 phosphorylation. JAK2 was associated with STAT5, and IL-12-induced STAT5 phosphorylation was elicited in the absence of JAK3 activation. These results indicate that IL-12 has the capacity to induce/maintain STAT4 and STAT5 proteins, and that TYK2 and JAK2 activation correlate with STAT4 phosphorylation/IFN-γ induction and STAT5 phosphorylation/cellular proliferation, respectively.
TNF/Lymphotoxin-α Double-Mutant Mice Resist Septic Arthritis but Display Increased Mortality in Response toStaphylococcus aureus
Authors: Hultgren Olof , Eugster Hans-Pietro , Sedgwick Jonathon D. , Körner Heinrich , Tarkowski Andrzej
Published: January 2023

To evaluate the importance of the proinflammatory cytokines TNF and lymphotoxin-α (LTα) in an experimental model of Staphylococcus aureus sepsis and arthritis, we used TNF/LTα-double-deficient mice raised on the C57BL/6 background. Mice were i.v. inoculated with a toxic shock syndrome toxin-1 (TSST-1)-producing S. aureus strain, LS-1. Intravenous inoculation of a high dose of bacteria (1 × 107/mouse) resulted in 67% mortality in TNF/LTα-deficient mice, whereas none of the controls died (p = 0.009). Those results correlated to a significantly decreased phagocytosis in vitro and inefficient bacterial clearance in vivo in mice lacking capacity to produce TNF/LTα. Thus, at day 6 after inoculation, S. aureus could not be found in the bloodstream of controls, but bacteremia developed in all TNF/LTα-deficient mice examined (p = 0.02). Interestingly, upon infection with a lower dose of staphylococci (3 × 106/mouse) the mortality was overall low, but the frequency of arthritis was clearly higher in the wild-type group as compared with the TNF/LTα-deficient mice (40% vs 13%). Histopathologic examination revealed a lower frequency of synovitis (38% vs 90%, p < 0.05) and erosivity (25% vs 60%, NS) in TNF/LTα-deficient mice as compared with wild-type counterparts. Our results show the importance of TNF/LTα in defense against systemic S. aureus infections and point out the detrimental role of these cytokines as mediators of inflammatory response in S. aureus arthritis.
MHC Class I Molecules Compete in the Endoplasmic Reticulum for Access to Transporter Associated with Antigen Processing
Authors: Knittler Michael R. , Gülow Karsten , Seelig Angela , Howard Jonathan C.
Published: January 2023

We have used the functionally distinct TAP alleles of the rat in cellular transfectants as tools to investigate how newly formed rat class I (RT1.A) molecules with distinct peptide requirements gain access to suitable peptides in the endoplasmic reticulum (ER). Normal maturation of RT1.Aa depends on the presence in the ER of peptides with C-terminal arginine, while restrictive TAP-B allelic group transporters fail to transport such peptides. In this situation, RT1.Aa is retained in the ER. We show that this retention is accompanied by accumulation of RT1.Aa in the ER, partly associated with TAP and partly free. In such cells, access to TAP of a second allelic product, RT1.Au, which does not require C-terminal arginine peptides, is competitively inhibited by the build-up of RT1.Aa. Nevertheless, RT1.Au loads and matures normally. Introduction of a permissive TAP-A allele competent to transport C-terminal arginine peptides releases RT1.Aa from the ER and restores RT1.Au interaction with TAP. Both class I alleles associate indiscriminately with permissive and restrictive TAP alleles. The data support the view that interaction with TAP is not a prerequisite for peptide loading by class I molecules, so long as suitable peptides are available in the ER. They further show that TAP association of a class I molecule depends on a competitive balance in the ER defined by the extent to which the peptide requirements of other class I molecules present are satisfied and not only by the intrinsic strength of the interaction with TAP.
Sequence Motifs in the Integrin α4 Cytoplasmic Tail Required for Regulation of In Vivo Expansion of Murine Lymphoma Cells
Authors: Bittner Michaela , Gosslar Uwe , Luz Arne , Holzmann Bernhard
Published: January 2023

