bluuebunny/crossref_metadata_2025_split huggingface dataset:
Desensitization of Formyl Peptide Receptors Is Abolished in Calcium Ionophore-Primed Neutrophils: An Association of the Ligand-Receptor Complex to the Cytoskeleton Is Not Required for a Rapid Termination of the NADPH-Oxidase Response
Authors: Liu Li , Harbecke Olle , Elwing Hans , Follin Per , Karlsson Anna , Dahlgren Claes 
Published: January 2023

AbstractBinding of ligands to N-formyl peptide chemoattractant receptors exposed on human neutrophils generates signals in the cells that induce an activation of the superoxide anion producing NADPH-oxidase. Ligand binding is followed by a rapid association of the ligand-receptor complex with the cytoskeleton, a process leading to desensitization of the cells with respect to NADPH-oxidase activation. We show that neutrophils that have experienced an intracellular calcium rise obtained through interaction with the calcium-specific ionophore ionomycin are “primed” with respect to the FMLP-induced production of superoxide anions. Mobilization of FMLP receptors from intracellular pools is one well-known mechanism behind the primed response. Based on our finding that ionomycin-treated neutrophils could not be desensitized, we suggest that the lack of association between the ligand-receptor complex and the cytoskeleton is an additional priming mechanism. Since in vivo-exudated neutrophils, which also had mobilized intracellular organelles, could be desensitized, we suggest that the abolished desensitization in ionomycin-treated neutrophils is not due to an inability of newly recruited receptors to couple to the cytoskeleton. We show that a rapid termination of FMLP-induced superoxide anion production is obtained in both desensitizable and nondesensitizable neutrophils, suggesting that the desensitization phenomenon is of limited importance in the oxidase termination process.
Increasing Immunogenicity of Antigens Fused to Ig-Binding Proteins by Cell Surface Targeting
Authors: Léonetti Michel , Thai Robert , Cotton Joël , Leroy Sandrine , Drevet Pascal , Ducancel Frédéric , Boulain Jean Claude , Ménez André 
Published: January 2023

AbstractFusion of antigenic proteins to Ig-binding proteins such as protein A from Staphylococcus aureus and its derived ZZ fragment is known to increase immunogenicity of the fused Ag in vivo. To shed light on the origin of this effect, we used snake toxins as Ags and observed that 1) fusion of toxins to ZZ enhanced their presentation to a toxin-specific T cell hybridoma (T1B2), using A20 B lymphoma cells, splenocytes, or peritoneal exudate cells as APCs; 2) this enhancement further increased when the number of fused Ig-binding domains varied from two with ZZ to five with protein A; and 3) the phenomenon vanished when the fusion protein was preincubated with an excess of free ZZ or when P388D1 monocytes cells were used as APCs. Therefore, ZZ-fused toxins are likely to be targeted to surface Igs of APCs by their ZZ moiety. Furthermore, ZZ-α and toxin α stimulated similar profiles of toxin-specific T cells in BALB/c mice, suggesting a comparable processing and presentation in vivo for both toxin forms. To improve the targeting efficiency, ZZ-α was noncovalently complexed to various Igs directed to different cell surface components of APCs. The resulting complexes were up to 103-fold more potent than the free toxin at stimulating T1B2. Also, they elicited both a T cell and an Ab response in BALB/c mice, without the need of any adjuvant. This simple approach may find practical applications by increasing the immunogenicity of recombinant proteins without the use of adjuvant.
Selective Inhibition of Expression of the Chemokine Receptor CCR2 in Human Monocytes by IFN-γ
Authors: Penton-Rol Giselle , Polentarutti Nadia , Luini Walter , Borsatti Alessandro , Mancinelli Roberta , Sica Antonio , Sozzani Silvano , Mantovani Alberto 
Published: January 2023

