bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR Engagement Regulates Differential Responsiveness of Human Memory T Cells to Fas (CD95)-Mediated Apoptosis
Authors: Di Somma M. Maddalena , Somma Francesca , Montani Maria Saveria Gilardini , Mangiacasale Rosamaria , Cundari Enrico , Piccolella Enza
Published: January 2023

In this work, we have tried to establish whether human memory T cells may be protected from Fas (CD95)-induced apoptosis when correctly activated by Ag, and not protected when nonspecifically or incorrectly activated. In particular, we wanted to investigate the molecular mechanisms that regulate the fate of memory T cells following an antigenic challenge. To address this issue, we chose an experimental system that closely mimics physiological T cell activation such as human T cell lines and clones specific for viral peptides or alloantigens. We demonstrate that memory T cells acquire an activation-induced cell death (AICD)-resistant phenotype when TCRs are properly engaged by specific Ag bound to MHC molecules. Ag concentration and costimulation are critical parameters in regulating the protective effect. The analysis of the mechanisms involved in the block of CD95 signal transduction pathways revealed that the crucial events are the inhibition of CD95-associated IL-1β-converting enzyme (ICE)-like protease (FLICE) activation and poly(ADP)-ribose polymerase cleavage, and the mRNA expression of FLICE-like inhibitory protein. Furthermore, we have observed that TCR-mediated neosynthesis of FLICE-like inhibitory protein mRNA is suppressed either by protein tyrosine kinase inhibitors or cyclosporin A. In conclusion, the present analysis of the effects of TCR triggering on the regulation of AICD suggests that AICD could be inhibited in human memory T cells activated in vivo by a foreign Ag, but may become operative when the Ag has been cleared.
T1/ST2 Expression Is Enhanced on CD4+ T Cells from Schistosome Egg-Induced Granulomas: Analysis of Th Cell Cytokine Coexpression Ex Vivo
Authors: Löhning Max , Grogan Jane L. , Coyle Anthony J. , Yazdanbakhsh Maria , Meisel Christian , Gutierrez-Ramos Jose-Carlos , Radbruch Andreas , Kamradt Thomas
Published: January 2023

Th cells are categorized into subsets based on the cytokine production of in vitro-differentiated Th populations. For in vivo-differentiated Th subsets, little is known about the heterogeneity of cytokine production in single cells. We recently described a molecule, T1/ST2, that is preferentially expressed on the surface of Th2 cells. Here we combined high-gradient magnetic cell separation with four-color single-cell cytometry to analyze simultaneously three intracellular cytokines and T1/ST2 surface expression on CD4+ cells from lungs containing granulomas induced by Schistosoma mansoni eggs. T1/ST2 was highly up-regulated on CD4+ T cells from hepatic granulomas and granulomatous lungs. T1/ST2+ cells from granulomatous lungs preferentially produced type 2 cytokines ex vivo. In the total CD4+ population, coexpression of type 1 and type 2 cytokines occurred frequently. However, such coproduction was drastically reduced in T1/ST2+ cells compared with T1/ST2− cells. Coexpression of type 1 and type 2 cytokines was also rare in cells simultaneously producing two cytokines of one type. These findings indicate that individual CD4+ T cells in vivo have different levels of commitment to a certain Th phenotype. Coexpression of two type 2 cytokines or production of one type 2 cytokine together with surface expression of T1/ST2 indicate advanced commitment to the Th2 phenotype.
Modulation of Monocytic Cell Activity and Virus Susceptibility During Differentiation into Macrophages
Authors: Basta Sameh , Knoetig Sonja M. , Spagnuolo-Weaver Martha , Allan Gordon , McCullough Kenneth C.
Published: January 2023

A major component of innate immune responses relies on monocytes and macrophages, virus infection of which will pose a particular problem for immunological defense. Consequently, the monocytic cell differentiation pathway was analyzed in terms of cellular modulations therein and their relation to monocytotropic virus infection. Differentiation was characterized by down-regulation of CD14, MHC Ags, the monocytic SWC1 marker, and p53; concomitant up-regulation of the SWC9 macrophage marker, a putative porcine CD80 (detected with anti-human CD80 Ab), and acid phosphatase secretion were also characteristic. Elevated phagocytic and endocytic activities as well as endosomal/lysosomal acidification were identified as being important to the macrophage. In contrast, monocytes possessed high accessory activity. This was multifactorial, concomitantly requiring 1) high MHC Ag expression; 2) enzyme activity of esterase, peroxidase, myeloperoxidase, and 5′ nucleotidase in preference to glucosidase, galactosidase, and glucuronidase; and 3) elevated capacity for spontaneous IL-1 production. Only with all parameters was efficient stimulation of Ag-specific lymphocytes possible. These results point to a continuous process during differentiation, involving inter-related characteristics linking the more accessory monocyte to the scavenger macrophage, both in vitro and in vivo. Of particular interest was how these characteristics related to monocytotropic virus infection, and how a particular virus could show a clear preference for the differentiating macrophages. Such results not only further our understanding of porcine immunology, but also provide evidence and a potential model for the determination and characterization of monocytotropic virus-host cell interactions.
Administration of an IL-12-Encoding DNA Plasmid Prevents the Development of Chronic Graft-Versus-Host Disease (GVHD)
Authors: Okubo Tadanobu , Hagiwara Eri , Ohno Shigeru , Tsuji Takashi , Ihata Atsushi , Ueda Atsuhisa , Shirai Akira , Aoki Ichiro , Okuda Kenji , Miyazaki Jun-ichi , Ishigatsubo Yoshiaki
Published: January 2023