The binding of integrins to cognate ligands is tightly controlled by intracellular signals. Conversely, integrin occupancy generates biochemical signals inside the cell. The present study examined whether concepts of integrin function established by in vitro analysis apply to regulation of receptor function in complex biologic settings in vivo using a mouse model of tumor metastasis. Integrin α4 subunits were truncated at amino acid Gln1014 (A4-1014), preserving the conserved GFFKR motif, and at position Glu1021 (A4-1021). In vitro adhesion assays revealed that cytoplasmic tail truncations did not affect constitutive ligand binding of α4 integrins, while agonist-induced adhesion was abolished by the A4-1014, but not by the A4-1021, mutation. Inducible ligand binding of α4 integrins was dependent on cytoskeletal function, whereas constitutive adhesion was not. In vivo metastasis formation assays demonstrated that expansion of murine T lymphoma cells in spleen is strongly inhibited by the wild-type α4 subunit and the A4-1021 mutant. In contrast, the in vivo phenotype of α4 integrin expression in lymphoma cells was completely abrogated by the A4-1014 mutation. Cross-linking of α4 integrins in vitro inhibited proliferation and induced apoptosis of LB cells expressing wild-type α4 subunits or the A4-1021 mutant, but not of LB-A4-1014 cells. In summary, these results demonstrate that sequence motifs regulating cytoskeleton-dependent α4 integrin activation in vitro are essential for the control of LB lymphoma cell expansion both in vitro and in vivo.
Recruitment of Hepatic NK Cells by IL-12 Is Dependent on IFN-γ and VCAM-1 and Is Rapidly Down-Regulated by a Mechanism Involving T Cells and Expression of Fas
Authors: Fogler William E. , Volker Kirk , Watanabe Morihiro , Wigginton Jon M. , Roessler Philip , Brunda Michael J. , Ortaldo John R. , Wiltrout Robert H.
Published: January 2023

NK cells have been shown to be important antitumor or antiviral effector cells in the liver. In the present study we have examined the factors that regulate the initial recruitment and subsequent fate of hepatic NK and T cells in mice treated with IL-12 or IL-2. Daily administration of IL-12 caused a rapid initial increase in NK cells followed by a subsequent decrease that coincided with an accumulation of T cells. The recruitment of hepatic NK cells by IL-12, but not the subsequent T cell infiltrate, was abrogated in IFN-γ−/− mice. In contrast, daily administration of IL-2 caused a sustained increase in liver-associated NK cells that was not diminished in IFN-γ−/− mice. The IL-12-induced recruitment in both hepatic NK and T cells was abrogated by in vivo treatment with anti-VCAM-1 mAbs, while treatment with anti-ICAM-1 Abs decreased only the recruitment of T cells in the IL-12-treated mice. The rapid loss of newly recruited hepatic NK cells in IL-12-treated mice did not occur in SCID mice or in B.MRL-Faslpr (Fas−) and B6Smn.C3H-Faslgld (FasL−) mutant mice, suggesting that T cells can actively eliminate hepatic NK cells through a Fas-dependent mechanism. These findings also imply that during the endogenous innate immune response to infectious agents or tumors or in the host response induced by cytokine therapies, the biologic effects of NK cells may be limited by T cell-mediated effects.
Modulation of the IL-7 Dose-Response Threshold During Pro-B Cell Differentiation Is Dependent on Pre-B Cell Receptor Expression
Authors: Marshall Aaron J. , Fleming Heather E. , Wu Gillian E. , Paige Christopher J.
Published: January 2023

The IL-7R and the pre-B cell receptor (pre-BCR) each provide critical signals during differentiation of B cell precursors. In this study we examine the interplay between signals dependent upon these receptors. We demonstrate that pre-BCR-deficient pro-B cells differ significantly from controls in their ability to use the IL-7R. We show that this difference, characterized by a failure to proliferate in response to IL-7, is narrowly restricted to IL-7 concentrations in the picogram per milliliter range and can be overcome with increasing amounts of IL-7. Restoration of Ig heavy chain to recombinase-activating gene-2-deficient pro-B cells leads to a restored response to picogram per milliliter levels of IL-7, providing strong evidence that modulation of the IL-7 dose-response threshold is dependent on pre-BCR. Culture of normal pro-B cells under low IL-7 conditions leads to selective outgrowth of cells expressing μ heavy chain, suggesting that modulation of IL-7 dose-response thresholds can allow for selective expansion of pre-BCR+ cells under conditions where IL-7 is limiting. We also provide evidence that expression of pre-BCR on pro-B cells limits the duration of IL-7 responsiveness by causing differentiation to an IL-7-unresponsive pre-B cell stage. Thus, the pre-BCR-dependent modulation of IL-7 responsiveness affects both the dose-response threshold and the duration of IL-7-induced clonal expansion. Our results suggest that positive selection of pre-BCR+ pro-B cells may be achieved through the fine tuning of IL-7 responses.
Cyclosporin A Inhibits Inositol 1,4,5-Trisphosphate Binding to Its Receptors and Release of Calcium from Intracellular Stores in Peritoneal Macrophages
Authors: Misra Uma K. , Gawdi Govind , Pizzo Salvatore V.
Published: January 2023

