bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal Role of Chemokines in a Murine Model of Cockroach Allergen-Induced Airway Hyperreactivity and Eosinophilia
Authors: Campbell Emma M. , Kunkel Steven L. , Strieter Robert M. , Lukacs Nicholas W.
Published: December 2022

The increase in inner-city asthma among children appears to be due to allergic responses to several allergens. Recent studies have demonstrated that Ags derived from cockroaches are especially prominent in these settings and a significant health concern for the induction of asthma in children. In the present study, we have outlined the development of a murine model of cockroach allergen-induced airway disease and assessed specific mechanisms of the response, which resembles atopic human asthma. The allergic responses in this model include allergen-specific airway eosinophilia and significantly altered airway physiology, which directly correlates with inflammation. We have further utilized this allergen to establish primary and secondary rechallenge stages of late phase hyperreactivity exacerbation. This latter stage is characterized by greater changes in airway physiology than the primary stage, and it is likely due to the preexisting peribronchial inflammation present at the time of the second allergen rechallenge. We have identified specific roles for CC chemokines during these stages, with MIP-1α being an important eosinophil attractant during the primary stage and eotaxin during the secondary rechallenge stage. The development of these models allows the evaluation of mediators involved in both stages of cockroach allergen challenge, as well as the testing of specific therapeutic modalities.
Cutting Edge: Transcutaneous Immunization with Cholera Toxin Protects Mice Against Lethal Mucosal Toxin Challenge
Authors: Glenn Gregory M. , Scharton-Kersten Tanya , Vassell Russell , Mallett Corey P. , Hale Thomas L. , Alving Carl R.
Published: December 2022

We recently reported that application of cholera toxin (CT) to the skin results in transcutaneous immunization and induces a systemic Ab response to both CT and coadministered Ags. In this paper, we demonstrate antitoxin IgG and IgA Abs in sera, lung washes, and stool samples from immunized mice as well as a broad spectrum of IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3) in the sera. Mice immunized with CT by the transcutaneous route exhibited significant protection from intranasal challenge with a lethal dose of CT. Thus, clinically relevant immunity against mucosal toxin challenge can be achieved via the transcutaneous route.
Identification of a Cytoplasmic Region of CD20 Required for Its Redistribution to a Detergent-Insoluble Membrane Compartment
Authors: Polyak Maria J. , Tailor Sweta H. , Deans Julie P.
Published: December 2022

CD20 is a B lymphocyte integral membrane protein with signal-transducing properties. Abs directed toward extracellular CD20 epitopes activate nonreceptor tyrosine kinases and modulate cell cycle progression of B lymphocytes. Recently, we demonstrated that binding of CD20 Abs to B cells induces the rapid redistribution of up to 95% of CD20 molecules to low density, detergent-insoluble membrane microdomains and induces the appearance of an approximately 50-kDa tyrosine-phosphorylated protein in the same compartment. Active relocalization of CD20 may thus be critical to the initiation of signaling events by CD20. The CD20 cDNA sequence predicts a nonglycosylated protein with four transmembrane-spanning regions and intracellular amino and carboxyl termini. Here we provide verification of the location of both the intracellular and extracellular regions of the CD20 molecule and identify a membrane-proximal sequence in the cytoplasmic carboxyl tail that is required for CD20 to redistribute to detergent-insoluble membrane microdomains.
Generation of Biologically Active IL-1β by Matrix Metalloproteinases: A Novel Caspase-1-Independent Pathway of IL-1β Processing
Authors: Schönbeck Uwe , Mach François , Libby Peter
Published: December 2022

Biologic activity of IL-1β requires processing of the inactive precursor, a function generally ascribed to IL-1β-converting enzyme (caspase-1). However, alternative mechanisms of IL-1β activation have been postulated in local inflammatory reactions. Expression of IL-1β and matrix metalloproteinases (MMPs) frequently occurs simultaneously at sites of inflammation. We describe here that stromelysin-1 (MMP-3), as well as the gelatinases A (MMP-2) and B (MMP-9), processes recombinant human IL-1β precursor (pIL-1β) into biologically active forms. Detection of both pIL-1β processing and biologic IL-1β activity demonstrated different processing capacities of the respective MMPs. Conversion of pIL-1β by stromelysin-1 required coincubation for at least 1 h, and biologic activity faded after 8 h to 24 h. Gelatinase A was less effective in processing pIL-1β, requiring at least 24 h of coincubation. In contrast, gelatinase B processed pIL-1β within minutes, resulting in immunoreactive products as well as biologic activity stable for 72 h. In addition, prolonged incubation of mature IL-1β with stromelysin-1, and to a lesser extent also with gelatinases, but not with interstitial collagenase, resulted in the degradation of mature IL-1β. None of the MMPs processed the second isoform of IL-1, IL-1α. The present study indicates a biphasic regulation of IL-1β activity by MMPs: a caspase-1-independent pathway of IL-1β activation and inhibition of IL-1β activity by degrading the mature cytokine. The balance of the respective MMPs and pIL-1β might regulate the long term appearance of IL-1β activity at sites of acute or chronic inflammation.
Steroid Hormone Regulation of Cytokine Secretion by Proteolipid Protein-Specific CD4+ T Cell Clones Isolated from Multiple Sclerosis Patients and Normal Control Subjects
Authors: Correale Jorge , Arias Magdalena , Gilmore Wendy
Published: December 2022