The transfer of DBA/2 spleen cells into (C57BL/10 × DBA/2)F1 mice induces chronic graft-vs-host disease (GVHD), which is characterized by the production of Th2 cytokines, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis like systemic lupus erythematosus. IL-12 strongly induces the production of Th1 cytokines and reduces Th2 activity in vivo. In this study, the effect of gene therapy on the development of murine chronic GVHD was examined using an IL-12-encoding plasmid (pCAGGSIL-12), with the expectation that it might regulate Th1/Th2 activity and have a beneficial impact on the clinical manifestations of disease. pCAGGSIL-12 or its p40 antagonist plasmid (pCAGGSp40) were injected i.m. every 3 wk in GVHD-induced (C57BL/10 × DBA/2)F1 mice. A total of 100 μg of pCAGGSIL-12 improved the Th1/Th2 balance in vivo, suppressed the production of IgG, and significantly reduced the development of glomerulonephritis. GVHD was exacerbated by injection of the pCAGGSp40 antagonist. Our results demonstrate that GVHD can be treated successfully by the administration of an IL-12-encoding plasmid, and that such therapy does not induce acute GVHD.
Similarities and Differences Between Human and Murine TNF Promoters in Their Response to Lipopolysaccharide
Authors: Kuprash Dmitry V. , Udalova Irina A. , Turetskaya Regina L. , Kwiatkowski Dominic , Rice Nancy R. , Nedospasov Sergei A.
Published: January 2023

Transcription of the TNF gene is rapidly and transiently induced by LPS in cells of monocyte/macrophage lineage. Previous data suggested that multiple NF-κB/Rel binding sites play a role in the transcriptional response to LPS of the murine gene. However, the relevance of homologous sites in the human TNF gene remained a matter of controversy, partly because the high affinity NF-κB/Rel site located at −510 in the murine promoter is not conserved in humans. Here we used two sets of similarly designed human and mouse TNF promoter deletion constructs and overexpression of IκB in the murine macrophage cell line ANA-1 to show remarkable similarity in the pattern of the transcriptional response to LPS, further demonstrating the functional role of the distal promoter region located between −600 and −650. This region was characterized by mutagenesis of protein binding sites, including two relatively low affinity NF-κB/Rel sites, #2 and 2a. Mutation in each of the NF-κB sites resulted in 2- to 3-fold lower transcriptional activity in response to LPS. In contrast to LPS activation, the response to PMA was substantially lower in magnitude and required only the proximal promoter region. In summary, the functional topography of human and murine promoters when assayed in the same system has some marked similarities. Our observations support the notion that full LPS response of TNF gene requires both NF-κB and non-NF-κB nuclear proteins. Our data also suggest that the functional activity of a given κB site depends on the entire DNA sequence context in the promoter region.
Repression of IL-4-Induced Gene Expression by IFN-γ Requires Stat1 Activation
Authors: Venkataraman Chandrasekar , Leung Stewart , Salvekar Anupama , Mano Hiroyuki , Schindler Ulrike
Published: January 2023

IFN-γ antagonizes many physiological responses mediated by IL-4, including the inhibition of IL-4-induced IgE production. This event is largely mediated at the level of transcription. We observed that the IL-4 response element of the germline epsilon promoter is sufficient to confer IFN-γ-mediated repression onto a reporter construct. The inhibitory effects were observed in both lymphoid and nonlymphoid cell lines. Stat1, which is activated by IFN-γ, cannot recognize the Stat6-specific IL-4 response element in the ε promoter. Hence, competitive DNA binding does not seem to be the underlying mechanism for the inhibitory effect. This is supported by the observation that inhibition is not seen at early time points, but requires prolonged IFN-γ treatment. IFN-γ stimulation results in a loss of IL-4-induced Stat6 tyrosine phosphorylation, nuclear translocation, and DNA binding. Using the fibrosarcoma cell line U3A, which lacks Stat1, we demonstrated that the transcription activation function of Stat1 is required for the IFN-γ-mediated repression. Repression was restored by overexpression of Stat1α, but not Stat1β, in U3A cells. Treatment with IFN-γ, but not IL-4, specifically up-regulates the expression of SOCS-1 (silencer of cytokine signaling), a recently characterized inhibitor of cytokine signaling pathways, such as IL-6 and IFN-γ. Overexpression of SOCS-1 effectively blocks IL-4-induced Stat6 phosphorylation and transcription. This suggests that IFN-γ-mediated repression of IL-4-induced transcription is at least in part mediated by SOCS-1.
CD40 Ligand-CD40 Interaction Induces Chemokines in Cervical Carcinoma Cells in Synergism with IFN-γ
Authors: Altenburg Andreas , Baldus Stephan E. , Smola Hans , Pfister Herbert , Hess Sigrun
Published: January 2023