We have studied the effects of the immunosuppressive drug cyclosporin A (CsA) on the generation of inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+ levels elicited upon ligation of murine macrophage receptors for α2-macroglobulin, bradykinin, epidermal growth factor, and platelet-derived growth factor. Preincubation of cells with CsA (500 ng/ml), either alone or with the various ligands, did not inhibit the synthesis of IP3. However, we observed 70–80% inhibition of the binding of [3H]IP3 to IP3 receptors on macrophage membranes isolated from CsA-treated macrophages. Preincubation of macrophages with CsA abolished IP3-mediated release of Ca2+ from intracellular stores and Ca2+ entry from the extracellular medium observed when macrophage receptors were stimulated with ligands in the absence of CsA. Preincubation of macrophages with CsA also significantly inhibited DNA synthesis induced by ligands for all four receptors studied. Thus in macrophages, as in T cells, CsA blocks receptor-activated signal transmission pathways characterized by an initial increase in intracellular Ca2+ concentration. This inhibition appears to result from a drug effect on IP3 receptors.
Antisense Oligonucleotides Targeting Protein Kinase C-α, -βI, or -δ But Not -η Inhibit Lipopolysaccharide-Induced Nitric Oxide Synthase Expression in RAW 264.7 Macrophages: Involvement of a Nuclear Factor κB-Dependent Mechanism
Authors: Chen Ching-Chow , Wang Jia-Kae , Lin Shwu-Bin
Published: January 2023

The signaling pathway for protein kinase C (PKC) activation and the role of PKC isoforms in LPS-induced nitric oxide (NO) release were studied in RAW 264.7 macrophages. The tyrosine kinase inhibitor genestein attenuated LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression, as did the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 and the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609. LPS stimulated phosphatidylinositol (PI) hydrolysis and PKC activity in RAW cells; both were inhibited by genestein. The PKC inhibitors (staurosporine, calphostin C, Ro 31-8220, or Go 6976) or long-term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment also resulted in inhibition of LPS-induced NO release and iNOS expression. Western blot analysis showed expression of PKC-α, -βI, -δ, -η, and -ζ in RAW cells; down-regulation of PKC-α, -βI, and -δ, but not -η, was seen after long-term TPA treatment, indicating the possible involvement of one or all of PKC-α, -βI, and -δ, but not -η, in LPS-mediated effects. Treatment with antisense oligonucleotides for these isoforms further demonstrated the involvement of PKC-α, -βI, and δ, but not -η, in LPS responses. Stimulation of cells with LPS for 1 h caused activation of NF-κB in the nuclei by detection of NF-κB-specific DNA-protein binding; this was inhibited by genestein, U73122, D609, calphostin C, or antisense oligonucleotides for PKC-α, -βI, and -δ, but not -η. These data suggest that LPS activates PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, resulting in the stimulation of NF-κB DNA-protein binding, then initiated the expression of iNOS and NO release. PKC isoforms α, βI, and δ were shown to be involved in the regulation of these LPS-induced events.
Lymphotactin Gene-Modified Bone Marrow Dendritic Cells Act as More Potent Adjuvants for Peptide Delivery to Induce Specific Antitumor Immunity
Authors: Cao Xuetao , Zhang Weiping , He Long , Xie Zhifang , Ma Shihua , Tao Qun , Yu Yizhi , Hamada Hirofumi , Wang Jianli
Published: January 2023

Dendritic cells (DC) are regarded as attractive candidates for cancer immunotherapy. Our aim is to improve the therapeutic efficacy of DC-based tumor vaccine by augmenting DC preferential chemotaxis on T cells. Mouse bone marrow-derived DC were transduced with lymphotactin (Lptn) gene by adenovirus vector. The supernatants from Lptn gene-modified DC (Lptn-DC) were capable of attracting CD4+ and CD8+ T cells in a chemotaxis assay, whereas their mock control could not. Lptn expression of Lptn-DC was further confirmed by RT-PCR. Lptn-DC were pulsed with Mut1 peptide and used for vaccination. Immunization with the low dose (1 × 104) of Mut1 peptide-pulsed DC induced weak CTL activity, whereas the same amounts of Mut1 peptide-pulsed Lptn-DC markedly induced specific CTL against 3LL tumor cells. A single immunization with 1 × 104 Mut1 peptide-pulsed Lptn-DC could render mice resistant to a 5 × 105 3LL tumor cell challenge completely, but their counterpart could not. The protective immunity induced by Mut1 peptide-pulsed Lptn-DC depends on both CD4+ T cells and CD8+ T cells rather than NK cells in the induction phase and depends on CD8+ T cells rather than CD4+ T cells and NK cells in the effector phase. Moreover, the involvement of CD28/CTLA4 costimulation pathway and IFN-γ are also necessary. When 3LL tumor-bearing mice were treated with 1 × 104 Mut1 peptide-pulsed Lptn-DC, their pulmonary metastases were significantly reduced, whereas the same low dose of Mut1 peptide-pulsed DC had no obvious therapeutic effects. Our data suggest that Lptn-DC are more potent adjuvants for peptide delivery to induce protective and therapeutic antitumor immunity.
Neutralizing Antibodies to IFN-γ-Inducing Factor Prevent Experimental Autoimmune Encephalomyelitis
Authors: Wildbaum Gizi , Youssef Sawsan , Grabie Nir , Karin Nathan
Published: January 2023