Steroid hormones have long been known to modulate immune function, and recent studies indicate that one of the means by which they do so involves effects on the secretion of immunoregulatory cytokines. Our laboratory has found recently that estradiol (E2) selectively modifies cytokine secretion in proteolipid protein (PLP)-specific, CD4+ T cell clones isolated from patients with the demyelinating disease, multiple sclerosis, and from normal control subjects. The data suggest that E2 may play a role in regulating the balance between pro- and antiinflammatory conditions, especially at concentrations typical of pregnancy. To determine whether other pregnancy-associated steroid hormones are capable of similar activity, we expanded our testing to include estrone (E1), estriol (E3), progesterone, and dexamethasone. The results indicate that E1 and E3 enhance secretion of Ag- or anti-CD3-stimulated IL-10 and IFN-γ in dose-dependent fashion, almost identical to that of E2. The effect on IL-10 was more potent than occurred with IFN-γ. In addition, E1 and E3, like E2, had a biphasic effect on TNF-αβ secretion, with low concentrations stimulatory, and high doses inhibitory. None of the estrogens influenced IL-4 or TGF-β secretion. Progesterone enhanced secretion of IL-4, without affecting any other tested cytokine. Finally, dexamethasone induced TGF-β secretion, but inhibited IFN-γ and TNF-αβ. This differential effect of steroid hormones on the secretion of cytokines by CD4+ human T cell clones is consistent with the possibility that, collectively, they promote antiinflammatory conditions at high concentrations typical of pregnancy.
Selective Inhibitors of Cytosolic or Secretory Phospholipase A2 Block TNF-Induced Activation of Transcription Factor Nuclear Factor-κB and Expression of ICAM-1
Authors: Thommesen Liv , Sjursen Wenche , Gåsvik Kathrine , Hanssen Wenche , Brekke Ole-Lars , Skattebøl Lars , Holmeide Anne Kristin , Espevik Terje , Johansen Berit , Lægreid Astrid
Published: December 2022

TNF signaling mechanisms involved in activation of transcription factor NF-κB were studied in the human keratinocyte cell line HaCaT. We show that TNF-induced activation of NF-κB was inhibited by the well-known selective inhibitors of cytosolic phospholipase A2 (cPLA2): the trifluoromethyl ketone analogue of arachidonic acid (AACOCF3) and methyl arachidonyl fluorophosphate. The trifluoromethyl ketone analogue of eicosapentaenoic acid (EPACOCF3) also suppressed TNF-induced NF-κB activation and inhibited in vitro cPLA2 enzyme activity with a similar potency as AACOCF3. The arachidonyl methyl ketone analogue (AACOCH3) and the eicosapentanoyl analogue (EPACHOHCF3), which both failed to inhibit cPLA2 enzyme activity in vitro, had no effect on TNF-induced NF-κB activation. TNF-induced NF-κB activation was also strongly reduced in cells stimulated in the presence of the secretory PLA2 (sPLA2) inhibitors 12-epi-scalaradial and LY311727. Addition of excess arachidonic acid suppressed the inhibitory effect of 12-epi-scalaradial and LY311727. Moreover, both methyl arachidonyl fluorophosphate and 12-epi-scalaradial blocked TNF-mediated enhancement of expression of ICAM-1. Activation of NF-κB by IL-1β was markedly less sensitive to both cPLA2 and sPLA2 inhibitors. The results indicate that both cPLA2 and sPLA2 may be involved in the TNF signal transduction pathway leading to nuclear translocation of NF-κB and to NF-κB-activated gene expression in HaCaT cells.
Oxidant Stress Incites Spreading of Macrophages via Extracellular Signal-Regulated Kinases and p38 Mitogen-Activated Protein Kinase
Authors: Ogura Makoto , Kitamura Masanori
Published: December 2022

Cultured macrophages exhibit spreading in response to external stimuli. It is relevant to in vivo morphologic changes of macrophages during extravasation, migration, and differentiation. The present study was performed to elucidate molecular mechanisms that regulate spreading of macrophages. Redox is a crucial factor that modulates a wide range of cell function. We found that macrophages undergo spreading in response to oxidant stress caused by hydrogen peroxide or an oxidant generating agent menadione. To identify signaling pathways involved, a role of mitogen-activated protein (MAP) kinases was investigated. Western blot analysis showed that treatment of macrophages with menadione rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK1, ERK2) and p38 MAP kinase, but not c-Jun N-terminal kinase (JNK). Pharmacologic inhibition of either ERK or p38 activation blunted the macrophage spreading. Similarly, transfection with dominant-negative mutants of ERKs or a mutant p38 significantly suppressed the oxidant-triggered spreading. ERKs and p38 are known to activate serum response element (SRE) via phosphorylation of the ternary complex factor Elk-1. To further identify downstream events, we focused on a role of SRE. Stimulation of macrophages with menadione induced activation of SRE. Intervention in the SRE activation by a dominant-negative mutant of Elk-1 inhibited the menadione-induced spreading. These results suggest that oxygen radical metabolites, the well-known mediators for tissue injury, incite spreading of macrophages via the MAP kinase-SRE signaling pathways.
A Breast and Melanoma-Shared Tumor Antigen: T Cell Responses to Antigenic Peptides Translated from Different Open Reading Frames
Authors: Wang Rong-Fu , Johnston Samuel L. , Zeng Gang , Topalian Suzanne L. , Schwartzentruber Douglas J. , Rosenberg Steven A.
Published: December 2022

