bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-17 Stimulates Granulopoiesis in Mice: Use of an Alternate, Novel Gene Therapy-Derived Method for In Vivo Evaluation of Cytokines
Authors: Schwarzenberger Paul , La Russa Vincent , Miller Allan , Ye Peng , Huang Weitao , Zieske Arthur , Nelson Steve , Bagby Gregory J. , Stoltz David , Mynatt Randall L. , Spriggs Melanie , Kolls Jay K.
Published: January 2023

IL-17 is a novel cytokine secreted principally by CD4+ T cells. It has been shown to support the growth of hemopoietic progenitors in vitro; however, its in vivo effects are presently unknown. Adenovirus-mediated gene transfer of the murine IL-17 cDNA targeted to the liver (5 × 109 plaque-forming units (PFU) intravenous) resulted in a transiently transgenic phenotype, with dramatic effects on in vivo granulopoiesis. Initially, there was a significant increase (fivefold) in the peripheral white blood count (WBC), including a 10-fold rise in the absolute neutrophil count. This was associated with a doubling in the spleen size over 7–14 days after gene transfer, which returned to near baseline by day 21, although the white blood cell count remained elevated. There was a profound stimulation of splenic hemopoiesis as demonstrated by an increase in total cellularity by 50% 7 days after gene transfer and an increase in hemopoietic colony formation. A maximal increase in frequency of high proliferative potential colonies (HPPC) (11-fold) and CFU-granulocyte-macrophage (GM) and CFU-granulocyte-erythrocyte-megakaryocyte-monocyte (GEMM) (CFU) (6-fold) was seen on day 3 after IL-17 gene transfer. Both CFU and HPPC remained significantly elevated in the spleen throughout day 21, but at reduced levels compared with day 3. Bone marrow CFU and HPPC were elevated on day 3 only by 75% and 25%, respectively, without changes in total cellularity. Thus, murine IL-17 is a cytokine that can stimulate granulopoiesis in vivo. Since IL-17 is principally produced by CD4+ T cells, this cytokine could have therapeutic implications in AIDS-related bone marrow failure and opportunistic infections.
Involvement of the IP-10 Chemokine in Sarcoid Granulomatous Reactions
Authors: Agostini Carlo , Cassatella Marco , Zambello Renato , Trentin Livio , Gasperini Sara , Perin Alessandra , Piazza Francesco , Siviero Marta , Facco Monica , Dziejman Michelle , Chilosi Marco , Qin Shixin , Luster Andrew D. , Semenzato Gianpietro
Published: January 2023

The accumulation of T cells and monocytes at sites of ongoing inflammation represents the earliest step in the series of events that lead to granuloma formation in sarcoidosis. In this study, we evaluated the pulmonary production of IFN-inducible protein 10 (IP-10), a CXC chemokine that stimulates the directional migration of activated T cells. Striking levels of IP-10 were demonstrated in the bronchoalveolar lavage (BAL) fluid of 24 patients with pulmonary sarcoidosis and lymphocytic alveolitis, as compared with patients with inactive disease or control subjects. A positive correlation was demonstrated between IP-10 levels and the number of sarcoid CD45R0+/CD4+ cells in the BAL. Immunochemistry, performed with an anti-human IP-10 polyclonal Ab in lymph nodes displaying prominent sarcoid granulomas, showed that cells bearing IP-10 were mainly epithelioid cells and CD68+ macrophages located inside granulomatous areas. Macrophages recovered from the BAL of sarcoid patients stained positive for IP-10 protein. Furthermore, alveolar macrophages isolated from sarcoid patients with T cell alveolitis and cultured for 24 h in presence of IFN-γ secreted definite levels of IP-10 capable of inducing T cell chemiotaxis. Interestingly, alveolar lymphocytes recovered from patients with active sarcoidosis were CD4+ T cells expressing Th1 cytokines (IL-2 and IFN-γ) and high levels of CXCR3. Taken together, these data suggest the potential role of IP-10 in regulating the migration and activation of T cells toward sites of sarcoid inflammatory process and the consequent granuloma formation.
Fibroblast Growth Factor-1 (FGF-1) Enhances IL-2 Production and Nuclear Translocation of NF-κB in FGF Receptor-Bearing Jurkat T Cells
Authors: Byrd Victor M. , Ballard Dean W. , Miller Geraldine G. , Thomas James W.
Published: January 2023

