bluuebunny/crossref_metadata_2025 huggingface dataset:
The Murine IL-13 Receptor α2: Molecular Cloning, Characterization, and Comparison with Murine IL-13 Receptor α1
Authors: Donaldson Debra D. , Whitters Matthew J. , Fitz Lori J. , Neben Tamlyn Yee , Finnerty Heather , Henderson Sheryl L. , O’Hara Richard M. , Beier David R. , Turner Katherine J. , Wood Clive R. , Collins Mary
Published: December 2022

Two components of a receptor complex for IL-13, the IL-4R and a low affinity IL-13-binding chain, IL-13Rα1, have been cloned in mice and humans. An additional high affinity binding chain for IL-13, IL-13Rα2, has been described in humans. We isolated a cDNA from the thymus that encodes the murine orthologue of the human IL-13Rα2. The predicted protein sequence of murine IL-13Rα2 (mIL-13Rα2) has 59% overall identity to human IL-13Rα2 and is closely related to the murine low affinity IL-13-binding subunit, IL-13Rα1. The genes for both mIL-13-binding chains map to the X chromosome. A specific interaction between mIL-13Rα2.Fc protein and IL-13 was demonstrated by surface plasmon resonance using a BIACORE instrument. Ba/F3 cells that were transfected with mIL-13Rα2 expressed 5000 molecules per cell and bound IL-13 with a single Kd of 0.5 to 1.2 nM. However, these cells did not proliferate in response to IL-13, and the IL-4 dose response was unaffected by high concentrations of IL-13. In contrast, the expression of mIL-13Rα1 by Ba/F3 cells resulted in a sensitive proliferative response to IL-13. Consistent with its lower affinity for IL-13, IL-13Rα1.Fc was 100-fold less effective than IL-13Rα2.Fc in neutralizing IL-13 in vitro. These results show that mIL-13Rα2 and mIL-13Rα1 are not functionally equivalent and predict distinct roles for each polypeptide in IL-13R complex formation and in the modulation of IL-13 signal transduction.
Protection from Lymphoma Cell Metastasis in ICAM-1 Mutant Mice: A Posthoming Event
Authors: Aoudjit Fawzi , Potworowski Edouard F. , Springer Timothy A. , St-Pierre Yves
Published: December 2022

It has been hypothesized that the intercellular adhesion receptors used by normal cells could also be operative in the spreading of circulating malignant cells to target organs. In the present work, we show that genetic ablation of the ICAM-1 gene confers resistance to T cell lymphoma metastasis. Following i.v. inoculation of LFA-1-expressing malignant T lymphoma cells, we found that ICAM-1-deficient mice were almost completely resistant to the development of lymphoid malignancy compared with wild-type control mice that developed lymphoid tumors in the kidneys, spleen, and liver. Histologic examinations confirmed that ICAM-1-deficient mice, in contrast to wild-type mice, had no evidence of lymphoid infiltration in these organs. The effect of ICAM-1 on T cell lymphoma metastasis was observed in two distinct strains of ICAM-1-deficient animals. Nonetheless, lymphoma cells migrated with the same efficiency to target organs in both normal and ICAM-1-deficient mice, indicating not only that ICAM-1 expression by the host is essential in lymphoma metastasis, but also that this is so at stages subsequent to homing and extravasation into target organs. These results point to posthoming events as a focus of future investigation on the control of metastasis mediated by ICAM-1.
Lung-Specific Transgenic Expression of KC Enhances Resistance toKlebsiella pneumoniaein Mice
Authors: Tsai Wan C. , Strieter Robert M. , Wilkowski Jodi M. , Bucknell Kathy A. , Burdick Marie D. , Lira Sergio A. , Standiford Theodore J.
Published: December 2022