Infusion of TIL586 along with IL-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. Here, we report that screening a cDNA library from the 586mel cell line using CTL clones derived from TIL586 resulted in the isolation of a gene, CAG-3 (cancer Ag gene 3). Sequence analysis revealed that CAG-3 encodes an open reading frame identical to NY-ESO-1, which was recently reported to be recognized by autologous serum from a patient with esophageal cancer. Thus, NY-ESO-1 appears to be an immune target for both Ab- and T cell-mediated responses. Significantly, NY-ESO-1-specific CTL clones were capable of recognizing two HLA-A31-positive fresh and cultured breast tumors. To our knowledge, this represents the first direct demonstration that tumor-specific CTL clones can recognize both breast and melanoma tumor cells. A 10-mer antigenic peptide ESO10–53 (ASGPGGGAPR) was identified from the normal open reading frame of NY-ESO-1 based on its ability to sensitize HLA-A31-positive target cells for cytokine release and specific lysis. Interestingly, two additional CTL clones that were sensitized with NY-ESO-1 recognized two overlapping antigenic peptides derived from an alternative open reading frame of the same gene. These findings indicate that CTLs simultaneously responded to two different gene products translated from the normal and alternative reading frames of the same gene. Understanding of this mechanism by which the alternative reading frame is translated may have important implications in tumor immunology.
A Synthetic Lipopolysaccharide-Binding Peptide Based on Amino Acids 27–39 of Serum Amyloid P Component Inhibits Lipopolysaccharide-Induced Responses in Human Blood
Authors: de Haas Carla J. C. , van der Tol Marijke E. , Van Kessel Kok P. M. , Verhoef Jan , Van Strijp Jos A. G.
Published: December 2022

LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27–39, a 13-mer peptide consisting of amino acids 27–39 of SAP, competitively inhibited the binding of LPS to SAP. In addition, pep27–39 significantly inhibited ReLPS-induced responses in phagocytes in the presence of serum, as well as in human whole blood. Carboxamidomethylated pep27–39 showed an even more pronounced reduction of the ReLPS-induced priming of phagocytes in human blood. Performing gel filtration of FITC-labeled ReLPS incubated with soluble CD14, we showed that SAP could not prevent binding of LPS to soluble CD14, in contrast to pep27–39. The ability of pep27–39 to antagonize specifically the effects of LPS in the complex environment of human blood suggests that pep27–39 may be a novel therapeutic agent in the treatment of Gram-negative sepsis.
Lipopolysaccharide-Induced Desensitization ofjunBGene Expression in a Mouse Macrophage-Like Cell Line, P388D1
Authors: Fujihara Mitsuhiro , Ikebuchi Kenji , Maekawa Takami L. , Wakamoto Shinobu , Ogiso Chikayo , Ito Takatoshi , Takahashi Tsuneo A. , Suzuki Tsuneo , Sekiguchi Sadayoshi
Published: December 2022

Treatment of a mouse macrophage cell line, P388D1, for 1 h with bacterial LPS caused a transient increase in the level of junB mRNA expression. These cells became refractory in terms of the junB gene response to exposure to a second round of LPS or lipid A, but not to PMA. The LPS-induced desensitized state was not due to the shortening of the half-life of junB mRNA, but was suggested, by nuclear run-on analysis, to be caused by reduction of junB gene transcription. Pretreating cells with herbimycin A, a tyrosine kinase inhibitor, substantially inhibited LPS-induced expression of junB mRNA and decreased tyrosine phosphorylation of 38- to 42-kDa proteins, which comigrated with p38 and p42 mitogen-activated protein (MAP) kinases. Parallel to down-regulation of junB mRNA expression, activation of the p38 MAP kinase was markedly reduced in LPS-tolerant cells, whereas activation of p42 MAP kinase was relatively constant. The specific p38 MAP kinase inhibitor, SB202190, potently inhibited LPS-induced junB mRNA expression. These results suggest that the LPS-induced desensitization of junB gene expression occurs at or upstream of the level of gene transcription and may be involved in a defective LPS-induced p38 MAP kinase pathway.
Modulation of Human Neutrophil Apoptosis by Immune Complexes
Authors: Gamberale Romina , Giordano Mirta , Trevani Analía S. , Andonegui Graciela , Geffner Jorge R.
Published: December 2022

In the present study we examined whether immune complexes (IC) are able to modulate human neutrophil apoptosis. We observed different effects depending on the type of IC employed. Precipitating IC (pIC) and Ab-coated erythrocytes (E-IgG) triggered a marked stimulation of apoptosis, while heat-aggregated IgG and soluble IC, significantly delayed spontaneous apoptosis. Blocking Abs directed to Fcγ receptor type II (FcγRII), but not to FcγRIII, markedly diminished the acceleration of apoptosis triggered by either pIC or E-IgG, supporting a critical role for FcγRII in apoptosis stimulation. This phenomenon, on the other hand, does not appear to involve IC phagocytosis or the participation of CR3. Acceleration of neutrophil apoptosis triggered by either pIC or E-IgG seems to require the activation of the respiratory burst, as suggested by 1) the ability of catalase to prevent apoptosis stimulation; 2) the effect of azide, an heme enzyme inhibitor, which dramatically enhanced apoptosis induced by pIC or E-IgG; and 3) the inability of pIC or E-IgG to accelerate apoptosis of neutrophils isolated from CGD patients. It is well established that IC affect the course of inflammation by inducing the release of inflammatory cytokines, proteolytic enzymes, oxidative agents, and other toxic molecules. Our results suggest that IC may also affect the course of inflammation by virtue of their ability to modulate neutrophil apoptosis.
The Regulation and Functional Consequence of Proinflammatory Cytokine Binding on Human Intestinal Epithelial Cells
Authors: Panja Asit , Goldberg Stan , Eckmann Lars , Krishen Priya , Mayer Lloyd
Published: December 2022