Fibroblast growth factors (FGFs) are heparin-binding proteins crucial to embryogenesis, angiogenesis, and wound healing. FGF-1 is abundantly expressed in the synovium in rheumatoid arthritis and in rejecting allografts, sites of chronic immune-mediated inflammation. The frequency of FGF-1-responsive T cells is increased in the peripheral blood of these disorders, and a high percentage of infiltrating T cells in rheumatoid arthritis synovium express receptors for FGF-1. To understand the action of FGF-1 in T cells, studies were initiated in Jurkat T cells that express the signaling isoform of FGF receptor-1. These experiments show that FGF-1 stimulation of Jurkat T cells provides a second signal that augments TCR-mediated IL-2 production. Analogous to costimulation via CD28, this activity is mediated through activation of Rel/κB, a family of transcription factors known to regulate IL-2 and other activation-inducible proteins. FGF-1 alone induces modest nuclear translocation of κB-binding proteins, and this translocation is enhanced by the combination of anti-CD3 and FGF-1. This NF-κB binding complex is composed of transcriptionally active p65(RelA)/p50 heterodimers and results primarily from the targeted degradation of IκB-α, an inhibitor that sequesters Rel/κB in the cytoplasm. These data are the first to show a connection between FGF-1 signaling and NF-κB activation outside of embryonic development. The signaling events that link FGF receptor-1 engagement and NF-κB activation in Jurkat are probably distinct from the CD28 costimulation pathway, since FGF-1-induced Rel/κB binding proteins do not contain significant levels of c-Rel and are not identical with the CD28 response complex.
Lack of Significant Differences in Association Rates and Affinities of Antibodies from Short-Term and Long-Term Responses to Hen Egg Lysozyme
Authors: Goldbaum Fernando A. , Cauerhff Ana , Velikovsky C. Alejandro , Llera Andrea S. , Riottot Marie-Madeleine , Poljak Roberto J.
Published: January 2023

The affinities (Ka) and association rate constants (kon) of 23 mouse (BALB/c) anti-lysozyme mAbs obtained after short and prolonged immunizations have been measured by plasmon resonance techniques. The affinities for the 23 Abs, measured using their Fab, range from Ka = 1.1 × 107 to 1.4 × 1010 M−1. There is no significant correlation between time or dose of immunization and affinity or association rates, indicating no time- or dose-dependent maturation of the response within the doses and times that were explored. IgMs are produced early and late in the response, with intrinsic affinities <105 M−1. Two independently derived mAbs, D44.1 (short term) and F10.6.6 (from a longer term response), result from identical or nearly identical somatic recombination events of germline gene segments. F10.6.6 has more mutations and a higher affinity constant (Ka = 1.4 × 1010 M−1) than D44.1 (Ka = 1.1 × 107 M−1). Although higher affinities may result from an accumulation of mutations, they do not correlate with the length and dose of immunogenic challenge.
Evidence for Fas-Dependent and Fas-Independent Mechanisms in the Pathogenesis of Experimental Autoimmune Encephalomyelitis
Authors: Dittel Bonnie N. , Merchant Raina M. , Janeway Charles A.
Published: January 2023

To determine whether Fas or Fas ligand (FasL) plays a role in susceptibility to experimental autoimmune encephalomyelitis (EAE), we bred a TCR transgenic mouse specific for the Ac1–11 peptide of myelin basic protein to mice with inactivating mutations in Fas (lpr) or FasL (gld). Disease induction by peptide immunization in such mice produced similar disease scores, demonstrating that Fas/FasL interactions were not necessary to generate EAE. However, adoptive transfer experiments showed evidence that these interactions can play a role in the pathogenesis of EAE, shown most dramatically by the absence of disease following transfer of cells from a normal myelin basic protein TCR transgenic mouse into a Fas-deficient lpr recipient. Furthermore, transfer of cells lacking FasL (gld) into normal or gld recipients gave a diminished disease score. Thus, Fas/FasL interactions can play a role in the pathogenesis of EAE, but they are not required for disease to occur.
Analysis of Adjuvant Function by Direct Visualization of Antigen Presentation In Vivo: Endotoxin Promotes Accumulation of Antigen-Bearing Dendritic Cells in the T Cell Areas of Lymphoid Tissue
Authors: Reis e Sousa Caetano , Germain Ronald N.
Published: January 2023

T cell activation requires exposure to processed Ag and signaling by cytokines and costimulatory ligands. Adjuvants are thought to enhance immunity primarily through up-regulation of the latter signals. Here, we explore the effect of the bacterial adjuvant, endotoxin, on Ag presentation by B cells and dendritic cells (DC). Using an mAb (C4H3) specific for the hen egg lysozyme (HEL) 46-61 determinant bound to I-Ak, we analyze processed Ag expression and the tissue distribution of presenting cells following systemic administration of soluble HEL to mice. In both LPS-responsive and -hyporesponsive mice given endotoxin-containing HEL, B cells rapidly display surface 46-61/I-Ak complexes. In marked contrast, in LPS-hyporesponsive mice, splenic DC show little gain in C4H3 staining. In LPS-responsive animals, interdigitating DC in T cell areas show no staining above background at early times after HEL administration, but C4H3+ DC rapidly accumulate in the outer periarteriolar lymphoid sheaths (PALS) and in follicular areas. Within a few hours, C4H3+ DC appear in the T cell areas, concomitant with a decline in C4H3+ cells in the outer PALS, suggesting migration between these two sites. Endotoxin enhancement of C4H3 staining is seen for both CD8α− and CD8α+ DC subsets. These data suggest that a major effect of adjuvants is to promote mobilization of Ag-bearing DC to the T areas of lymphoid tissue, and possibly also to enhance Ag processing by these DC. Thus, microbial products promote T cell immunity not only through DC activation for cosignaling, but through improvement in signal 1 delivery.
Characterization of scFv-Ig Constructs Generated from the Anti-CD20 mAb 1F5 Using Linker Peptides of Varying Lengths
Authors: Shan Daming , Press Oliver W. , Tsu Theta T. , Hayden Martha S. , Ledbetter Jeffrey A.
Published: January 2023