Specific oligonucleotide primers were used to identify and isolate IFN-γ-inducing factor (IGIF) from the brain of rats with developing experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as a model for multiple sclerosis. IGIF was highly transcribed in the brain at the onset and during the course of active EAE. PCR products encoding rat IGIF were used to generate the recombinant protein that was used to induce anti-IGIF neutralizing Abs. These Abs significantly reduced the production of IFN-γ by primed T cells proliferating in response to their target myelin basic protein epitope and by Con A-activated T cells from naive donors. When administered to rats during the development of either active or transferred EAE, these Abs significantly blocked the development of disease. Splenic T cells from protected rats were cultured with the encephalitogenic myelin basic protein epitope and evaluated for production of IL-4 and IFN-γ. These cells, which proliferated, exhibited a profound increase in IL-4 production that was accompanied by a significant decrease in IFN-γ and TNF-α production. Thus, we suggest that perturbation of the Th1/Th2 balance toward Th2 cells is the mechanism underlying EAE blockade by anti-IGIF immunotherapy.
A Model for the Origin of TCR-αβ+ CD4−CD8− B220+ Cells Based on High Affinity TCR Signals
Authors: Mixter Philip F. , Russell Jennifer Q. , Morrissette Gregory J. , Charland Colette , Aleman-Hoey Deborah , Budd Ralph C.
Published: January 2023

The origin of TCR-αβ+ CD4−CD8− cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-αβ+ CD4−CD8− cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-αβ+ CD4−CD8− thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rβ and the absence of surface CD2. The CD4−CD8− B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-αβ+ CD2− CD4−CD8− B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.
Limiting TCR Expression Leads to Quantitative But Not Qualitative Changes in Thymic Selection
Authors: Dave Vibhuti P. , Allman David , Wiest David L. , Kappes Dietmar J.
Published: January 2023

Thymic selection is controlled in part by the avidity of the interaction between thymocytes and APCs. In agreement, the selective outcome can be modulated by altering the expression levels of selecting ligands on APCs. Here we test the converse proposition, i.e., whether changing TCR levels on thymocytes can alter the selective outcome. To this end, we have generated mice in which all thymocytes express two transgenic TCRs simultaneously (dual TCR-expressing (DTE) mice), the class I-restricted HY TCR and the class II-restricted AND TCR. Due to mutual dilution, surface expression levels of the two individual transgenic TCRs are diminished in DTE relative to single TCR-expressing mice. We find that thymic selection is highly sensitive to these reductions in TCR surface expression. Positive selection mediated by the AND and HY TCRs is severely impaired or abolished, respectively. Negative selection of the HY TCR in male DTE mice is also partly blocked, leading to the appearance of significant numbers of double positive thymocytes. Also, in the periphery of male, but not female, DTE mice, substantial numbers of single positive CD8bright cells accumulate, which are positively selected in the thymus but by a highly inefficient hemopoietic cell-dependent process. Overall our results favor the interpretation that the outcome of thymic selection is not determined solely by avidity and the resulting signal intensity, but is also constrained by other factors such as the nature of the ligand and/or its presentation by different subsets of APCs.
Costimulation Reverses the Defect in IL-2 But Not Effector Cytokine Production by T Cells with Impaired IκBα Degradation
Authors: Aune Thomas M. , Mora Ana L. , Kim Somee , Boothby Mark , Lichtman Andrew H.
Published: January 2023