Products of an activated immune system may affect cells within the immune system as well as nonlymphoid cells in the local environment. Given the immunologically activated state of the intestinal tract, it is conceivable that locally produced cytokines could regulate epithelial cell function. To assess whether epithelial cells are targets for particular cytokines, we initiated studies on the binding of a panel of proinflammatory cytokines in freshly isolated epithelial cells from normal and inflammatory bowel disease (IBD) patients as well as in cell lines. Isolated intestinal epithelial cells (IEC) were stained with phycoerythrin-conjugated or biotinylated cytokines to determine the expression and density of receptors for IL-1β, IL-6, granulocyte-macrophage CSF (GM-CSF), and TNF-α. Receptors for IL-1β, IL-6, and GM-CSF were readily detectable in all epithelial cell preparations at levels equal to (GM-CSFR) or lower than those seen on monocytes. However TNFα-R were not detectable on freshly isolated IECs. Receptor density was greater in surface vs crypt epithelial cells, but no significant differences were seen between normal and IBD epithelial cells. Expression of IL-1R and IL-6R was enhanced by LPS and IFN-γ. Functionally, IL-1β enhanced proliferation of the IEC cell line, DLD1, whereas GM-CSF treatment of de-differentiated crypt-like DLD1 and HT29 cells resulted in enhanced expression of ICAM-1. Furthermore, TNF-α treatment enhanced the secretion of IL-8 and GRO-α in HT29 cells, but not in freshly isolated IEC cultures. The differential binding and function of proinflammatory cytokines on IEC support the hypothesis that these cytokines may be involved in normal physiological processes as well as in regulating mucosal immune responses.
Essential Roles of Lyn in Fibronectin-Mediated Filamentous Actin Assembly and Cell Motility in Mast Cells
Authors: Suzuki Takeshi , Shoji Shunsuke , Yamamoto Kazuhiko , Nada Shigeyuki , Okada Masato , Yamamoto Tadashi , Honda Zen-ichiro
Published: December 2022

Although the requirement for c-Src in extracellular matrix (ECM)-mediated fibroblast motility has been well established, the roles of hemopoietic Src family protein tyrosine kinases in leukocyte migration have not been fully elucidated. To address the issue, we analyzed fibronectin (Fn)-mediated adhesion signaling in rat basophilic leukemia (RBL) 2H3 cells overexpressing 1) Csk, 2) a membrane-anchored, gain-of-function Csk (mCsk), and 3) a kinase-defective mCsk (mCsk(−)). Parent RBL2H3 cells, expressing autoactivated c-kit, readily adhered to Fn-coated surface, developed typical leukocyte adhesion machinery (podosome), and migrated toward Fn without cytokine priming, thus provided a simple experimental system to analyze Fn-mediated outside-in signaling. While overexpression of Csk or the Csk mutants did not significantly affect cell adhesion to the Fn surface or α5 integrin recruitment to the attachment sites, Csk suppressed and mCsk almost abolished Fn-mediated tyrosine phosphorylation of paxillin, filamentous actin assembly to podosomes, and cell migration, but mCsk(−) did not. Coexpression of LynA devoid of C-terminal negative regulatory tyrosine in mCsk cells successfully restored Fn-mediated podosome formation and cell migration. Coexpression of c-Src lacking the C-terminal tyrosine reconstructed podosomes, but could not restore the cell migration regardless of its expression level. Collectively, these observations provide evidence that Src family protein tyrosine kinases are required, and that Lyn could transmit sufficient signal for Fn-mediated cytoskeletal changes leading to cell locomotion in RBL2H3 cells, and they suggest that Lyn and c-Src are differentially involved in cell motility.
Membrane Cofactor Protein: Importance ofN- andO-Glycosylation for Complement Regulatory Function
Authors: Liszewski M. Kathryn , Leung Marilyn K. , Atkinson John P.
Published: December 2022

Membrane cofactor protein (MCP; CD46) is a type 1 membrane glycoprotein that inhibits complement activation on host cells. It also is a measles virus (MV) receptor, an adherence factor for group A Streptococcus pyogenes, and a cellular pilus receptor for pathogenic Neisseria. The amino terminus of MCP consists of four complement control protein (CCP) repeats, three of which (CCP-1, -2, and -4) possess N-glycans. Immediately following the CCP modules is an alternatively spliced region for extensive O-glycosylation (termed the STP domain). Previous studies established that the N-glycan of CCP-2 is essential for MV binding and infection and that the splicing variants of the STP domain not only affect MV binding and fusion, but also differentially protect against complement-mediated cytolysis. In this report, we dissect the role of these carbohydrates on complement regulatory function. We constructed, expressed, and characterized proteins deleting these carbohydrates. For MCP-mediated protection against cytolysis, the N-glycans of CCP-2 and -4 were necessary, the STP segment influenced but was not essential, and the N-glycan of CCP-1 was not required. In addition, the rate and magnitude of cell surface cleavage of C4b to C4c and C4d by MCP and factor I correlated with cytoprotection. These studies expand the structure-function understanding of the active sites of MCP and elucidate an important role for carbohydrates in its function, a finding consistent with their conservation in the MCP of other species.
Prostaglandin E2 Stimulates IL-8 Gene Expression in Human Colonic Epithelial Cells by a Posttranscriptional Mechanism
Authors: Yu Yi , Chadee Kris
Published: December 2022