A vigorous host response is required to effectively clear pathogenic bacteria from the lungs and is dependent upon the recruitment and activation of neutrophils and macrophages. A family of chemotactic cytokines, referred to as chemokines, have been shown to participate in this complex protective response. In this study, we assessed the role of the C-X-C chemokine KC in lung antibacterial host defense using wild-type (wt) B6D2 mice or transgenic mice that had been bred on a B6D2 background expressing KC under the control of a Clara cell-specific promoter within the lung. The administration of Klebsiella pneumoniae to both wt and KC-transgenic mice resulted in a time-dependent expression of KC protein within the lung that peaked at 24 to 48 h postinoculation. When infected with K. pneumoniae, the KC-transgenic mice showed a striking improvement in survival compared with wt control mice. This improved survival was due to an increase in bacterial clearance, which occurred in association with a vigorous recruitment of neutrophils in the KC-transgenic mice compared with their wt control counterparts. No differences in the lung levels of the specific cytokines TNF-α, IFN-γ, IL-12, and IL-10 were noted. However, inducible macrophage inflammatory protein-2 levels were significantly decreased in the KC-transgenic mice compared with the wt mice. This study indicates that the compartmentalized overexpression of KC in vivo results in increased lung bacterial clearance and improved survival, which occurs in association with enhanced polymorphonuclear leukocyte influx to the lung.
IFN-γ Inhibits Activation-Induced Expression of E- and P-Selectin on Endothelial Cells
Authors: Melrose Jennifer , Tsurushita Naoya , Liu Gao , Berg Ellen L.
Published: December 2022

E- and P-selectin are cell surface lectins that mediate leukocyte-endothelial cell adhesion and thereby participate in neutrophil recruitment into inflammatory sites. E-selectin can be induced on endothelial cells by various activators, including TNF-α, IL-1β, and PMA. Induction of E-selectin is blocked by pretreatment of endothelial cells with IL-4 or TGF-β, both of which have antiinflammatory properties in vivo. In addition to its well-known proinflammatory activities, IFN-γ also has antiinflammatory effects in vivo, one of which is inhibition of neutrophil recruitment. To determine whether IFN-γ inhibits neutrophil recruitment by inhibiting adhesion molecule expression, the effect of IFN-γ on activation-induced cell adhesion molecule expression by cultured HUVEC was evaluated. Pretreatment of endothelial cells with IFN-γ for 24 to 72 h before 6- to 24-h activation with IL-1β, TNF-α, or PMA resulted in significantly reduced levels of cell surface E-selectin, although levels of ICAM-1 and VCAM-1 were the same or increased. The reduction of cell surface E-selectin levels under these conditions was reflected in reduced levels of E-selectin mRNA, indicating an effect at the transcription level or RNA stability. Interestingly, the increase of cell surface P-selectin expression due to IL-4 treatment of HUVEC was also inhibited by IFN-γ, while constitutive levels of P-selectin were not. These results suggest that the inhibition of neutrophil recruitment by IFN-γ in vivo may be due, in part, to the ability of IFN-γ to inhibit E- and P-selectin up-regulation. Furthermore, these findings emphasize the process of leukocyte recruitment as an important step through which IFN-γ can direct the character of inflammatory reactions.
Subcellular Site of Expression and Route of Vaccination Influence Pulmonary Eosinophilia Following Respiratory Syncytial Virus Challenge in BALB/c Mice Sensitized to the Attachment G Protein
Authors: Bembridge Gary P. , Garcia-Beato Regina , López Juan A. , Melero Jose A. , Taylor Geraldine
Published: December 2022

The attachment glycoprotein (G) of respiratory syncytial virus (RSV) is synthesized as two mature forms: a membrane-anchored form and a smaller secreted form. Mutant cDNAs were constructed that encoded one or the other form of the protein and were expressed in recombinant vaccinia viruses (rVV). Mice were immunized with rVV by dermal scarification or i.p. injection to determine the contribution of the membrane-anchored and secreted forms of the G protein on the augmentation of pulmonary pathology seen following RSV challenge. Mice scarified with rVV expressing the membrane-anchored G protein had a markedly reduced pulmonary eosinophilic response following RSV challenge compared with mice scarified with rVV expressing either wild-type or secreted G protein. The induction of pulmonary eosinophilia in rVV-primed mice was also dependent upon the route of vaccination. An eosinophilic response was not observed in any groups of mice immunized i.p. with rVV expressing any of the different forms of the G protein The difference in pulmonary pathology observed between dermal scarification or i.p. vaccinated mice was not reflected in a difference in cytokine production by splenocytes from vaccinated and challenged mice restimulated with RSV in vitro. Both groups produced significant levels of IL-4 and IL-5. These data suggest that the local APCs and lymphoid environment, together with the form of the G protein, influence pulmonary pathology following RSV challenge.
Important Contributions of P-Selectin Glycoprotein Ligand-1-Mediated Secondary Capture to Human Monocyte Adhesion to P-Selectin, E-Selectin, and TNF-α-Activated Endothelium Under Flow In Vitro
Authors: Lim Yaw-Chyn , Snapp Karen , Kansas Geoffrey S. , Camphausen Raymond , Ding Han , Luscinskas Francis W.
Published: December 2022