Cellular immunity plays a major role in controlling human papilloma virus infection and development of cervical carcinoma. Mononuclear cell infiltration possibly due to the action of chemokines becomes prominent in the tumor tissue. In fact, the macrophage chemoattractant protein-1, MCP-1, was detected in cervical squamous cell carcinoma in situ, whereas absent in cultured cells. From this, unknown environmental factors were postulated regulating chemokine expression in vivo. In this study, we show high CD40 expression on cervical carcinoma cells and CD40 ligand (CD40L) staining on attracted T cells in tumor tissue, suggesting a paracrine stimulation mechanism via CD40L-CD40 interactions. We therefore investigated chemokine synthesis in nonmalignant and malignant human papilloma virus-positive cell lines after CD40L exposure. Constitutive expression of MCP-1, MCP-3, RANTES, and IFN-γ-inducible protein-10 was almost undetectable in all cell lines tested. CD40L was able to induce MCP-1 production; however, despite much higher CD40 expression in malignant cells, MCP-1 induction was significantly lower compared with nontumorigenic cells. After sensitization with IFN-γ, another T cell-derived cytokine showing minimal effects on CD40 expression levels, CD40 ligation led to a more than 20-fold MCP-1 induction in carcinoma cell lines. An even stronger effect was observed for IFN-γ-inducible protein-10. Our study highlights the synergism of T cell-derived mediators such as CD40L and IFN-γ for chemokine responses in cervical carcinoma cells, helping to understand the chemokine expression patterns observed in vivo.
Overexpression of Bcl-2 in Transgenic Mice Decreases Apoptosis and Improves Survival in Sepsis
Authors: Hotchkiss Richard S. , Swanson Paul E. , Knudson C. Michael , Chang Katherine C. , Cobb J. Perren , Osborne Dale F. , Zollner Kimberly M. , Buchman Timothy G. , Korsmeyer Stanley J. , Karl Irene E.
Published: January 2023

In sepsis there is extensive apoptosis of lymphocytes, which may be beneficial by down-regulating the accompanying inflammation. Alternatively, apoptosis may be detrimental by impairing host defense. We studied whether Bcl-2, a potent antiapoptotic protein, could prevent lymphocyte apoptosis in a clinically relevant model of sepsis. Transgenic mice in which Bcl-2 was overexpressed in T cells had complete protection against sepsis-induced T lymphocyte apoptosis in thymus and spleen. Surprisingly, there was also a decrease in splenic B cell apoptosis in septic Bcl-2 overexpressors compared with septic HeJ and HeOuJ mice. There were marked increases in TNF-α, IL-1β, and IL-10 in thymic tissue in sepsis in the three species of mice, and the increase in TNF-α and IL-10 in HeOuJ mice was greater than that in Bcl-2 mice. Mitotracker, a mitochondrial membrane potential indicator, demonstrated a sepsis-induced loss of membrane potential in T cells in HeJ and HeOuJ mice but not in Bcl-2 mice. Importantly, Bcl-2 overexpressors also had improved survival in sepsis. To investigate the potential impact of loss of lymphocytes on survival in sepsis, Rag-1−/− mice, which are totally deficient in mature T and B cells, were also studied. Rag-1−/− mice had decreased survival compared with immunologically normal mice with sepsis. We conclude that overexpression of Bcl-2 provides protection against cell death in sepsis. Lymphocyte death may be detrimental in sepsis by compromising host defense.
Dose Dependence of CTL Precursor Frequency Induced by a DNA Vaccine and Correlation with Protective Immunity Against Influenza Virus Challenge
Authors: Fu Tong-Ming , Guan Liming , Friedman Arthur , Schofield Timothy L. , Ulmer Jeffrey B. , Liu Margaret A. , Donnelly John J.
Published: January 2023