Intestinal mucosal epithelial cells produce IL-8, a neutrophil chemoattractant that contributes to mucosal inflammation in various infectious and inflammatory diseases. However, the mediators involved and the molecular regulation of IL-8 production are poorly understood. As PGE2 is central in gut inflammation and modulates a variety of mucosal epithelial cell functions, we determined whether PGE2 can affect the expression of IL-8. Exogenous PGE2 induced the accumulation of IL-8 mRNA and protein production in a dose- and time-dependent manner in T84 human colonic epithelial cells. Forskolin and dibutyryl cAMP, which increase intracellular cAMP, stimulated IL-8 in a fashion similar to that of PGE2. PGE2 and PGE2 receptor agonists coupling through EP4 receptors elevated intracellular cAMP and up-regulated IL-8 mRNA expression by activating protein kinase A. Unlike PMA, PGE2 and forskolin did not increase IL-8 gene transcription. However, PGE2, forskolin, and PMA enhanced the stability of IL-8 mRNA transcripts, suggesting the involvement of posttranscriptional regulation. Chloramphenicol acetyltransferase reporter gene transfection studies confirmed the presence of a PGE2 responsive cis-element(s) in the IL-8 3′ untranslated region. Furthermore, dexamethasone inhibited PGE2-, forskolin-, and dibutyryl cAMP-induced, but not PMA-induced, IL-8 protein production. These results highlight a novel role for PGE2 in up-regulating IL-8 gene expression by colonic epithelial cells, which may contribute to exacerbation of inflammation in the gastrointestinal tract.
Cutting Edge: Expression of Functional CD94/NKG2A Inhibitory Receptors on Fetal NK1.1+Ly-49− Cells: A Possible Mechanism of Tolerance During NK Cell Development
Authors: Sivakumar P. V. , Gunturi A. , Salcedo M. , Schatzle J. D. , Lai W. C. , Kurepa Z. , Pitcher L. , Seaman M. S. , Lemonnier F. A. , Bennett M. , Forman J. , Kumar V.
Published: December 2022

Fetal liver- and thymus-derived NK1.1+ cells do not express known Ly-49 receptors. Despite the absence of Ly-49 inhibitory receptors, fetal and neonatal NK1.1+Ly-49− cells can distinguish between class Ihigh and class Ilow target cells, suggesting the existence of other class I-specific inhibitory receptors. We demonstrate that fetal NK1.1+Ly-49− cell lysates contain CD94 protein and that a significant proportion of fetal NK cells are bound by Qa1b tetramers. Fetal and adult NK cells efficiently lyse lymphoblasts from Kb−/−Db−/− mice. Qa1b-specific peptides Qdm and HLA-CW4 leader peptide specifically inhibited the lysis of these blasts by adult and fetal NK cells. Qdm peptide also inhibited the lysis of Qa1b-transfected human 721.221 cells by fetal NK cells. Taken together, these results suggest that the CD94/NKG2A receptor complex is the major known inhibitory receptor for class I (Qa1b) molecules on developing fetal NK cells.
Cutting Edge: Human 2B4, an Activating NK Cell Receptor, Recruits the Protein Tyrosine Phosphatase SHP-2 and the Adaptor Signaling Protein SAP
Authors: Tangye Stuart G. , Lazetic Sasha , Woollatt Erica , Sutherland Grant R. , Lanier Lewis L. , Phillips Joseph H.
Published: December 2022

The genetic defect in X-linked lymphoproliferative syndrome (XLP) is the Src homology 2 domain-containing protein SAP. SAP constitutively associates with the cell surface molecule, signaling lymphocytic activation molecule (SLAM), and competes with SH2-domain containing protein tyrosine phosphatase-2 (SHP-2) for recruitment to SLAM. SLAM exhibits homology with the mouse cell surface receptor 2B4. The human homologue of 2B4 has now been identified. It is recognized by the c1.7 mAb, a mAb capable of activating human NK cells. Human 2B4 became tyrosine phosphorylated following pervanadate-treatment of transfected cells and recruited SHP-2. SAP was also recruited to 2B4 in activated cells. Importantly, the 2B4-SAP interaction prevented the association between 2B4 and SHP-2. These results suggest that the phenotype of XLP may result from perturbed signaling not only through SLAM, but also other cell surface molecules that utilize SAP as a signaling adaptor protein.
The MHC Class I Molecule H-2Dp Inhibits Murine NK Cells via the Inhibitory Receptor Ly49A
Authors: Olsson-Alheim Mats Y. , Sundbäck Jonas , Kärre Klas , Sentman Charles L.
Published: December 2022

MHC class I molecules strongly influence the phenotype and function of mouse NK cells. NK cell-mediated lysis is prevented through the interaction of Ly49 receptors on the effector cell with appropriate MHC class I ligands on the target cell. In addition, host MHC class I molecules have been shown to modulate the in vivo expression of Ly49 receptors. We have previously reported that H-2Dd and H-2Dp MHC class I molecules are able to protect (at the target cell level) from NK cell-mediated lysis and alter the NK cell specificity (at the host level) in a similar manner, although the mechanism behind this was not clear. In this study, we demonstrate that the expression of both H-2Dd and H-2Dp class I molecules in target cells leads to inhibition of B6 (H-2b)-derived Ly49A+ NK cells. This inhibition could in both cases be reversed by anti-Ly49A Abs. Cellular conjugate assays showed that Ly49A-expressing cells indeed bind to cells expressing H-2Dp. The expression of Ly49A and Ly49G2 receptors on NK cells was down-regulated in H-2Dp-transgenic (B6DP) mice compared with nontransgenic B6 mice. However, B6DP mice expressed significantly higher levels of Ly49A compared with H-2Dd-transgenic (D8) mice. We propose that both H-2Dd and H-2Dp MHC class I molecules can act as ligands for Ly49A.
Diverse Fine Specificity and Receptor Repertoire of T Cells Reactive to the Major VP1 Epitope (VP1230–250) of Theiler’s Virus: Vβ Restriction Correlates with T Cell Recognition of the C-Terminal Residue
Authors: Kim Byung S. , Bahk Young Y. , Kang Hee-Kap , Yauch Robert L. , Kang Jeong-Ah , Park Mi-Jung , Ponzio Nicholas M.
Published: December 2022