Although the transcriptional basis for states of unresponsiveness in primary T cells is unclear, tolerant B lymphocytes exhibit inhibition of both c-Jun N-terminal kinase induction and IκBα (inhibitor of NF-κBα) degradation, leading to lower levels of both nuclear AP-1 and NF-κB. Expression of an IκBα mutant resistant to signal-induced degradation in transgenic T cells caused markedly deficient effector cytokine (IL-4, IFN-γ) production after primary TCR stimulation despite a detectable level of nuclear NF-κB. A TCR response element from the IFN-γ promoter, despite lacking detectable NF-κB/Rel sites, was also unresponsive to TCR ligation. Nuclear induction of AP-1 proteins in response to T cell activation was diminished in transgenic T cells. Costimulation induced by anti-CD28 mAb increased IL-2 production, but failed to reverse the defects in effector cytokine production. Taken together, these data indicate that impaired NF-κB/Rel signaling in T cells interferes with the signal transduction pathways required for efficient induction of effector cytokine production.
CTLA-4-Mediated Inhibition of Early Events of T Cell Proliferation
Authors: Brunner Monika C. , Chambers Cynthia A. , Chan Francis Ka-Ming , Hanke Jeff , Winoto Astar , Allison James P.
Published: January 2023

CTLA-4 engagement by mAbs inhibits, while CD28 enhances, IL-2 production and proliferation upon T cell activation. Here, we have analyzed the mechanisms involved in CTLA-4-mediated inhibition of T cell activation of naive CD4+ T cells using Ab cross-linking. CTLA-4 ligation inhibited CD3/CD28-induced IL-2 mRNA accumulation by inhibiting IL-2 transcription, which appears to be mediated in part through decreasing NF-AT accumulation in the nuclei. However, CTLA-4 ligation did not appear to affect the CD28-mediated stabilization of IL-2 mRNA. Further, CTLA-4 engagement inhibited progression through the cell cycle by inhibiting the production of cyclin D3, cyclin-dependent kinase (cdk)4, and cdk6 when the T cells were stimulated with anti-CD3/CD28 and with anti-CD3 alone. These results indicate that CTLA-4 signaling inhibits events early in T cell activation both at IL-2 transcription and at the level of IL-2-independent events of the cell cycle, and does not simply oppose CD28-mediated costimulation.
Antigen-Specific T Cell Activation and Proliferation During Oral Tolerance Induction
Authors: Sun Jiaren , Dirden-Kramer Bernadette , Ito Komei , Ernst Peter B. , Van Houten Nancy
Published: January 2023

One of several routes of achieving immunologic tolerance is through functional inactivation of Ag-specific T cells. Oral administration of Ag can allow survival of the Ag-specific T cells that are functionally anergic. The aim of this study was to investigate whether functional inactivation of Ag-specific T cells is directed through an activation process and to further define the differentiative pathways and functional characteristics of anergic T cells. Mice were transplanted with OVA-specific TCR-transgenic T cells and either fed OVA or immunized s.c. with the OVA peptide 323–339 in CFA. OVA-specific T cells from OVA-fed mice were unresponsive to restimulation in vitro within 48–72 h after treatment. In vivo, however, T cell proliferation was detected by 5,6-carboxy-succinimidyl-fluoresceine-ester intensity changes in OVA-specific T cells. The mesenteric lymph nodes (LNs) from OVA-fed mice more frequently contained OVA-specific dividing cells in vivo than those in the peripheral LNs, and the reciprocal was observed following s.c. immunization of the OVA peptide in CFA. The induction of anergy in OVA-fed mice was accompanied by rapid up-regulation of CD69 and CTLA-4, later down-regulation of CD45RB on OVA-specific T cells, and a marked decrease in T cell secretion of IL-2, IL-10, and IFN-γ after OVA restimulation in vitro. Results from this study indicate that the inductive phase of oral tolerance is preceded by Ag-specific T cell activation in vivo, proliferation in the regional draining LNs, and differentiation into a memory-like state. These results indicate that Ag-directed differentiation occurs as a part of T cell tolerance through anergy.
Syndecan-4 Is Expressed by B Lineage Lymphocytes and Can Transmit a Signal for Formation of Dendritic Processes
Authors: Yamashita Yoshio , Oritani Kenji , Miyoshi Erina K. , Wall Randolph , Bernfield Merton , Kincade Paul W.
Published: January 2023