AbstractIFN-γ is a potent activator of mononuclear phagocyte function and promotes the development of Th1 responses. Moreover, it induces and modulates chemokine production in a variety of cell types, including mononuclear phagocytes. In the present study, we examined the effect of IFN-γ on the expression of CC chemokine receptors in human monocytes. IFN-γ selectively and rapidly inhibited expression of the monocyte chemotactic protein (MCP) receptor CCR2 with an ED50 of ∼50 U/ml. The effect was rapid (detectable after 1 h) and reversible. Other chemokine receptors (CCR1, CCR3, CCR4, and CCR5) were not substantially affected, and CXCR4 was reduced. IFN-γ acted in concert with LPS, TNF-α, and IL-1β in inhibiting CCR2 expression. IFN-γ-treated monocytes showed a shorter half-life of CCR2 mRNA compared with untreated cells, whereas the rate of nuclear transcription was unaffected. The inhibition of CCR2 mRNA expression by IFN-γ was associated with a lower number of surface receptors and lower chemotactic responsiveness. Thus, IFN-γ, an inducer of MCP-1 and MCP-3 in mononuclear phagocytes, selectively inhibits expression of the MCP receptor CCR2 in monocytes. These results are consistent with an emerging paradigm of divergent regulation by several agents of chemokine production and receptor expression in monocytes. The inhibition of MCP-1R expression may serve as a means of retaining mononuclear phagocytes at sites of inflammation and as a feedback mechanism in the regulation of recruitment from the blood.
Target Recognition of β2-Glycoprotein I (β2GPI)-Dependent Anticardiolipin Antibodies: Evidence for Involvement of the Fourth Domain of β2GPI in Antibody Binding
Authors: George Jacob , Gilburd Boris , Hojnik Maja , Levy Yair , Langevitz Pnina , Matsuura Eiji , Koike Takao , Shoenfeld Yehuda 
Published: January 2023

Abstractβ2-glycoprotein I (β2GPI) is an absolute requirement for the binding of autoimmune anticardiolipin Abs (aCL) to cardiolipin (CL). We evaluated the target recognition of human β2GPI by IgG derived from two patients with primary and two with secondary antiphospholipid syndrome. The total IgG serum fractions and β2GPI affinity-purified IgGs were assessed by using various domain-deleted mutants (DM) of human β2GPI (DMs: I-III, I-IV, II-V, III-V, IV-V, and V) and mouse mAbs against individual β2GPI domains. The four IgGs bound slightly to CL in the absence of β2GPI and showed increased binding in the β2GPI presence. Following affinity purification of the IgGs on a β2GPI column, reactivity toward CL was absent. DMs containing domain V inhibited the binding of biotinylated β2GPI to CL. The addition to CL-coated plates of DM V, but not the other DMs, reduced the binding of all four IgGs. The anti-β2GPI IgGs bound only to complete β2GPI and DM I-IV coated on the plates. The binding to plate-adsorbed β2GPI could be inhibited by complete β2GPI and DM I-IV, the latter being a more efficient inhibitor. Further, the human anti-β2GPI IgGs could compete with the binding to β2GPI of Cof-21 mouse mAb (directed at domain IV), but not with the two other mouse mAbs. The results suggest that some “autoimmune:” β2GPI-dependent anticardiolipin Abs recognize a β2GPI target that is distinct from the CL-binding site in domain V. The target site for some antiphospholipid syndrome IgGs appear to reside in domain IV of β2GPI.
Cutting Edge: Characterization of an Associated 16-kDa Tyrosine Phosphoprotein Required for Ly-49D Signal Transduction
Authors: Mason Llewellyn H. , Willette-Brown Jami , Anderson Stephen K. , Gosselin Pierre , Shores Elizabeth W. , Love Paul E. , Ortaldo John R. , McVicar Daniel W. 
Published: January 2023

AbstractLy-49D is an activating receptor on NK cells that does not become tyrosine phosphorylated upon activation. This report demonstrates that immunoprecipitation of Ly-49D, following pervanadate treatment or specific Ab cross-linking, coprecipitates a 16-kDa tyrosine-phosphorylated protein (pp16). Immunoblotting experiments and data from TCR-ζ/FcεRIγ double knockout mice confirm that pp16 is not TCR-ζ, TCR-η, or FcεRIγ. Association of pp16 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49DR54L) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49DR54L transfectants fail to mediate Ca2+ mobilization following Ab cross-linking. Therefore, signaling through Ly-49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families.
Mechanisms of CD8β-Mediated T Cell Response Enhancement: Interaction with MHC Class I/β2-Microglobulin and Functional Coupling to TCR/CD3
Authors: Wheeler Christopher J. , Chen Jing-Yi , Potter Terry A. , Parnes Jane R. 
Published: January 2023