Intramuscular injection of BALB/c mice with a DNA plasmid encoding nucleoprotein (NP) from influenza virus A/PR/8/34 (H1N1) provides cross-strain protection against lethal challenge with influenza virus A/HK/68 (H3N2). CTL specific for the H-2Kd-restricted epitope NP147–155 are present in these mice and are thought to play a role in the protection. To assess the effectiveness of NP DNA immunization in comparison with influenza virus infection in the induction of CTL responses, we monitored the frequency of CTL precursors (CTLp) in mice following i.m. injection with NP DNA or intranasal infection with influenza virus and showed that the CTLp frequency in NP DNA-immunized mice can reach levels found in mice that had been infected with influenza virus. We also measured the CTLp frequency, anti-NP Ab titers, and T cell proliferative responses in mice that were injected with titrated dosages of NP DNA and documented a correlation of the CTLp frequency and the Ab titers, but not proliferative responses, with the injection dose. Furthermore, we observed a positive correlation between the frequency of NP147–155 epitope-specific CTLp and the extent of protective immunity against cross-strain influenza challenge induced by NP DNA injection. Collectively, these results and our early observations from adoptive transfer experiments of in vitro activated lymphocytes from NP DNA-immunized mice suggest a protective function of NP-specific CTLp in mice against cross-strain influenza virus challenge.
Extracellular Signal-Regulated Kinases Regulate Leukotriene C4 Generation, But Not Histamine Release or IL-4 Production from Human Basophils
Authors: Miura Katsushi , Schroeder John T. , Hubbard Walter C. , MacGlashan Donald W.
Published: January 2023

Human basophils secrete histamine and leukotriene C4 (LTC4) in response to various stimuli, such as Ag and the bacterial product, FMLP. IgE-mediated stimulation also results in IL-4 secretion. However, the mechanisms of these three classes of secretion are unknown in human basophils. The activation of extracellular signal-regulated kinases (ERKs; ERK-1 and ERK-2) during IgE- and FMLP-mediated stimulation of human basophils was examined. Following FMLP stimulation, histamine release preceded phosphorylation of ERKs, whereas phosphorylation of cytosolic phospholipase A2 (cPLA2), and arachidonic acid (AA) and LTC4 release followed phosphorylation of ERKs. The phosphorylation of ERKs was transient, decreasing to baseline levels after 15 min. PD98059 (MEK inhibitor) inhibited the phosphorylation of ERKs and cPLA2 without inhibition of several other tyrosine phosphorylation events, including phosphorylation of p38 MAPK. PD98059 also inhibited LTC4 generation (IC50 = ∼2 μM), but not histamine release. Stimulation with anti-IgE Ab resulted in the phosphorylation of ERKs, which was kinetically similar to both histamine and LTC4 release and decreased toward resting levels by 30 min. Similar to FMLP, PD98059 inhibited anti-IgE-mediated LTC4 release (IC50, ∼2 μM), with only a modest effect on histamine release and IL-4 production at higher concentrations. Taken together, these results suggest that ERKs might selectively regulate the pathway leading to LTC4 generation by phosphorylating cPLA2, but not histamine release or IL-4 production, in human basophils.
CD8 T Cells Are Essential in the Development of Respiratory Syncytial Virus-Induced Lung Eosinophilia and Airway Hyperresponsiveness
Authors: Schwarze Jürgen , Cieslewicz Grzegorz , Joetham Anthony , Ikemura Toshihide , Hamelmann Eckard , Gelfand Erwin W.
Published: January 2023

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways. The immune cell requirements for these responses to RSV infection are not well defined. To delineate the role of CD8 T cells in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice depleted of CD8 T cells to develop these symptoms of RSV infection. BALB/c mice were depleted of CD8 T cells using anti-CD8 Ab treatment before intranasal administration of infectious RSV. Six days postinfection, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and levels of IFN-γ, IL-4, and IL-5 in bronchoalveolar lavage fluid were monitored. RSV infection resulted in airway eosinophilia and AHR in control mice, but not in CD8-depleted animals. Further, whereas RSV-infected mice secreted increased amounts of IL-5 into the airways as compared with noninfected controls, no IL-5 was detectable in both bronchoalveolar lavage fluid and culture supernatants from CD8-depleted animals. Treatment of CD8-depleted mice with IL-5 fully restored both lung eosinophilia and AHR. We conclude that CD8 T cells are essential for the influx of eosinophils into the lung and the development of AHR in response to RSV infection.
Lipopolysaccharide-Coated Erythrocytes Activate Human Neutrophils Via CD14 While Subsequent Binding Is Through CD11b/CD18
Authors: Troelstra Annet , de Graaf-Miltenburg Lia A. M. , van Bommel Toon , Verhoef Jan , Van Kessel Kok P. M. , Van Strijp Jos A. G.
Published: January 2023

Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP enhanced binding twofold, and this surplus was dependent upon CD14 but not on CR3. We conclude that ELPS interact with neutrophils via CD14, initially giving rise to cell activation; subsequently, binding is solely mediated by activated CR3.
Nuclear Factor-κB Mediates TNF-α Inhibitory Effect on α2(I) Collagen (COL1A2) Gene Transcription in Human Dermal Fibroblasts
Authors: Kouba David J. , Chung Kee-Yang , Nishiyama Takafumi , Vindevoghel Laurence , Kon Atsushi , Klement John F. , Uitto Jouni , Mauviel Alain
Published: January 2023