The heavy (VH) and light (VL) chain variable regions of the murine anti-human CD20 mAb 1F5 were cloned, and four single-chain Ab (scFv) molecules were constructed using linker peptides of variable lengths to join the VH and VL domains. Three constructs were engineered using linker peptides of 15, 10, and 5 aa residues consisting of (GGGGS)3, (GGGGS)2, and (GGGGS)1 sequences, respectively, whereas the fourth was prepared by joining the VH and VL domains directly. Each construct was fused to a derivative of human IgG1 (hinge plus CH2 plus CH3) to facilitate purification using staphylococcal protein A. The aggregation and CD20 binding properties of these four 1F5 scFv-Ig derivatives produced were investigated. Both size-exclusion HPLC column analysis and Western blots of proteins subjected to nonreducing SDS-PAGE suggested that all four 1F5 scFv-Ig were monomeric with m.w. of ∼55 kDa. The CD20 binding properties of the four 1F5 scFv-Ig were studied by ELISA and flow cytometry. The 1F5 scFv-Ig with the 5-aa linker (GS1) demonstrated significantly superior binding to CD20-expressing target cells, compared with the other scFv-Ig constructs. Scatchard analysis of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity of 1.35 × 108 M−1 compared with an avidity of 7.56 × 108 M−1 for the native bivalent 1F5 Ab. These findings suggest that the GS1 scFv-Ig with a short linker peptide of ∼5 aa is the best of the engineered constructs for future studies.
Dose-Dependent Induction of Distinct Anergic Phenotypes: Multiple Levels of T Cell Anergy
Authors: Taams Leonie S. , van Eden Willem , Wauben Marca H. M.
Published: January 2023

T cell anergy has been proposed as one of the mechanisms underlying peripheral T cell tolerance. In recent years, the functional relevance of T cell anergy has been studied extensively in vitro and in vivo, using different species, cell systems, and ways to induce anergy. Although these studies concurred about the induction of unresponsiveness, conflicting findings were obtained with respect to the function of anergic T cells and to the persistence of T cell anergy. In the present study, T cell anergy was induced through T-T presentation of the specific Ag by rat MHC class II+ T cells in the absence of professional APC. We show that, depending on the Ag dose with which T cells were incubated, distinct anergic phenotypes were induced. Incubation of T cell clones with a low (suboptimal) Ag dose induced hyporesponsiveness. Incubation with a higher (optimal) Ag dose induced an anergic state capable of exerting immunoregulatory effects. Incubation with a high (supraoptimal) Ag dose led to an anergic suppressive phenotype that was persistent and was not reversed by APC, Ag, and rIL-2. These findings demonstrate that T cell anergy is not confined to a single state of functional inactivation. Instead, multiple levels of T cell anergy exist. Thus, anergic T cells can contribute to the regulation of the immune response either in a persistent and active manner or in a passive manner, depending on their level of T cell anergy.
Lysophosphatidic Acid and Sphingosine 1-Phosphate Protection of T Cells from Apoptosis in Association with Suppression of Bax
Authors: Goetzl Edward J. , Kong Yvonne , Mei Baisong
Published: January 2023

Members of a subfamily of G protein-coupled receptors (GPCRs), encoded by five different endothelial differentiation genes (edgs), specifically mediate effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on cellular proliferation and differentiation. Mechanisms of suppression of apoptosis by LPA and S1P were studied in the Tsup-1 cultured line of human T lymphoblastoma cells, which express Edg-2 and Edg-4 GPCRs for LPA and Edg-3 and Edg-5 GPCRs for S1P. At 10−10 M to 10−7 M, both LPA and S1P protected Tsup-1 cells from apoptosis induced by Abs to Fas, CD2, and CD3 plus CD28 in combination. Apoptosis elicited by C6 ceramide was inhibited by S1P, but not by LPA, in part because ceramide suppressed expression of Edg-2 and Edg-4 surface receptors for LPA without affecting Edg-3 surface receptors for S1P. At 10−9 M to 10−7 M, LPA and S1P significantly suppressed cellular levels of the apoptosis-promoting protein Bax, without altering the levels of Bcl-xL or Bcl-2 assessed by Western blots and immunoassays. Transfections of pairs of antisense plasmids for Edg-2 plus Edg-4 and Edg-3 plus Edg-5, and hygromycin selection of transfectants with reduced expression of the respective Edg R proteins in Western blots, inhibited both protection from apoptosis and reduction in cellular levels of Bax by LPA and S1P. Thus, LPA and S1P protection from apoptosis is mediated by distinct Edg GPCRs and may involve novel effects on Bax regulatory protein.
Mitogen-Activated Protein Kinase Activation Through Fcε Receptor I and Stem Cell Factor Receptor Is Differentially Regulated by Phosphatidylinositol 3-Kinase and Calcineurin in Mouse Bone Marrow-Derived Mast Cells
Authors: Ishizuka Tamotsu , Chayama Kosuke , Takeda Katsuyuki , Hamelmann Eckard , Terada Naohiro , Keller Gordon M. , Johnson Gary L. , Gelfand Erwin W.
Published: January 2023