Abstract
CD8β expression results in enhanced IL-2 production and/or altered specificity in allogeneic MHC class I-restricted T cell hybridomas. Expression of chimeric CD8β-α molecules (extracellular CD8β, transmembrane and cytoplasmic CD8α) also results in enhancement of T hybridoma responses to alloantigen, suggesting that at least part of CD8β’s ability to influence responses similar to those of mature CD8+ T cells is mediated by its extracellular domain. Current data suggest that CD8β-mediated response enhancement proceeds through mechanisms similar to those mediated by CD8α, i.e., interacting with MHC class I and stabilizing CD8-associated Lck activity. In this study we present evidence that the extracellular portion of CD8β is capable of independent interaction with MHC class I/β2m dimers in the absence of CD8α. In addition, CD8β may enhance interaction with MHC class I/β2m when associated with CD8α. We also present evidence from T hybridoma responses suggesting that the extracellular portion of CD8β is uniquely capable of efficient interaction with the TCR/CD3 complex and may couple the TCR/CD3 complex to other surface components capable of enhancing TCR-mediated signals. This represents the first evidence that a critical coreceptor function can be preferentially associated with the CD8β subunit.
Oligoclonal Development of B Cells Bearing Discrete Ig Chains in Chicken Single Germinal Centers
Authors: Arakawa Hiroshi , Kuma Kei-ichi , Yasuda Masahiro , Furusawa Shuichi , Ekino Shigeo , Yamagishi Hideo 
Published: January 2023

Abstract
Chicken single germinal centers enable us to analyze the postbursal diversifications of B cells due to their easy isolation. Germinal center formation has peaked by day 7 of primary responses and begins to wane 14 days after immunization. To detail the kinetics of Ig mutation and selection, we analyzed Ig light chain sequences recovered from single germinal centers at 7 and 11 days postimmunization with an artificial Ag. Our observations show that multiple, Ag-activated B cells migrating into single germinal centers are diversified by gene conversion in the very early phase of the germinal center reaction and are subsequently subjected to point mutations and selection for oligoclonality.
Presentation of Proteolipid Protein Epitopes and B7-1-Dependent Activation of Encephalitogenic T Cells by IFN-γ-Activated SJL/J Astrocytes
Authors: Tan Litjen , Gordon Kenneth B. , Mueller John P. , Matis Louis A. , Miller Stephen D. 
Published: January 2023

AbstractThere is controversy regarding the possible role of glial cells as APCs in the pathogenesis of central nervous system (CNS) demyelinating diseases such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Microglia have been clearly shown to present Ag in the CNS, and due to the proximity of activated astroglial cells to infiltrating T cells and macrophages in demyelinating lesions, it is also possible that astrocytes positively or negatively regulate disease initiation and/or progression. We examined the capacity of IFN-γ-treated astrocytes from EAE-susceptible SJL/J mice to process and present myelin epitopes. IFN-γ activation up-regulated ICAM-1, VCAM-1, MHC class II, invariant chain, H2-M, CD40, and B7-1 as determined by FACS and/or RT-PCR analyses. B7-2 expression was only marginally enhanced on SJL/J astrocytes. Consistent with the expression of these accessory molecules, IFN-γ-treated SJL/J astrocytes induced the B7-1-dependent activation of Th1 lines and lymph node T cells specific for the immunodominant encephalitogenic proteolipid protein (PLP) epitope (PLP139–151) as assessed by proliferation and activation for the adoptive transfer of EAE. Interestingly, IFN-γ-activated astrocytes efficiently processed and presented PLP139–151, but not the subdominant PLP178–191, PLP56–70, or PLP104–117 epitopes, from intact PLP and a recombinant variant fusion protein of PLP (MP4). The data are consistent with the hypothesis that astrocytes in the proinflammatory CNS environment have the capability of activating CNS-infiltrating encephalitogenic T cells specific for immunodominant epitopes on various myelin proteins that may be involved in either the initial or the relapsing stages of EAE.
The Glycosylphosphatidylinositol-Anchored Form and the Transmembrane Form of CD58 Associate with Protein Kinases
Authors: Itzhaky Dganit , Raz Nava , Hollander Nurit 
Published: January 2023