Among its plethora of activities as an inflammatory mediator, TNF-α has potent regulatory control on extracellular matrix production and degradation. Earlier studies have documented that TNF-α inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5′ end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-α response element of the COL1A2 promoter. The TNF-α response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-κB (AGAGTTTCCC) binding sites. TNF-α effect was eliminated by a 2-bp substitution mutation in the NF-κB1 binding half site of the NF-κB cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-κB heterodimers as well as NF-κB1 and RelA homodimers, but not AP-1, were capable of binding this element. Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-α stimulation, which reached a plateau by 1 h and was not affected by preincubation of cells with cycloheximide. Gel supershift assays identified the complex as the NF-κB (p50/p65) heterodimer, whereas Abs to nuclear factor of activated T cells (NF-AT) and Jun family members failed to recognize the complex. These data suggest that in fibroblasts TNF-α activates and initiates the nuclear translocation of NF-κB that binds a divergent NF-κB element and plays a critical role in the observed inhibition of α2(I) collagen gene transcription.
Differential Expression and Activation of p38 Mitogen-Activated Protein Kinase α, β, γ, and δ in Inflammatory Cell Lineages
Authors: Hale Karin K. , Trollinger David , Rihanek Marynette , Manthey Carl L.
Published: January 2023

Four p38 mitogen-activated protein kinases (p38α, β, γ, δ) have been described. To understand the role of p38 family members in inflammation, we determined their relative expression in cells that participate in the inflammatory process. Expression was measured at the level of mRNA by reverse-transcriptase PCR and protein by Western blot analysis. p38α was the dominant form of p38 in monocytes; expression of p38δ was low and p38β was undetected. In macrophages, p38α and p38δ were abundant, but p38β was undetected. p38α and p38δ were also expressed by neutrophils, CD4+ T cells, and endothelial cells. Again, p38β was not detected in neutrophils, although low amounts were present in CD4+ T cells. In contrast, p38β was abundant in endothelial cells. p38γ protein was not detected in any cell type, although p38γ mRNA was present in endothelial cells. Immunokinase assays showed a strong activation of p38α and a lesser activation of p38δ in LPS-stimulated macrophages. Abs specific for mono- and dual-phophorylated forms of p38 suggested that LPS induces dual phosphorylation of p38α, but primarily mono-phosphorylation of p38δ. IL-1β activated p38α and p38β in endothelial cells. However, p38α was the more activated form based on kinase assays and phosphorylation analysis. Expression and activation patterns of p38α in macrophages and endothelial cells suggest that p38α plays a major role in the inflammatory response. Additional studies will be needed to define the contribution of p38δ to macrophage, neutrophil, and T cell functions, and of p38β to signaling in endothelial cells and T cells.
Importance of CD23 for Collagen-Induced Arthritis: Delayed Onset and Reduced Severity in CD23-Deficient Mice
Authors: Kleinau Sandra , Martinsson Pernilla , Gustavsson Susanne , Heyman Birgitta
Published: January 2023

Increased expression of the low affinity receptor for IgE, FcεRII/CD23 has been observed in rheumatoid arthritis. In view of this, we have investigated the expression and influence of CD23 in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. CD23+ cells were analyzed in lymph nodes of DBA/1 mice immunized with bovine collagen type II (BCII) in CFA or with CFA only. The percentage of CD23+ lymph node cells was increased in both BCII/CFA- and CFA-immunized mice at 1, 3, and 7 wk after immunization compared with unimmunized mice, indicating a role for the adjuvant to trigger general inflammation and CD23 expression. To investigate the functional role of CD23 in CIA, CD23-deficient mice on the DBA/1 genetic background were studied. After immunization with BCII/CFA, these mice developed CIA with delayed onset and reduced severity compared with wild-type mice. These findings suggest that an increased number of CD23+ cells is part of an inflammatory response and that CD23 expression is of pathogenic importance in the arthritic process.
A Novel Glycosylphosphatidyl Inositol-Anchored Protein on Human Leukocytes: A Possible Role for Regulation of Neutrophil Adherence and Migration
Authors: Suzuki Kichiya , Watanabe Tadashi , Sakurai Shin-ichi , Ohtake Kazuhisa , Kinoshita Taroh , Araki Akemi , Fujita Teizo , Takei Hiroshi , Takeda Yuji , Sato Yukiko , Yamashita Takao , Araki Yoshihiko , Sendo Fujiro
Published: January 2023