Aggregation of high affinity FcR for IgE (FcεRI) on mast cells activates intracellular signal transduction pathways, including the activation of protein tyrosine kinases, phosphatidylinositol 3-kinase (PI3-kinase), and protein kinase C. Binding of stem cell factor (SCF) to its receptor (SCFR, c-Kit) on mast cells also induces increases in intrinsic tyrosine kinase activity and activation of PI3-kinase. Although ligation of both receptors induces Ras and Raf-1 activation, the downstream consequences of these early activation events are not well defined, except for the activation of extracellular signal-regulated kinases (ERK). Addition of Ag (OVA) to mouse bone marrow-derived mast cells (BMMC) sensitized with anti-OVA IgE triggers the activation of three members of the mitogen-activated protein (MAP) kinase family, c-Jun amino-terminal kinase (JNK), p38 MAP kinase (p38), and extracellular signal-regulated kinases. SCF similarly activates all three MAP kinases. Wortmannin, an inhibitor of PI3-kinase, inhibited both FcεRI- and SCFR-mediated JNK activation and partially inhibited FcεRI, but not SCFR-mediated p38 activation. Cyclosporin A inhibited FcεRI-mediated JNK and p38 activation, but did not affect the activation of these kinases when stimulated through the SCFR. Wortmannin and cyclosporin A inhibited FcεRI-mediated production of TNF-α and IL-4 in addition to serotonin release in BMMC. These results indicate that both PI3-kinase and calcineurin may contribute to the regulation of cytokine gene transcription and the degranulation response by modulating JNK activity in BMMC.
Chronic Modulation of the TCR Repertoire in the Lymphoid Periphery
Authors: Blish Catherine A. , Gallay Brian J. , Turk Gail L. , Kline Khristina M. , Wheat William , Fink Pamela J.
Published: January 2023

Using TCR Vβ5 transgenic mice as a model system, we demonstrate that the induction of peripheral tolerance can mold the TCR repertoire throughout adult life. In these mice, three distinct populations of peripheral T cells are affected by chronic selective events in the lymphoid periphery. First, CD4+Vβ5+ T cells are deleted in the lymphoid periphery by superantigens encoded by mouse mammary tumor viruses-8 and -9 in an MHC class II-dependent manner. Second, mature CD8+Vβ5+ T cells transit through a CD8lowVβ5low deletional intermediate during tolerance induction by a process that depends upon neither mouse mammary tumor virus-encoded superantigens nor MHC class II expression. Third, a population of CD4−CD8−Vβ5+ T cells arises in the lymphoid periphery in an age-dependent manner. We analyzed the TCR Vα repertoire of each of these cellular compartments in both Vβ5 transgenic and nontransgenic C57BL/6 mice as a function of age. This analysis revealed age-related changes in the expression of Vα families among different cellular compartments, highlighting the dynamic state of the peripheral immune repertoire. Our work indicates that the chronic processes maintaining peripheral T cell tolerance can dramatically shape the available TCR repertoire.
Regulation of Lymphotoxin Production by the p21ras-raf-MEK-ERK Cascade in PHA/PMA-Stimulated Jurkat Cells
Authors: Li Yong Q. , Hii Charles S. T. , Costabile Maurizio , Goh David , Der Channing J. , Ferrante Antonio
Published: January 2023