Our previous studies indicated that stromal cell-derived syndecan-4 might mediate some form of communication with pre-B cells in bone marrow. We now report additional aspects of this recognition and show that syndecan-4 is also present on pre-B cells. Indeed, the molecule is acquired at an early stage of differentiation and retained until mature B cells undergo Ig isotype switching. mAbs developed to two portions of the syndecan-4 protein core were used to probe possible functions on B lineage lymphocytes. Syndecan-4 ligation had no obvious influence on B lymphocyte formation or activation, but this treatment caused a dramatic morphological change in appropriately stimulated leukocytes. Extended filopodia appeared on transfected Ba/F3 or FDCP-1 cells, as well as activated B cell blasts that were placed on syndecan-4 Ab-coated surfaces. The dendritic processes contained polymerized actin as well as pp52(LSP1), a prominent F-actin binding protein in lymphocytes. The cytoplasmic domain of syndecan-4 was not required for this response. Shape changes of this type could facilitate interactions between B lymphocytes and other components of the immune system. Not only is syndecan-4 a useful marker for discriminating normal B lineage lymphocyte subsets, but our results suggest new ways for the syndecans to participate in immune responses.
The Substance P Receptor Is Necessary for a Normal Granulomatous Response in Murine Schistosomiasis Mansoni
Authors: Blum Arthur M. , Metwali Ahmed , Kim-Miller Mindy , Li Jie , Qadir Khurram , Elliott David E. , Lu Bao , Fabry Zsuzsa , Gerard Norma , Weinstock Joel V.
Published: January 2023

Immune cells within the granulomas of murine schistosomiasis mansoni make the neuropeptide substance P (SP) and express neurokine 1 receptor, which is the specific receptor for substance P (SPr). It was determined if mice with deletion of the SPr (SPr−/−) would develop a normal granulomatous response to schistosome ova during the course of natural infection. Mean liver granuloma size was smaller in SPr−/− mice compared with that of wild-type control animals. Although flow analysis revealed little difference in the cellular composition of the granulomas, both splenocytes and granuloma cells from SPr−/− mice produced much less IFN-γ and IgG2a and less IgE. The expression of Th2 cytokines (IL-4/IL-5) and IgG1 was comparable to the wild-type control. The mouse with targeted disruption of its SPr had the nonmammalian gene encoding the enzyme β-galactosidase inserted in exon 1 of the SPr gene. There was β-galactosidase activity in many mononuclear cells scattered throughout the schistosome granulomas of SPr−/− mice. Also, a granuloma T cell line derived from this transgenic mouse produced β-galactosidase. These results provide further evidence that in murine schistosomiasis SPr is displayed commonly on granuloma inflammatory cells and is important for granuloma development and expression of IFN-γ circuitry in this natural infection.
Glucocorticoid Suppresses Autocrine Survival of Mast Cells by Inhibiting IL-4 Production and ICAM-1 Expression
Authors: Yoshikawa Hideshi , Nakajima Yasuo , Tasaka Kachio
Published: January 2023

When mast cells are activated through their high affinity IgE receptors (FcεRI), release of chemical mediators is followed by secretion of multiple cytokines. In this work, we report that IL-3-dependent mast cell line MC9 undergoes apoptosis when IL-3 is withdrawn. However, cross-linking of FcεRI prevents apoptosis of MC9 by an autocrine mechanism, producing IL-3, IL-4, and GM-CSF. Although stimulated MC9 synthesizes mRNAs and proteins of these cytokines, secretion of endogenous IL-3 and GM-CSF is not enough for cell survival, whereas IL-4 itself does not have survival effect on MC9, but it induces cell aggregation by expressing LFA-1 and makes it reactive to endogenous growth factors. Addition of dexamethazone (DXM) to MC9 results in significant down-regulation of IL-4 mRNA in activated MC9. However, mRNA levels of IL-3 and GM-CSF are not changed by DXM. DXM also directly down-regulates the expression of ICAM-1 that is the high affinity ligand of LFA-1, by which the self-aggregation of MC9 is inhibited. Thus, glucocorticoids suppress autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression.
Regulation of Monocyte Survival In Vitro by Deposited IgG: Role of Macrophage Colony-Stimulating Factor
Authors: Marsh Clay B. , Pomerantz Richard P. , Parker Jennifer M. , Winnard Alissa V. , Mazzaferri Ernest L. , Moldovan Nicanor , Kelley Todd W. , Beck Eric , Wewers Mark D.
Published: January 2023