Theiler’s murine encephalomyelitis virus induces chronic demyelinating disease in genetically susceptible mice. The histopathological and immunological manifestation of the disease closely resembles human multiple sclerosis, and, thus, this system serves as a relevant infectious model for multiple sclerosis. The pathogenesis of demyelination appears to be mediated by the inflammatory Th1 response to viral epitopes. In this study, T cell repertoire reactive to the major pathogenic VP1 epitope region (VP1233–250) was analyzed. Diverse minimal T cell epitopes were found within this region, and yet close to 50% of the VP1-reactive T cell hybridomas used Vβ16. The majority (8/11) of the Vβ16+ T cells required the C-terminal amino acid residue on the epitope, valine at position 245, and every T cell hybridoma recognizing this C-terminal residue expressed Vβ16. However, the complementarity-determining region 3 sequences of the Vβ16+ T cell hybridomas were markedly heterogeneous. In contrast, such a restriction was not found in the Vα usage. Only restricted residues at this C-terminal position allowed for T cell activation, suggesting that Vβ16 may recognize this terminal residue. Further functional competition analysis for TCR and MHC class II-contacting residues indicate that many different residues can be involved in the class II and/or TCR binding depending on the T cell population, even if they recognize the identical minimal epitope region. Thus, recognition of the C-terminal residue of a minimal T cell epitope may associate with a particular Vβ (but not Vα) subfamily-specific sequence, resulting in a highly restricted Vβ repertoire of the epitope-specific T cells.
IL-7 Up-Regulates IL-4 Production by Splenic NK1.1+ and NK1.1− MHC Class I-Like/CD1-Dependent CD4+ T Cells
Authors: Hameg Agathe , Gouarin Christine , Gombert Jean-Marc , Hong Seokmann , Van Kaer Luc , Bach Jean-François , Herbelin André
Published: December 2022

NK T cells are an unusual subset of T lymphocytes. They express NK1.1 Ag, are CD1 restricted, and highly skewed toward Vβ8 for their TCR usage. They express the unique potential to produce large amounts of IL-4 and IFN-γ immediately upon TCR cross-linking. We previously showed in the thymus that the NK T subset requires IL-7 for its functional maturation. In this study, we analyzed whether IL-7 was capable of regulating the production of IL-4 and IFN-γ by the discrete NK T subset of CD4+ cells in the periphery. Two hours after injection of IL-7 into mice, or after a 4-h exposure to IL-7 in vitro, IL-4 production by CD4+ cells in response to anti-TCR-αβ is markedly increased. In contrast, IFN-γ production remains essentially unchanged. In β2-microglobulin- and CD1-deficient mice, which lack NK T cells, IL-7 treatment does not reestablish normal levels of IL-4 by CD4+ T cells. Moreover, we observe that in wild-type mice, the memory phenotype (CD62L−CD44+) CD4+ T cells responsible for IL-4 production are not only NK1.1+ cells, but also NK1.1− cells. This NK1.1−IL-4-producing subset shares three important characteristics with NK T cells: 1) Vβ8 skewing; 2) CD1 restriction as demonstrated by their absence in CD1-deficient mice and relative overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of IL-4 production. In conclusion, the present study provides evidence that CD4+MHC class I-like-dependent T cell populations include not only NK1.1+ cells, but also NK1.1− cells, and that these two subsets are biased toward IL-4 production by IL-7.
Regulation of Integrin-Mediated T Cell Adhesion by the Transmembrane Protein Tyrosine Phosphatase CD45
Authors: Shenoi Hemanth , Seavitt John , Zheleznyak Alexander , Thomas Matthew L. , Brown Eric J.
Published: December 2022

The transmembrane protein tyrosine phosphatase CD45 is required for Ag receptor signal transduction in lymphocytes. Recently, a role for CD45 in the regulation of macrophage adhesion has been demonstrated as well. To investigate further the role of CD45 in the regulation of adhesion, we examined integrin-mediated adhesion to fibronectin of two T cell lines and their CD45-deficient variants. The absence of CD45 correlated with enhanced adhesion to fibronectin via integrin α5β1 (VLA-5), but not α4β1 (VLA-4) in both cell lines. Adhesion returned to normal levels upon transfection of wild-type CD45 into the CD45-deficient lines. Transfection of chimeric or mutant molecules expressing some, but not all, CD45 domains and activities demonstrated that both the transmembrane domain and the tyrosine phosphatase activity of CD45 were required for regulation of integrin-dependent adhesion, but the highly glycosylated extracellular domain was dispensable. In contrast, only a catalytically active CD45 cytoplasmic domain was required for TCR signaling. Transfectants that restored normal levels of adhesion to fibronectin coimmunoprecipitated with the transmembrane protein known as CD45-associated protein. These studies demonstrate a novel role for CD45 in adhesion regulation and suggest a possible function for its association with CD45-associated protein.
IgMhighCD21high Lymphocytes Enriched in the Splenic Marginal Zone Generate Effector Cells More Rapidly Than the Bulk of Follicular B Cells
Authors: Oliver Alyce M. , Martin Flavius , Kearney John F.
Published: December 2022