Although the production of lymphotoxin (LT) from activated Th1 lymphocytes has been reported extensively, the intracellular signaling mechanisms that regulate this T cell function remain totally undefined. We have examined whether the p21ras-raf-1-mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase (MEK)-ERK cascade plays a role in regulating the production of LT, because the activity of these signaling molecules is up-regulated in activated T lymphocytes. Transfection of Jurkat leukemic T cells with a dominant negative mutant of p21ras (ras17N or ras15A), raf-1 (raf 1–130), or ERK1 (Erk1-K71R) resulted in the suppression of the mitogen/phorbol ester-stimulated production/secretion of LT. This suppression was accompanied by a parallel inhibition of mitogen-stimulated ERK activation. The selective antagonist of MEK1 activation, PD98059, also attenuated the mitogen-stimulated or anti-CD3 Ab and phorbol ester-stimulated production of LT from Jurkat cells or peripheral blood T lymphocytes. This study provides, for the first time, direct evidence that the p21ras-raf-MEK-ERK cascade plays a vital role in regulating the production of LT.
Differential Activation of c-Jun NH2-Terminal Kinase and p38 Pathways During FTY720-Induced Apoptosis of T Lymphocytes That Is Suppressed by the Extracellular Signal-Regulated Kinase Pathway
Authors: Matsuda Satoshi , Minowa Akiko , Suzuki Seiichi , Koyasu Shigeo
Published: January 2023

FTY720 is a novel immunosuppressive drug derived from a metabolite from Isaria sinclairii that is known to induce apoptosis of rat splenic T cells. In this study, we examined the intracellular signaling pathway triggered by FTY720. Treatment of human Jurkat T lymphocytes with FTY720-induced apoptosis characterized by DNA fragmentation. The same treatment induced activation of protein kinases such as c-Jun NH2-terminal kinase (JNK), p38/CSBP (CSAID-binding protein), and a novel 36-kDa myelin basic protein (MBP) kinase, but not extracellular signal-regulated kinase (ERK). Pretreatment of Jurkat cells with DEVD-CHO blocked FTY720-induced DNA fragmentation as well as the activation of p38/CSBP. However, DEVD-CHO treatment failed to inhibit FTY720-induced activation of JNK and the 36-kDa MBP kinase. We have also demonstrated that activation of the ERK signaling pathway completely suppressed the FTY720-induced apoptotic process including activation of caspase 3 and activation of JNK and the 36-kDa MBP kinase. Furthermore, transient expression of constitutively active mitogen-activated protein kinase/ERK kinase (MEK) protected the cells from FTY720-induced cell death. The effect of MEK was canceled by coexpression of a mitogen-activated protein kinase phosphatase, CL100. These results indicate that JNK and p38 pathways are differentially regulated during FTY720-induced apoptosis and that activation of ERK pathway alone is sufficient to cancel the FTY720-induced death signal.
Toward a Role of Dendritic Cells in the Germinal Center Reaction: Triggering of B Cell Proliferation and Isotype Switching
Authors: Dubois Bertrand , Barthélémy Clarisse , Durand Isabelle , Liu Yong-Jun , Caux Christophe , Brière Francine
Published: January 2023

We have reported previously that in vitro generated dendritic cells (DC) can directly regulate B cell responses. Recently, germinal center DC (GCDC) were identified within B cell follicles. Due to their particular localization, we have tested in the present study whether GCDC could contribute to key events characteristic of the GC reaction. Our present results demonstrate that 1) ex vivo GCDC induce a dramatic GC B cell expansion upon CD40 and IL-2 activation and drive plasma cell differentiation, 2) this property is shared by GCDC and blood DC, but not by Langerhans cells, 3) IL-12 production by GCDC is critical in GC B cell expansion and differentiation, and 4) importantly, GCDC also induce IL-10-independent isotype switching toward IgG1. These observations support the novel concept that GCDC directly contribute to the germinal center reaction.
Sodium Dodecyl Sulfate Stability of HLA-DR1 Complexes Correlates with Burial of Hydrophobic Residues in Pocket 1
Authors: Natarajan Sateesh K. , Stern Lawrence J. , Sadegh-Nasseri Scheherazade
Published: January 2023

Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306–318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue βGly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an α/β heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.
Modulation of Endocytosis in Nuclear Factor IL-6(−/−) Macrophages Is Responsible for a High Susceptibility to Intracellular Bacterial Infection
Authors: Pizarro-Cerdá Javier , Desjardins Michel , Moreno Edgardo , Akira Shizuo , Gorvel Jean-Pierre
Published: January 2023

Activated macrophages kill bacteria, a function known to depend on the expression of NF-IL-6. Here, it is demonstrated that the attenuated Brucella abortus vaccine strain 19 replicates much better in NF-IL-6−/− than in NF-IL-6(+/+) and NF-IL-6(+/+)-activated murine macrophages and at levels comparable to those observed in normal macrophages infected with the pathogenic strain 2308. The role of NF-IL-6 in the inhibition of intracellular bacterial replication is related to its control of endocytosis and membrane fusion between endosomes and Brucella-containing phagosomes. Addition of the granulocyte-CSF (G-CSF), whose induction is impaired in NF-IL-6(−/−) macrophages, restores both endocytosis and the morphology of endosomes, together with bactericidal activity. Regulation of membrane traffic in endocytosis by G-CSF whose expression is controlled by NF-IL-6 may explain how a host cell can control intracellular bacterial replication.
Regulatory Effects of Endogenous Protease Inhibitors in Acute Lung Inflammatory Injury
Authors: Gipson Teletha S. , Bless Nicolas M. , Shanley Thomas P. , Crouch Larry D. , Bleavins Michael R. , Younkin Ellen M. , Sarma Vidya , Gibbs Douglas F. , Tefera Wongelawit , McConnell Patrick C. , Mueller William T. , Johnson Kent J. , Ward Peter A.
Published: January 2023

Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-α and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a.
Implication of TNF Receptor-I-Mediated Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) Activation in Growth of AIDS-Associated Kaposi’s Sarcoma Cells: A Possible Role of a Novel Death Domain Protein MADD in TNF-α-Induced ERK1/2 Activation in Kaposi’s Sarcoma Cells
Authors: Murakami-Mori Kaoru , Mori Shunsuke , Bonavida Benjamin , Nakamura Shuji
Published: January 2023

TNF-α is a key pathogenic mediator of infectious and inflammatory diseases. HIV infection stimulates and dysregulates the immune system, leading to abnormal production of TNF-α. Despite its cytotoxic effect on some tumor cell lines, TNF-α functions as a growth stimulator for Kaposi’s sarcoma (KS), a common malignancy in HIV-infected patients. However, signaling pathways linked to TNF-α-induced mitogenic responses are not well understood. We found that extracellular signal-regulated kinases 1 and 2 (ERK1/2) in KS cells were significantly activated by TNF-α through tyrosine/threonine phosphorylation. Using neutralizing anti-TNFR-I and TNFR-II mAbs, we have now obtained evidence that TNF-α-induced KS cell growth and ERK1/2 activation are mediated exclusively by TNFR-I, not by TNFR-II. A selective inhibitor for ERK1/2 activator kinases, PD98059, profoundly inhibited not only the activation of ERK1/2, but also the TNF-α-induced KS cell proliferation. We therefore propose that the TNFR-I-ERK1/2 pathway plays a pivotal role in transmitting to KS cells the mitogenic signals of TNF-α. TNFR-I possesses no intrinsic kinase activity, suggesting that TNFR-I-associated proteins may provide a link between TNFR-I and ERK1/2 activation. We found that actinomycin D treatment of KS cells selectively abolished expression of mitogen-activated protein kinase-activating death domain protein (MADD), a novel TNFR-I-associated death domain protein. TNF-α failed to induce ERK1/2 activation in the actinomycin D-treated cells. MADD may couple TNFR-I with the ERK1/2 signaling pathway required for KS cell proliferation.
Deficiency of Human Complement Protein C4 Due to Identical Frameshift Mutations in the C4A and C4B Genes
Authors: Lokki Marja-Liisa , Circolo Antonella , Ahokas Pirkko , Rupert Kristi L. , Yu C. Yung , Colten Harvey R.
Published: January 2023

The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected. This defect in C4 expression was specific in that synthesis of two other complement proteins was normal. Analysis of genomic DNA showed that the proposita had both deleted and nonexpressed C4 genes. Each of her nonexpressed genes, a C4A null gene inherited from the mother, a C4A null gene, and a C4B null gene inherited from the father, all contained an identical 2-bp insertion (TC) after nucleotide 5880 in exon 29, providing the first confirmatory proof of the C4B pseudogene. This mutation has been previously found only in C4A null genes. Although the exon 29/30 junction is spliced accurately, this frameshift mutation generates a premature stop at codon 3 in exon 30. These truncated C4A and C4B gene products were confirmed through RT-PCR and sequence analysis. Among the possible genetic mechanisms that produce identical mutations in both genes, the most likely is a mutation in C4A followed by a gene conversion to generate the mutated C4B allele.
JAK3, STAT, and MAPK Signaling Pathways as Novel Molecular Targets for the Tyrphostin AG-490 Regulation of IL-2-Mediated T Cell Response
Authors: Wang Li Hua , Kirken Robert A. , Erwin Rebecca A. , Yu Cheng-Rong , Farrar William L.
Published: January 2023

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor, these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study, evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation, a non-JAK2-dependent signal, in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates, STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1, -3, -5a, and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT, JAK3/AP-1, and JAK3/MAPK pathways and their cellular consequences. Taken together, these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways.
Selection of CD8+ T Cells with Highly Focused Specificity During Viral Persistence in the Central Nervous System
Authors: Marten Norman W. , Stohlman Stephen A. , Smith-Begolka Wendy , Miller Stephen D. , Dimacali Emmanuel , Yao Qin , Stohl Sheldon , Goverman Joan , Bergmann Cornelia C.
Published: January 2023

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR α/β-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-γ enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.
CD69 Expression Discriminates MHC-Dependent and -Independent Stages of Thymocyte Positive Selection
Authors: Hare Katherine J. , Jenkinson Eric J. , Anderson Graham
Published: January 2023