AbstractThe significance of the glycosylphosphatidylinositol (GPI) anchor is unknown. Since GPI-anchored proteins mediate signaling, it has been suggested that the GPI structure serves as a signal-transducing element. However, the division of signaling functions between transmembrane and GPI-anchored proteins is unclear. Studies of distinct membrane-anchored forms of the same protein may resolve this issue. The adhesion molecule CD58 is expressed on the cell surface in both a transmembrane and a GPI-anchored form and hence provides a useful model. We studied CD58 in the human B lymphoblastoid cell line JY. In addition to mediating adhesion, CD58 is involved in signal transduction. Incubation of JY cells with immobilized anti-CD58 Abs results in extensive tyrosine phosphorylation and in secretion of TNF-α. We demonstrate that CD58 is associated with protein kinase(s) and with several kinase substrates. We further demonstrate that both CD58 isoforms are involved. CD58 in JY variant cells, which express only the transmembrane form, as well as CD58 in JY variant cells, which express only the GPI-anchored form, are associated with kinase activity. This association results in a phosphorylation pattern that is common to the variant and to wild-type JY cells. Thus, these findings suggest that the capacity of GPI-anchored proteins to interact with kinases is not always dependent on the GPI anchor itself.
Selective Inhibition of IL-5 Receptor α-Chain Gene Transcription by IL-5, IL-3, and Granulocyte-Macrophage Colony-Stimulating Factor in Human Blood Eosinophils
Authors: Wang Peng , Wu Ping , Cheewatrakoolpong Boonlert , Myers Joyce G. , Egan Robert W. , Billah M. Motasim 
Published: January 2023

AbstractHigh affinity receptor for IL-5 (IL-5R), a predominant eosinophil maturation factor, is composed of an IL-5-binding α-chain (IL-5Rα) and a signal-transducing β-chain that is shared by IL-3 and granulocyte-macrophage CSF (GM-CSF) receptors (IL-3R and GM-CSFR). By Northern blot analysis of mRNAs obtained from normal human blood eosinophils, we show in this report that the hematopoietic cytokines IL-5, IL-3, and GM-CSF down-regulate IL-5Rα mRNA while up-regulating α-chain mRNAs for both IL-3R and GM-CSFR as well as the β-chain mRNA. More detailed characterization reveals that the down-regulation of IL-5Rα mRNA is specific to IL-3, IL-5, and GM-CSF; occurs very rapidly (reaching maximum inhibition within 2 h); is cytokine dose dependent; and does not require protein synthesis. Nuclear run-on and mRNA stability experiments demonstrate that cytokine-induced inhibition of IL-5Rα mRNA accumulation occurs at the level of IL-5Rα gene transcription, whereas enhanced accumulation of mRNAs for IL-3Rα and the β-chain results from reduced mRNA degradation. We suggest from these experiments that in human blood eosinophils, IL-5Rα gene transcription and IL-5Rα mRNA metabolism can be regulated by mechanisms that are distinct from those used for IL-3Rα and GM-CSFRα.
Endogenous IL-12 Is Required for Induction and Expression of Experimental Autoimmune Uveitis
Authors: Tarrant Teresa K. , Silver Phyllis B. , Chan Chi-Chao , Wiggert Barbara , Caspi Rachel R. 
Published: January 2023

AbstractExperimental autoimmune uveitis (EAU) has been associated with a Th1 response. However, in IFN-γ-deficient mice, EAU develops in the context of an effector response having Th2-like elements, and administration of IL-12 to mice immunized for EAU induction can be protective. We, therefore, investigated whether endogenous IL-12 is required for development of EAU. IL-12 p40-deficient mice (12KO) were resistant to EAU induced with the uveitogenic retinal Ag interphotoreceptor retinoid binding protein (IRBP). Delayed hypersensitivity to IRBP was marginally reduced, whereas Ag-specific proliferation was enhanced. Primed lymphocytes of wild-type (wt) mice, cultured with IRBP, produced a Th1-like cytokine profile and transferred EAU to syngeneic wt recipients. Interestingly, the same cells were inefficient in transferring EAU to 12KO recipients, unless IL-12 was included in the culture. Primed cells of the 12KO mice produced a Th2-like cytokine profile and failed to transfer EAU. However, when IL-12 was added to the culture, 12KO cells produced large amounts of IFN-γ and transferred EAU to naive 12KO recipients. We conclude that resistance to EAU of 12KO mice is not due to an inherent inability of these mice to develop ocular disease. Despite an apparent similarity in Ag-specific cytokine responses to IFN-γ-deficient mice, 12KO mice have inhibited generation of uveitogenic effector cells, a situation that can be reversed even after priming, by adding exogenous IL-12 ex vivo. Lastly, the diminished ability of primed wt lymphocytes to induce EAU in 12KO mice indicates a role for endogenous IL-12 in the efferent phase of disease expression that is distinct from its role during Ag priming.
Both PU.1 and Nuclear Factor-κB Mediate Lipopolysaccharide-Induced HIV-1 Long Terminal Repeat Transcription in Macrophages
Authors: Lodie Tracey A. , Reiner Marshall , Coniglio Salvatore , Viglianti Gregory , Fenton Matthew J. 
Published: January 2023