We report here a novel glycosylphosphatidyl-inositol (GPI)-anchored glycoprotein on human leukocytes. Treatment of neutrophils with a mAb (3H9) to this molecule sequentially up-regulates and down-regulates β2 integrin-dependent adhesion of these cells as well as their transendothelial migration in vitro. In addition, this mAb simultaneously modulates the avidity of β2 integrin for its ligand, iC3b, with kinetics similar to those observed in 3H9 modulation of neutrophil adherence. This mAb also induces β2 integrin-dependent cytoskeletal remodeling. This novel GPI-anchored protein (GPI-80) is highly homologous with Vanin-1, a recently reported GPI-anchored protein that is expressed on perivascular thymic stromal cells and is involved in thymus homing in mice. The finding that both GPI-80 and Vanin-1 are 40% homologous with human biotinidase suggests the existence of a biotinidase superfamily of molecules that may be involved in the regulation of leukocyte trafficking.
Contribution of Renal Secreted Complement C3 to the Circulating Pool in Humans
Authors: Tang Sydney , Zhou Wuding , Sheerin Neil S. , Vaughan Robert W. , Sacks Steven H.
Published: January 2023

Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3–13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.
IL-12-Mediated NKRP1A Up-Regulation and Consequent Enhancement of Endothelial Transmigration of Vδ2+ TCRγδ+ T Lymphocytes from Healthy Donors and Multiple Sclerosis Patients
Authors: Poggi Alessandro , Zocchi Maria Raffaella , Costa Paola , Ferrero Elisabetta , Borsellino Giovanna , Placido Roberta , Galgani Simona , Salvetti Marco , Gasperini Claudio , Ristori Giovanni , Brosnan Celia F. , Battistini Luca
Published: January 2023

γδ T lymphocytes are thought to play a role in the pathogenesis of multiple sclerosis (MS) contributing to demyelinization and fibrosis in the central nervous system. In this study, we show that, in MS patients with active disease, the percentage of circulating Vδ2+ γδ T cells coexpressing NKRP1A is significantly increased compared with healthy donors. Vδ2+ and Vδ1+ T cells were sorted from MS patients and healthy volunteers and cloned. At variance with Vδ1+ clones, all Vδ2+ clones expressed NKRP1A, which was strongly up-regulated upon culture with IL-12; this effect was neutralized by specific anti-IL-12 Abs. No up-regulation of NKRP1A by IL-12 was noted on Vδ1+ clones. RNase protection assay showed that IL-12R β2 subunit transcript was significantly less represented in Vδ1+ than Vδ2+ clones. This finding may explain the different effect exerted by IL-12 on these clones. In transendothelial migration assays, Vδ2+ NKRP1A+ clones migrated more effectively than Vδ1+ clones, and this migratory potential was enhanced following culture with IL-12. Migration was strongly inhibited by the F(ab′)2 of an anti-NKRP1A Ab, suggesting that this lectin is involved in the migration process. We also show that, in freshly isolated PBMC from MS patients, the migrated population was enriched for Vδ2+ NKRP1A+ cells. We conclude that the expression of NKRP1A on Vδ2+ cells is associated with increased ability to migrate across the vascular endothelium and that this phenomenon may be regulated by IL-12 present in the microenvironment.
The Dependence for Leukocyte Function-Associated Antigen-1/ICAM-1 Interactions in T Cell Activation Cannot Be Overcome by Expression of High Density TCR Ligand
Authors: Abraham Clara , Griffith Justin , Miller Jim
Published: January 2023

The leukocyte-specific integrin, LFA-1, can enhance T cell activation. However, it is unclear whether the binding of LFA-1 to its ligand, ICAM-1, functions through intercellular adhesion alone, resulting in an augmentation of the TCR signal, or involves an additional LFA-1-mediated cellular signal transduction pathway. We have previously shown that naive CD4+ lymph node T cells, isolated from DO11.10 TCR transgenic mice, are activated by increasing doses of exogenous OVA peptide presented by transfectants expressing both class II and ICAM-1, but not by cells expressing class II alone. To determine whether LFA-1/ICAM-1 interactions were simply enhancing the presentation of low concentrations of specific MHC/peptide complexes generated from exogenously added peptide, we transfected cells with class II that is covalently coupled to peptide, alone or in combination with ICAM-1. These cells express 100-fold more specific class II/peptide complexes than can be loaded onto class II-positive cells at maximum concentrations of exogenous peptide. Despite this high density of TCR ligand, activation of naive CD4+ T cells still requires the coexpression of ICAM-1. LFA-1/ICAM-1 interactions are not required for effective conjugate formation and TCR engagement because presentation of class II/peptide complexes in the absence of ICAM-1 does induce up-regulation of CD25 and CD69. Thus, high numbers of engaged TCR cannot compensate for the lack of LFA-1/ICAM-1 interactions in the activation of naive CD4+ T cells.
Antigen Receptor Engagement Selectively Induces Macrophage Inflammatory Protein-1α (MIP-1α) and MIP-1β Chemokine Production in Human B Cells
Authors: Krzysiek Roman , Lefèvre Eric A. , Zou Weiping , Foussat Arnaud , Bernard Jérôme , Portier Alain , Galanaud Pierre , Richard Yolande
Published: January 2023