In the thymus, phenotypically and functionally mature single positive cells are generated from immature CD4+8+ precursors by a process known as positive selection. Although this event is known to involve αβTCR ligation by peptide/MHC complexes expressed on thymic stromal cells, it is clear that positive selection is a multistage process involving transition through an intermediate CD4+8+69+ phase as well as subsequent postselection phases. By analyzing the development of preselection CD4+8+69− and intermediate CD4+8+69+ thymocytes in the presence of MHC class I-deficient, MHC class II-deficient, and MHC double-deficient thymic stromal cells, we investigated the role of MHC molecules at three distinct points during positive selection. Although the initiation of positive selection is critically dependent upon MHC interactions, we find the that later stages of maturation, involving the differentiation of CD4+8− and CD4−8+ cells from CD4+8+69+ thymocytes, occur in the absence of MHC molecules. Moreover, an analysis of the postselection proliferation of newly generated CD4+8− and CD4−8+ thymocytes shows that this also occurs independently of MHC molecules. Thus, our data provide direct evidence that, although positive selection is a multistage process initiated by TCR-MHC interactions, continuation of this process and subsequent postselection events are independent of ongoing engagement of the TCR.
Structural Requirements for CD43 Function
Authors: Walker Joy , Green Jonathan M.
Published: January 2023

The regulation of T cell activation and adhesion by CD43 (leukosialin, sialophorin) has been thought to be mainly a function of the large size and negative charge of the extracellular domain of the protein. In this work, we demonstrate that the cytoplasmic tail is both necessary and sufficient for the negative regulatory effect of CD43 on cell-cell adhesion. Expression of mutant CD43 proteins in primary T cells from CD43-deficient mice demonstrated that the antiproliferative effect of CD43 is also dependent upon the cytoplasmic tail. In contrast, Ab-mediated costimulation through CD43 does not require the intracellular domain of CD43. These data demonstrate that CD43 primarily serves as a negative regulator of T cell activation and adhesion, and that this is mediated not exclusively by passive effects of the extracellular domain, but requires participation of the cytoplasmic tail, perhaps through interactions with the cytoskeleton, or alternatively, active regulation of intracellular signaling pathways.
Protective Role of β-Chemokines Associated with HIV-Specific Th Responses Against Perinatal HIV Transmission
Authors: Wasik Thomas J. , Bratosiewicz Jolanta , Wierzbicki Andrzej , Whiteman Valerie E. , Rutstein Richard R. , Starr Stuart E. , Douglas Steven D. , Kaufman David , Sison Antonio V. , Polansky Marcia , Lischner Harold W. , Kozbor Danuta
Published: January 2023

To examine the protective role of cellular immunity in the vertical transmission of HIV, we analyzed HIV-specific IL-2 and CTL responses, as well as β-chemokine expression in HIV-infected and uninfected infants of HIV+ mothers. Our results showed that HIV envelope (env) peptide-specific IL-2 responses associated with β-chemokine production were detectable at birth in the majority of uninfected infants of HIV+ mothers. The responses falling to background before the infants were 1 yr old were rarely associated with HIV-specific CTL activity. Conversely, HIV-specific Th and CTL cellular responses were absent at birth in HIV-infected infants. Infants with AIDS-related symptoms exhibited undetectable or very low levels of HIV-specific cellular immunity during the first year of life, whereas those with a slowly progressive disease showed evidence of such immunity between their second and ninth month. The latter group of infected infants tested negative for plasma HIV RNA levels shortly after birth, suggesting lack of intrauterine exposure to HIV. The presence of HIV-specific Th responses at birth in uninfected newborns of HIV+ mothers, but absence of such activities in HIV-infected infants without evidence of intrauterine HIV infection, suggests that in utero development of HIV-specific Th responses associated with β-chemokines could mediate nonlytic inhibition of infection during vertical transmission of HIV.
Role of Peptide Backbone in T Cell Recognition
Authors: Calbo Sébastien , Guichard Gilles , Bousso Philippe , Muller Sylviane , Kourilsky Philippe , Briand Jean-Paul , Abastado Jean-Pierre
Published: January 2023

T cells recognize self and nonself peptides presented by molecules of the MHC. Amino acid substitutions in the antigenic peptide showed that T cell specificity is highly degenerate. Recently, determination of the crystal structure of several TCR/MHC-peptide complexes suggested that the peptide backbone may significantly contribute to the interaction with the TCR. To directly investigate the role of the peptide backbone in T cell recognition, we performed a methylene-amino scan on the backbone of an antigenic peptide and measured the capacity of such pseudopeptides to bind their cognate MHC molecule, to sensitize target cells for T cell lysis, and to stimulate IL-2 secretion by two T cell hybridomas. For one of these pseudopeptides, we prepared fluorescent tetramers of MHC molecules and compared the staining of two T cell hybridomas. Our results demonstrate that the peptide backbone has an important contribution to TCR binding and suggest that some interactions between the peptide backbone and the TCR may be partially conserved. We discuss this finding in the perspective of TCR plasticity and T cell function.
Sodium Dodecyl Sulfate-Resistant HLA-DR “Superdimer” Bands Are in Some Cases Class II Heterodimers Bound to Antibody
Authors: Hitzel Christoph , Grüneberg Ulrike , van Ham Marieke , Trowsdale John , Koch Norbert
Published: January 2023