IgG deposition at tissue sites characteristically leads to macrophage accumulation and organ injury. Although the mechanism by which deposited IgG induces tissue injury is not known, we have recently demonstrated that deposited IgG stimulates the release of IL-8 and monocyte chemoattractant protein-1 from normal human monocytes, which may drive inflammation. Since IgG also induces macrophage accumulation in these diseases, we hypothesized that deposited IgG protects monocytes from apoptosis. As an in vitro model of the effect of deposited IgG on monocyte survival, monocyte apoptosis was studied after FcγR cross-linking. Monocytes cultured on immobilized IgG, which induces FcγR cross-linking, were protected from apoptosis, whereas monocytes cultured with equivalent concentrations of F(ab′)2 IgG or 50 times higher concentrations of soluble IgG, neither of which induces FcγR cross-linking, were not protected. Moreover, this protection was transferable, as supernatants from immobilized IgG-stimulated monocytes protected freshly isolated monocytes from apoptosis and contained functional M-CSF, a known monocyte survival factor. M-CSF mediated the monocyte survival induced by FcγR cross-linking, as neutralizing anti-human M-CSF Abs blocked the monocyte protection provided by either immobilized IgG or IgG-stimulated monocyte supernatants. These findings demonstrate a novel mechanism by which deposited IgG targets tissue macrophage accumulation through FcγR-mediated M-CSF release. This pathway may play an important role in promoting and potentiating IgG-mediated tissue injury.
Cutting Edge: Clonally Restricted Production of the Neurotrophins Brain-Derived Neurotrophic Factor and Neurotrophin-3 mRNA by Human Immune Cells and Th1/Th2-Polarized Expression of Their Receptors
Authors: Besser Michal , Wank Rudolf
Published: January 2023

Neurotrophins, such as neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), are potent regulators of neuronal functions. Here we show that human immune cells also produce NT-3 mRNA, secrete BDNF, and express their specific receptors trkB and trkC. The truncated trkB receptor, usually expressed in sensory neurons of the central nervous system, was also constitutively expressed in unstimulated Th cells. Full-length trkB was detectable in stimulated PBMC, B cell lines, and Th1, but not in Th2 and Th0 cell clones. Clonally restricted expression was also observed for trkC, until now not detected on blood cells. The Th1 cytokine IL-2 stimulated production of trkB mRNA but not of trkC, whereas the Th2 cytokine IL-4 enhanced NT-3 but not BDNF mRNA expression. Microbial Ags, which influence the Th1/Th2 balance, could therefore modulate the neurotrophic system and thereby affect neuronal synaptic activity of the central nervous system.
Cutting Edge: Apoptosis of Superantigen-Activated T Cells Occurs Preferentially After a Discrete Number of Cell Divisions In Vivo
Authors: Renno Toufic , Attinger Antoine , Locatelli Sabrina , Bakker Talitha , Vacheron Sonia , MacDonald H. Robson
Published: January 2023

Staphylococcal enterotoxins are bacterial products that display superantigen activity in vitro as well as in vivo. For instance, staphylococcal enterotoxin B (SEB) polyclonally activates T cells that bear the Vβ8 gene segment of the TCR. SEB-activated T cells undergo a burst of proliferation that is followed by apoptosis. Using an in vivo adaptation of a fluorescent cell division monitoring technique, we show here that SEB-activated T cells divide asynchronously, and that apoptosis of superantigen-activated T cells is preferentially restricted to cells which have undergone a discrete number of cell divisions. Collectively, our data suggest that superantigen-activated T cells are programmed to undergo a fixed number of cell divisions before undergoing apoptosis. A delayed death program may provide a mechanistic compromise between effector functions and homeostasis of activated T cells.
Inhibition of B Cell Receptor-Mediated Apoptosis by IFN
Authors: Su Leon , David Michael
Published: January 2023

IFNs are a family of cytokines that are involved in the regulation of immune and inflammatory responses. Clinical use of IFN-α/β encompasses treatment for a variety of diseases; however, prolonged exposure to IFN-α/β results in elevated levels of autoreactive Abs. In this study, we investigated the potential of IFNs to modulate apoptotic signals in B cells. We demonstrate that IFN-α or IFN-β inhibit Ag receptor-mediated apoptosis in a dose-dependent manner. Inhibition of phosphatidylinositol 3′ (PI3)-kinase did not abolish the effect of IFN, indicating that the antiapoptotic mechanism is PI3-kinase- and protein kinase B/Akt-independent. Instead, IFN-α and IFN-β, but not IFN-γ, significantly increase the levels of the survival protein Bcl-2, and to a lesser extent, Bcl-xL expression. Thus, IFN-α/β-mediated inhibition of B cell Ag receptor-triggered apoptosis may offer a model for the process that leads to the escape of self-reactive B cells from negative selection and consequently results in autoantibody production.
Cross-Linking of Membrane CD43 Mediates Dendritic Cell Maturation
Authors: Corinti Silvia , Fanales-Belasio Emanuele , Albanesi Cristina , Cavani Andrea , Angelisova Pavla , Girolomoni Giampiero
Published: January 2023