We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1β (MIP-1β) and MIP-1α, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1α/β in response to BCR triggering. In contrast to MIP-1α/β, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1α/β production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1β and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1α Ab suggesting that MIP-1β plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1α/β, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.
Virus-Induced CD8+ T Cell Clonal Expansion Is Associated with Telomerase Up-Regulation and Telomere Length Preservation: A Mechanism for Rescue from Replicative Senescence
Authors: Maini Mala K. , Soares Maria Vieira D. , Zilch Christian F. , Akbar Arne N. , Beverley Peter C. L.
Published: January 2023

In acute infectious mononucleosis (AIM), very large clones of Ag-specific CD8+ effector T cells are generated. Many clones persist as memory cells, although the clone size is greatly reduced. It would be expected that the large number of cell divisions occurring during clonal expansion would lead to shortening of telomeres, predisposing to replicative senescence. Instead, we show that clonally expanded CD8+ T cells in AIM have paradoxical preservation of telomere length in association with marked up-regulation of telomerase. We postulate that this allows a proportion of responding T cells to enter the memory pool with a preserved capacity to continue dividing so that long-term immunological memory can be maintained.
Pancreatic Infiltration But Not Diabetes Occurs in the Relative Absence of MHC Class II-Restricted CD4 T Cells: Studies Using NOD/CIITA-Deficient Mice
Authors: Mora Conchi , Wong F. Susan , Chang Cheong-Hee , Flavell Richard A.
Published: January 2023

The NOD (nonobese diabetic) mouse is a good animal model for human IDDM. MHC class II-restricted CD4 T cells are necessary for the onset of diabetes in NOD mice. Here, we demonstrate that NOD mice lacking the CIITA (class II transactivator) molecule, and hence deficient in MHC class II expression and peripheral CD4 T cells, show significant pancreatic infiltration but do not develop diabetes. CD4 T cell deficiency, then, does not prevent initial pancreatic infiltration, but does stop progression to insulitis. Adoptive transfer studies show that the paucity of CD4 T cells in NOD-CIITA knockout mice is responsible for the absence of diabetes, since the CD8 T cell and B cell compartments are functional. An autoaggressive CD8+ T cell clone can, however, transfer diabetes in CIITA knockout recipient mice without CD4 T cell help, albeit with some delay compared with that in CIITA-sufficient recipients. This highlights the fact that a high number of in vitro activated autoaggressive CD8 T cells can over-ride the requirement for CD4 T cell help for the onset of diabetes.
TNF Receptor 1-Dependent β Cell Toxicity as an Effector Pathway in Autoimmune Diabetes
Authors: Kägi David , Ho Alexandra , Odermatt Bernhard , Zakarian Arsen , Ohashi Pamela S. , Mak Tak W.
Published: January 2023

Autoimmune diabetes is characterized by a chronic progressive inflammatory autoimmune reaction that ultimately causes the selective elimination of pancreatic β cells. To address the question of whether the cell death-inducing cytokines TNF and lymphotoxin α are involved in this process, we generated nonobese diabetic (NOD) mice that are deficient for TNF receptor 1 (TNFR1 or TNFRp55). Insulitis developed in these mice similarly to that in normal control NOD mice, but progression to diabetes was completely abrogated. Since this was probably due to the complex immunomodulatory effects of TNF and lymphotoxin α signaled via TNFR1 on lymphohemopoietic cells, adoptive transfer experiments with spleen cells from diabetic NOD mice were conducted. It was found that the absence of TNFR1 in recipients delayed diabetes induced by normal control and precluded diabetes induced by perforin-deficient spleen cells. In a CD8+ T cell-mediated model of diabetes, however, diabetes induced by adoptive transfer of TCR transgenic lymphocytic choriomeningitis virus glycoprotein-specific CD8+ T cells was not delayed by the absence of TNFR1 in recipient mice. Together with the described expression patterns of perforin and TNF in the mononuclear islet infiltrates of NOD mice, these results indicate that two diabetogenic effector mechanisms are delivered by distinct cell populations: CD8+ T cells lyse β cells via perforin-dependent cytotoxicity, whereas CD4+ T cells, macrophages, and dendritic cells contribute to diabetes development via TNFR1-dependent β cell toxicity.
Type 1 and Type 2 Cytokine Regulation of Macrophage Endocytosis: Differential Activation by IL-4/IL-13 as Opposed to IFN-γ or IL-10
Authors: Montaner Luis J. , da Silva Rosangela P. , Sun Junwei , Sutterwala Shaheen , Hollinshead Michael , Vaux David , Gordon Siamon
Published: January 2023

Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-γ (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-γ decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-γ stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity.
TAP Association Influences the Conformation of Nascent MHC Class I Molecules
Authors: Owen Barbara A. L. , Pease Larry R.
Published: January 2023