Ag encounter will recruit Ag-specific cells from the pool of mature B lymphocytes in the spleen and activate them to perform effector functions: generation of Ab-forming cells (plasma cells) and presentation of Ag to T cells. We have compared the ability of mature follicular and marginal zone cells to develop into effector B cells. The generation of marginal zone B cells and their localization in the marginal sinus area are T cell and CD40 ligand independent, suggesting that they do not represent a postgerminal center population. Compared with mature recirculating follicular B cells, they express several characteristics of previous antigenic experience, including higher levels of B7.1 (CD80) and B7.2 (CD86) when freshly isolated and following in vitro stimulation. After a brief 6- to 8-h in vitro stimulation with LPS or anti-CD40 Abs, marginal zone B cells become potent APCs. In addition, their ability to proliferate and differentiate into plasma cells in response to low doses of T-independent polyclonal stimuli (LPS) is far greater than that of follicular B cells. These findings indicate a functional heterogeneity within splenic mature B lymphocytes, with marginal zone B cells having the capacity to generate effector cells in early stages of the immune response against particulate Ags scavenged efficiently in this special anatomical site.
Fyn Membrane Localization Is Necessary to Induce the Constitutive Tyrosine Phosphorylation of Sam68 in the Nucleus of T Lymphocytes
Authors: Lang Valérie , Semichon Monique , Michel Frédérique , Brossard Cédric , Gary-Gouy Hélène , Bismuth Georges
Published: December 2022

A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src protein tyrosine kinases (PTK) has already been established, also in T lymphocytes. A constitutive phosphorylation of the molecule has also been documented in various transformed T cells, which probably reflects an increased expression of PTK of the Src family. Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the respective contribution of the two Src kinases Fyn and Lck, expressed in T cells, in this phenomenon. By overexpressing the two proteins, we show that the constitutive phosphorylation of Sam68 in vivo directly correlates with cellular Fyn levels, but not with Lck expression, despite the capacity of the PTK to strongly phosphorylate the molecule in vitro. Overexpressed Fyn is mainly localized at the cell membrane. We find that Sam68 phosphorylation, including in the nuclear fraction in which the molecule is predominantly expressed, is lost with a delocalized Fyn mutant deleted of its N-terminal membrane-anchoring domain. Finally, we demonstrate, using a construct encoding a Sam68 molecule without its nuclear localization signal, that nuclear expression of Sam68 is not required for phosphorylation. We conclude that the constitutive phosphorylation of Sam68 in T cells is a Fyn-dependent process occurring in a cell-membrane compartment from which phospho-Sam68 molecules can thereafter accumulate into the nucleus.
A Central Role for αβ T Cells in the Pathogenesis of Murine Lupus
Authors: Seery John P. , Wang Eddie C. Y. , Cattell Victoria , Carroll Joseph M. , Owen Michael J. , Watt Fiona M.
Published: December 2022

We have previously shown that female transgenic mice expressing IFN-γ in the epidermis, under the control of the involucrin promoter, develop inflammatory skin disease and a form of murine lupus. To investigate the pathogenesis of this syndrome, we generated female IFN-γ transgenic mice congenitally deficient in either αβ or γδ T cells. TCRδ−/− transgenics continued to produce antinuclear autoantibodies and to develop severe kidney lesions. In contrast, TCRβ−/− IFN-γ transgenic mice failed to produce antinucleosome, anti-dsDNA, or antihistone autoantibodies, and kidney disease was abolished. Both αβ- and γδ-deficient transgenics continued to develop IFN-γ-associated skin disease, lymphadenopathy, and splenomegaly. The data show that the autoantibody-mediated pathology of murine lupus in IFN-γ transgenic mice is completely αβ T cell dependent and that γδ T cells cannot drive autoantibody production. These results imply that production of antinuclear autoantibodies in IFN-γ transgenic animals is Ag driven, and we identified clusters of apoptotic cells in the epidermis of the mice as a possible source of self Ags. Our findings emphasize the relevance of this murine lupus model to the human disease.
HLA-B15 Peptide Ligands Are Preferentially Anchored at Their C Termini
Authors: Prilliman Kiley R. , Jackson Kenneth W. , Lindsey Mark , Wang Jihua , Crawford David , Hildebrand William H.
Published: December 2022

Therapies to elicit protective CTL require the selection of pathogen- and tumor-derived peptide ligands for presentation by MHC class I molecules. Edman sequencing of class I peptide pools generates “motifs” that indicate that nonameric ligands bearing conserved position 2 (P2) and P9 anchors provide the optimal search parameters for selecting immunogenic epitopes. To determine how well a motif represents its individual constituents, we used a hollow-fiber peptide production scheme followed by the mapping of endogenously processed class I peptide ligands through reverse-phase HPLC and mass spectrometry. Systematically mapping and characterizing ligands from B*1508, B*1501, B*1503, and B*1510 demonstrate that the peptides bound by these B15 allotypes i) vary in length from 7 to 12 residues, and ii) are more conserved at their C termini than their N-proximal P2 anchors. Comparative peptide mapping of these B15 allotypes further pinpoints endogenously processed ligands that bind to the allotypes B*1508, B*1501, and B*1503, but not B*1510. Overlapping peptide ligands are successful in binding to B*1501, B*1503, and B*1508 because these B15 allotypes share identical C-terminal anchoring pockets whereas B*1510 is divergent in the C-terminal pocket. Therefore, endogenous peptide loading into the B15 allotypes requires that a conserved C terminus be anchored in the appropriate specificity pocket while N-proximal anchors are more flexible in their location and sequence. Queries for overlapping and allele-specific peptide ligands may thus be contingent on a conserved C-terminal anchor.
Generation of Recombinant Fragments of CD11b Expressing the Functional β-Glucan-Binding Lectin Site of CR3 (CD11b/CD18)
Authors: Xia Yu , Ross Gordon D.
Published: December 2022