The detection of dimers of dimers in MHC class II crystals has excited speculation about their possible functions in T cell Ag recognition. Biochemical evidence for the existence of DR superdimers falls short of proof and is controversial. To monitor B lymphoma cells for high m.w. complexes of HLA-DR molecules, membrane preparations and cell lysates were screened by one- and two-dimensional Western blotting. Under these conditions, in which DRαβ heterodimers were readily detected, no DR complexes with an (αβ)2-chain composition could be identified. Two mAbs (L243 and D1-12) immunoprecipitated high m.w. DR complexes suspected to be superdimers. However, biochemical analysis revealed that, rather than superdimers, these were SDS-stable complexes of DR in combination with the Abs. Thus, previous observations of HLA-DR superdimer bands may also reflect complexes of DR molecules with bound Ab.
Binding Motifs of Copolymer 1 to Multiple Sclerosis- and Rheumatoid Arthritis-Associated HLA-DR Molecules
Authors: Fridkis-Hareli Masha , Neveu John M. , Robinson Renee A. , Lane William S. , Gauthier Laurent , Wucherpfennig Kai W. , Sela Michael , Strominger Jack L.
Published: January 2023

Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant “empty” HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.
Human Cytomegalovirus Binding to Human Monocytes Induces Immunoregulatory Gene Expression
Authors: Yurochko Andrew D. , Huang Eng-Shang
Published: January 2023

To continue our investigation of the cellular events that occur following human CMV (HCMV) infection, we focused on the regulation of cellular activation following viral binding to human monocytes. First, we showed that viral binding induced a number of immunoregulatory genes (IL-1β, A20, NF-κB-p105/p50, and IκBα) in unactivated monocytes and that neutralizing Abs to the major HCMV glycoproteins, gB (UL55) and gH (UL75), inhibited the induction of these genes. Next, we demonstrated that these viral ligands directly up-regulated monocyte gene expression upon their binding to their appropriate cellular receptors. We then investigated if HCMV binding also resulted in the translation and secretion of cytokines. Our results showed that HCMV binding to monocytes resulted in the production and release of IL-1β protein. Because these induced gene products have NF-κB sites in their promoter regions, we next examined whether there was an up-regulation of nuclear NF-κB levels. These experiments showed that, in fact, NF-κB was translocated to the nucleus following viral binding or purified viral ligand binding. Changes in IκBα levels correlated with the changes in NF-κB translocation. Lastly, we demonstrated that p38 kinase activity played a central role in IL-1β production and that it was rapidly up-regulated following infection. These results support our hypothesis that HCMV initiates a signal transduction pathway that leads to monocyte activation and pinpoints a potential mechanism whereby HCMV infection of monocytes can result in profound pathogenesis, especially in chronic inflammatory-type conditions.
Role of the C-C Chemokine, TCA3, in the Protective Anticryptococcal Cell-Mediated Immune Response
Authors: Doyle Hester A. , Murphy Juneann W.
Published: January 2023

Activated T lymphocytes play a crucial role in orchestrating cellular infiltration during a cell-mediated immune (CMI) reaction. TCA3, a C-C chemokine, is produced by Ag-activated T cells and is chemotactic for neutrophils and macrophages, two cell types in a murine CMI reaction. Using a gelatin sponge model for delayed-type hypersensitivity (DTH), we show that TCA3 is a component of the expression phase of an anticryptococcal CMI response in mice. TCA3 mRNA levels are augmented in anticryptococcal DTH reactions at the same time peak influxes of neutrophils and lymphocytes are observed. Neutralization of TCA3 in immunized mice results in reduced numbers of neutrophils and lymphocytes at DTH reaction sites. However, when rTCA3 is injected into sponges in naive mice, only neutrophils are attracted into the sponges, indicating TCA3 is chemotactic for neutrophils, but not lymphocytes. We show that TCA3 is indirectly attracting lymphocytes into DTH-reactive sponges by affecting at least one other chemokine that is chemotactic for lymphocytes. Of the two lymphocyte-attracting chemokines assessed, monocyte-chemotactic protein-1 and macrophage-inflammatory protein-1α (MIP-1α), only MIP-1α was reduced when TCA3 was neutralized, indicating that TCA3 affects the levels of MIP-1α, which attracts lymphocytes into the sponges. TCA3 also plays a role in protection against Cryptococcus neoformans in the lungs and brains of infected mice, as evidenced by the fact that neutralization of TCA3 results in increased C. neoformans CFU in those two organs.