The influence of TAP-MHC class I interactions on peptide binding to the class I heavy chain is assessed during TAP-dependent assembly using Kb-specific Abs that recognize conformational changes induced by assembly with β2-microglobulin (β2m) and by peptide binding. A significant portion (45%) of Kb molecules in TAP+, RMA-derived microsomes are associated with the TAP complex as measured by coimmunoisolation of Kb using anti-TAP1 Abs, while only 20% of the Kb heavy chain molecules are isolated as Kbβ2m complexes with the α-Kb-specific Abs, Y-3 or K-10-56. The amount of Kb isolated with Y-3 and K-10-56 increases in proportion to transport and binding of peptide to the Kb molecules within the RMA microsomes. In contrast, less than 5% of the Kb within TAP2-RMA-S microsomes associated with the remaining TAP1 subunit. However, greater than 60% of Kb heavy chain is isolated as K-10-56- and Y-3-reactive Kbβ2m complexes. We propose that a TAP-MHC class I interaction serves to stabilize the MHC class I:β2m complex in an immature conformation (Y-3 and K-10-56 nonreactive) prior to high affinity peptide binding, preventing the export of class I molecules complexed with low affinity peptide ligands from the ER.
An 11-Amino Acid Sequence in the Cytoplasmic Domain of CD40 Is Sufficient for Activation of c-Jun N-Terminal Kinase, Activation of MAPKAP Kinase-2, Phosphorylation of IκBα, and Protection of WEHI-231 Cells from Anti-IgM-Induced Growth Arrest
Authors: Sutherland Claire L. , Krebs Danielle L. , Gold Michael R.
Published: January 2023

We have previously shown that CD40 causes strong activation of the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinases (MAPK) and MAPKAP kinase-2, a downstream target of p38 MAPK. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of these kinases, we have created a set of 11 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. We found that amino acids 35–45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAPK pathways, as well as for induction of IκBα phosphorylation and degradation. Amino acids 35–45 were also sufficient to protect WEHI-231 cells from anti-IgM-induced growth arrest. This is the same region of CD40 required for binding the TNF receptor-associated factor-2 (TRAF2), TRAF3, and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-κB, JNK, and p38 MAPK.
MHC-Restricted, Glycopeptide-Specific T Cells Show Specificity for Both Carbohydrate and Peptide Residues
Authors: Deck M. Brian , Sjölin Petter , Unanue Emil R. , Kihlberg Jan
Published: January 2023

We examined the antigenic specificity of two T cell hybridomas elicited against the disaccharide galabiose attached to the fifth residue of the I-Ak binding peptide 52–61 of lysozyme. By making changes in the saccharide molecule and in the peptide, we conclude that the outer galactose residue of the galabiose moiety is directly recognized by the T cells together with the exposed side chains of the peptide. The overall spatial display of this galactose moiety on the 52–61 peptide is likewise important.
Human Glioma-Induced Immunosuppression Involves Soluble Factor(s) That Alters Monocyte Cytokine Profile and Surface Markers
Authors: Zou Jian-Ping , Morford Lorri A. , Chougnet Claire , Dix Amy R. , Brooks Andrew G. , Torres Naomi , Shuman Jon D. , Coligan John E. , Brooks William H. , Roszman Thomas L. , Shearer Gene M.
Published: January 2023

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-γ, and TNF-α, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-β1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.
Characterization of CD44 Induction by IL-1: A Critical Role for Egr-1
Authors: Fitzgerald Katherine A. , O’Neill Luke A. J.
Published: January 2023

The adhesion molecule CD44 is a multifunctional, ubiquitously expressed glycoprotein that participates in the process of leukocyte recruitment to sites of inflammation and to their migration through lymphatic tissues. In this study, we have investigated the effect of the proinflammatory cytokine IL-1α on CD44 gene expression in the human immortalized endothelial cell line ECV304. Immunoblotting of cell extracts showed constitutive expression of a 85-kDa protein corresponding to the standard form of CD44, which was potently up-regulated following IL-1α treatment. Furthermore, IL-1α induced expression of v3- and v6-containing isoforms of CD44, which migrated at 110 and 140–180 kDa, respectively. The effect of IL-1α on CD44 standard, v3- and v6-containing isoforms was dose and time dependent and was inhibited in the presence of IL-1 receptor antagonist. To elucidate the molecular mechanisms regulating CD44 expression in response to IL-1α, we investigated the effect of IL-1α on CD44 mRNA expression. Reverse-transcriptase PCR and Northern analysis demonstrated an increase in CD44 mRNA expression indicating a transcriptional mechanism of control by IL-1α. Furthermore, IL-1α increased expression of a reporter gene under the control of the CD44 promoter (up to −1.75 kb). The effect of IL-1α was critically dependent on the site spanning −151 to −701 of the promoter. This effect required the presence of an Egr-1 motif at position −301 within the CD44 promoter since mutation of this site abolished responsiveness. IL-1α also induced Egr-1 expression in these cells. These studies therefore identify Egr-1 as a critical transcription factor involved in CD44 induction by IL-1α.