CR3 (Mac-1; αMβ2 integrin) functions as both a receptor for the opsonic iC3b fragment of C3 triggering phagocytosis or cytotoxicity and an adhesion molecule mediating leukocyte diapedesis. Recent reports have suggested that a CR3 lectin site may be required for both cytotoxic responses and adhesion. Cytotoxic responses require dual recognition of iC3b via the I domain of CD11b and specific microbial surface polysaccharides (e.g., β-glucan) via a separate lectin site. Likewise, adhesion requires a lectin-dependent membrane complex between CR3 and CD87. To characterize the lectin site further, a recombinant baculovirus (rBv) system was developed that allowed high level expression of rCD11b on membranes and in the cytoplasm of Sf21 insect cells. Six rBv were generated that contained truncated cDNA encoding various CD11b domains. Immunoblotting of rBv-infected Sf21 cells showed that some native epitopes were expressed by five of six rCD11b fragments. Lectin activity of rCD11b proteins was evaluated by both flow cytometry with β-glucan-FITC and radioactive binding assays with [125I]β-glucan. Sf21 cells expressing rCD11b that included the C-terminal region, with or without the I-domain, exhibited lectin activity that was inhibited by unlabeled β-glucan or anti-CR3 mAbs. The smallest rCD11b fragment exhibiting lectin activity included the C-terminus and part of the divalent cation binding region. The β-glucan binding affinities of the three C-terminal region-containing rCD11bs expressed on Sf21 cell membranes were not significantly different from each other and were similar to that of neutrophil CR3. These data suggest that the lectin site may be located entirely within CD11b, although lectin site-dependent signaling through CD18 probably occurs with the heterodimer.
Differences Between IL-4- and IL-4 Receptor α-Deficient Mice in Chronic Leishmaniasis Reveal a Protective Role for IL-13 Receptor Signaling
Authors: Mohrs Markus , Ledermann Birgit , Köhler Gabriele , Dorfmüller Andreas , Gessner Andre , Brombacher Frank
Published: December 2022

IL-4 receptor α-chain-deficient (IL-4Rα−/−) mice were generated by homologous and site-specific recombination, using the Cre/loxP system in BALB/c-derived embryonic stem cells. In vitro analysis of cells from these mice revealed impaired IL-4- and IL-13-mediated functions, demonstrating that the IL-4Rα-chain is an essential component of both the IL-4 and the IL-13 receptor. Whereas Leishmania major-infected BALB/c mice developed fatal progressive disease with type 2 Ab responses within 3 mo, both IL-4Rα−/− and IL-4−/− BALB/c mice contained infection with reduced footpad swelling, parasite load, moderate histopathology, and type 1 Ab responses during this time period. Conclusively, these results demonstrate an IL-4-dependent mechanism of susceptibility in BALB/c mice. Nevertheless, in contrast to mutant mice, infected C57BL/6 mice healed completely within 3 mo, indicating that additional factors are necessary for subsequent healing and elimination of the pathogen. During the further course of infection, IL-4Rα−/− mice developed progressive disease with massive footpad swelling. Lesions became ulcerative and necrotic with subsequent destruction of connective tissue and bones, as well as dissemination into organs and consequent mortality within the monitored 6 mo of chronic infection. In striking contrast, IL-4−/− mice maintained control of infection on a moderate level, but were unable to clear the pathogen. The distinct phenotypes of the BALB/c embryonic stem cell-derived IL-4−/− and IL-4Rα−/− mouse strains identify previously unsuspected mechanisms for maintaining host immunity to chronic infection with L. major, mediated by a functional IL-13 receptor.
Complex Requirements for Nascent and Memory Immunity in Pulmonary Histoplasmosis
Authors: Allendörfer Ruth , Brunner George D. , Deepe George S.
Published: December 2022

The presence of functional T cells is often required for successful resolution of infections with intracellular pathogens, yet the mechanisms by which they contribute to elimination of the invading pathogen in primary and secondary immunity are only partly understood. We report that increased mortality of naive α/β TCR+ or CD4+ T cell-depleted mice infected with the fungus Histoplasma capsulatum is associated with impairment of IFN-γ production. Upon secondary infection, mice concomitantly depleted of CD4+ and CD8+ cells exhibit decreased survival beyond day 25 of rechallenge, whereas elimination of either T cell subset or B cell deficiency does not result in accelerated mortality compared with controls. Remarkably, despite a decrease of H. capsulatum CFU in lungs of CD4+ plus CD8+-deficient mice, a progressive increase in spleen CFU is observed. The ability to control fungus growth in lungs is associated with vigorous TNF-α, but not IFN-γ, production by bronchoalveolar lavage cells. In contrast, spleen cells from CD4+ plus CD8+-deficient mice are unable to produce TNF-α. Thus, the cellular and molecular requirements for protective immunity vary between primary and secondary infection. Furthermore, in secondary histoplasmosis, a sharp contrast can be drawn between lungs and spleens in their reliance upon T cells to control fungal replication. The opposing activities of these organs can be ascribed in part to differential production of TNF-α.