CD43/leukosialin is a major sialoglycoprotein of the dendritic cell (DC) surface, which can regulate cell adhesion and has the potential to mediate cell activation signals. Monocyte-derived DC transiently incubated with the anti-CD43 mAb, MEM-59, or with F(ab′)2 fragments, but not with monovalent Fab fragments or control IgG, 24 h later showed increased levels of membrane HLA-DR, CD54, CD40, CD80, CD86, and CD83. In parallel, CD43 cross-linking induced synthesis and release of IL-1β, IL-6, TNF-α, IL-12, and IL-10. CD43 ligation inhibited the endocytic activity of DC, and enhanced the capacity of DC to stimulate T cell proliferation in the primary allogeneic and autologous MLR assay. In addition, anti-CD43-treated DC were less efficient at presenting native HIV-1 reverse transcriptase to a specific CD4+ T cell clone, whereas presentation of the reverse transcriptase 55–72 peptide to the same clone was increased. Finally, MEM-59 or its F(ab′)2 fragments elicited a rise in intracellular free calcium and tyrosine phosphorylation of a 25-kDa protein in DC. The results thus indicate that CD43 cross-linking with specific ligands induces activation and functional maturation of DC.
Maturation of Dendritic Cells Accompanies High-Efficiency Gene Transfer by a CD40-Targeted Adenoviral Vector
Authors: Tillman Bryan W. , Gruijl Tanja D. de , Bakker Sylvia A. Luykx-de , Scheper Rik J. , Pinedo Herbert M. , Curiel Tyler J. , Gerritsen Winald R. , Curiel David T.
Published: January 2023

Important therapeutic applications of genetically modified dendritic cells (DC) have been proposed; however, current vector systems have demonstrated only limited gene delivery efficacy to this cell type. By means of bispecific Abs, we have dramatically enhanced gene transfer to monocyte derived DC (MDDC) by retargeting adenoviral (Ad) vectors to a marker expressed on DC, CD40. Adenovirus targeted to CD40 demonstrated dramatic improvements in gene transfer relative to untargeted Ad vectors. Fundamental to the novelty of this system is the capacity of the vector itself to modulate the immunological status of the MDDC. This vector induces DC maturation as demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a MLR. In comparing this vector to other Ad-based gene transfer systems, we have illustrated that the features of DC maturation are not a function of the Ad particle, but rather a consequence of targeting to the CD40 marker. This vector approach may thus mediate not only high-efficiency gene delivery but also serve a proactive role in DC activation that could ultimately strengthen the utility of this vector for immunotherapy strategies.
Induction of T Cell Anergy by Low Numbers of Agonist Ligands
Authors: Korb Laura C. , Mirshahidi Saied , Ramyar Kasra , Sadighi Akha Amir A. , Sadegh-Nasseri Scheherazade
Published: January 2023

Engagement of TCR by its ligand, the MHC/peptide complex, causes T cell activation. T cells respond positively to stimulation with agonists, and are inhibited by antagonist MHC/peptide ligands. Failure to induce proper conformational changes in the TCR or fast TCR/MHC dissociation are the leading models proposed to explain anergy induction by antagonist ligands. In this study, we demonstrate that presentation of between 1 and 10 complexes of agonist/MHC II by unfixed APC induces T cell anergy that persists up to 7 days and has characteristics similar to anergy induced by antagonist ligand or TCR occupancy without costimulation. Furthermore, anergy-inducing doses of hemagglutinin 306–318 peptide led to the engagement of less than 1000 TCR/CD3 complexes. Thus, engagement of a subthreshold number of TCR by either a low density of agonist/MHC or a 2–3 orders of magnitude higher density of antagonist/MHC causes anergy. Moreover, we show that anergy induced by low agonist concentrations is inhibited in the presence of IL-2 or cyclosporin A, suggesting involvement of the calcineurin signaling pathway.
A Critical Role for Antigen-Specific Th1 Cells in Acute Liver Injury in Mice
Authors: Nishimura Takashi , Ohta Akio
Published: January 2023

A novel liver injury model was established in mice by targeting of OVA-containing liposomes into the liver, followed by adoptive transfer of OVA-specific Th1 cells. Combined treatment of mice with OVA-containing liposomes and Th1 cell transfer caused an increase in serum transaminase activity that was paralleled with an elevation of serum IFN-γ levels. In sharp contrast, OVA-specific Th2 cell transfer resulted in an increase of serum IL-4 levels, but did not induce liver injury. Neither NK, NK T, nor CD8+ T cells were required for the Th1-induced liver injury. The liver injury was blocked by anti-IFN-γ mAb and anti-TNF-α mAb, but not by anti-Fas ligand mAb. The Fas/Fas ligand independency was also demonstrated using Fas-deficient lpr mice. These findings indicate that Th1 cells are the major effector cells in acute liver injury.