Abstract
We recently reported that LPS stimulation of monocytic cells leads to the activation of PU.1, a member of the Ets family of transcription factors. Phosphorylation of PU.1 by protein kinase CK2 was found to up-regulate its trans-activation function, but not its DNA binding activity. Previous studies suggested that Ets proteins could bind to NF-κB motifs at the tetrameric core sequence TTCC. In macrophages, LPS-inducible HIV-1 gene expression is mediated in part by binding of NF-κB to identical tandem binding sites located within the long terminal repeat (LTR). Thus, we performed additional studies to determine whether PU.1 also played a role in regulating HIV-1 gene expression in macrophages. Our functional studies revealed that activation of the HIV-1 LTR in LPS-stimulated cells requires both NF-κB and PU.1. Extensive mutagenesis of the HIV-1 LTR revealed that PU.1-dependent activation requires the Ets motif within the upstream NF-κB site, whereas NF-κB itself binds to the downstream site. We also found that insertion of five additional nucleotides between the NF-κB sites abolished LPS inducibility, suggesting a direct interaction between factors that bind these sites. Lastly, we found that mutation of PU.1 at serine 148, which prevents its phosphorylation by CK2, blocked its ability to activate the HIV-1 LTR in response to LPS. These effects were promoter specific because PU.1 did not affect LPS-inducible activation of a distinct NF-κB-dependent promoter. While these data do not demonstrate direct binding of PU.1 to the HIV-1 LTR, they illustrate a novel role for PU.1 in activation of the HIV-1 LTR by LPS.
Crucial Role of TCRγ Chain Junctional Region in Prenyl Pyrophosphate Antigen Recognition by γδ T Cells
Authors: Bukowski Jack F. , Morita Craig T. , Band Hamid , Brenner Michael B. 
Published: January 2023

AbstractHuman γδ T cells recognize prenyl pyrophosphate Ags and their analogues in a Vγ2Vδ2 TCR-dependent manner. Few data are available regarding the TCR structural requirements for recognition of such prenyl pyrophosphate Ags by γδ T cells. Presently, we made chain pair switch, chimeric, and site mutant γδ TCRs and transfected them into TCR− mutant Jurkat T cells to examine the effects of changing the TCRγ junctional region sequences on reactivity to prenyl pyrophosphate Ags. Substitution of the TCRγ junctional region (N and J) sequences from an Ag-reactive TCR with TCRγ junctional region sequences from an Ag-nonreactive TCR abrogated reactivity to the prenyl pyrophosphate Ag isopentenyl pyrophosphate and to its synthetic analogue ethyl pyrophosphate but not to a mycobacterial supernatant containing a mixture of prenyl pyrophosphate Ags. Substitution of only the TCRγ N nucleotide region with that from this Ag-nonreactive TCR destroyed reactivity to isopentenyl pyrophosphate and to the mycobacterial supernatant. Substitution of the entire Vδ2 chain from the Ag-reactive TCR with a Vδ1 chain from an Ag-nonreactive TCR yielded a prenyl pyrophosphate Ag-nonreactive TCR. Thus, using TCR mutagenesis and TCR transfectants, we show that γδ TCR reactivity to prenyl pyrophosphate Ags is dependent upon the junctional region of the TCRγ chain and upon pairing of Vγ2 and Vδ2 TCR chains. These structural requirements of TCRγδ recognition of prenyl pyrophosphates distinguish this reactivity from that of protein superantigens and emphasize the importance of the TCRγ CDR3 loop and adjacent residues.
A λ 3′ Enhancer Drives Active and Untemplated Somatic Hypermutation of a λ1 Transgene
Authors: Kong Qingzhong , Zhao Lisa , Subbaiah Sathish , Maizels Nancy 
Published: January 2023

AbstractSomatic hypermutation is a highly regulated process that targets mutations to the rearranged Ig genes. Little is known about the cis-elements required for somatic hypermutation of the λ light chain gene. We have studied somatic hypermutation of a rearranged λ1 transgene under the control of either a λ2-4 or κ 3′ enhancer. The mutations in the transgenes were analyzed by sequencing DNA amplified from hypermutating Peyer’s patch B cells. The results indicate that the λ 3′ enhancer can drive active hypermutation of a λ1 transgene in Peyer’s patch cells. The λ1 transgene under analysis carried two marked Vλ2 genes immediately upstream that could serve as sequence donors in possible gene conversion events. There was no evidence of sequence transfer to the hypermutated λ1 gene, suggesting that gene conversion is not a major mechanism for somatic hypermutation in mice.