In this study, an in vitro flow model and a blocking mAb to P-selectin glycoprotein ligand-1 (PSGL-1) were used to define the role of PSGL-1 in monocyte attachment and rolling on E- and P-selectin and in attachment and accumulation on 6-h TNF-α-activated HUVEC. KPL1, an adhesion-blocking mAb directed against the tyrosine sulfate motif of PSGL-1, abolished monocyte-adhesive interactions with P-selectin, but only partially blocked monocyte interaction with E-selectin. Further analysis showed that on E-selectin, KPL1 blocked only secondary (i.e., monocyte/monocyte) interactions, but did not block primary (i.e., monocyte/E-selectin) interactions, with secondary adhesion accounting for 90% of the total adhesive interactions on either E- or P-selectin. On cytokine-activated HUVEC, monocytes initially attached and formed linear strings of adherent cells, which involved both primary and secondary adhesion. PSGL-1 or L-selectin mAb reduced string formation, and the combination of PSGL-1 and L-selectin mAb prevented monocyte strings and inhibited 86% of accumulation. Monocyte attachment and rolling on purified adherent monocytes were also critically dependent on PSGL-1 on the adherent monocytes. These studies document that secondary interactions between monocytes, mediated by PSGL-1, are crucial for monocyte initial attachment, rolling, and accumulation on activated endothelium under laminar shear flow.
The Influence of Mast Cells on Pathways of Transepithelial Antigen Transport in Rat Intestine
Authors: Berin M. Cecilia , Kiliaan Amanda J. , Yang Ping-Chang , Groot Jack A. , Kitamura Yukihiko , Perdue Mary H.
Published: December 2022

Luminal Ag challenge of intestinal segments from sensitized rats results in a rapid (∼3 min) secretory response. We previously showed in horseradish peroxidase (HRP)-sensitized rats that the initial phase of transepithelial Ag transport occurred via a transcellular route and was enhanced by sensitization. However, following the hypersensitivity reaction, Ag also crossed between epithelial cells. The aim of this study was to determine the role of mast cells in the altered transepithelial Ag transport. White spotting mast cell-deficient rats and +/+ littermate controls were sensitized to HRP. After 10 to 14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. Electron microscopy of jejunum fixed at 2 min showed a similarly enhanced endocytic transport of HRP in sensitized +/+ and Ws/Ws rats compared with naive controls. In sensitized +/+ rats, a secretory response occurred ∼3 min after challenge, and tissue conductance increased thereafter. Naive +/+ and sensitized Ws/Ws rats did not demonstrate a secretory response to HRP challenge, and conductance remained at baseline levels. The flux of HRP was elevated across tissue from sensitized +/+ rats but not across tissue from naive controls or sensitized Ws/Ws rats. The results indicate that sensitization enhances the initial phase of transepithelial uptake of Ag by transcytosis in a mast cell-independent manner. However, subsequent recruitment of the paracellular pathway for Ag transport in sensitized rats is dependent upon the presence of mast cells and occurs after the activation of such cells.
Expression and Functional Activity of the IL-8 Receptor Type CXCR1 and CXCR2 on Human Mast Cells
Authors: Lippert Undine , Artuc Metin , Grützkau Andreas , Möller Annelie , Kenderessy-Szabo Anna , Schadendorf Dirk , Norgauer Johannes , Hartmann Karin , Schweitzer-Stenner Reinhard , Zuberbier Thorsten , Henz Beate M. , Krüger-Krasagakes Sabine
Published: December 2022

To further elucidate mechanisms involved in mast cell accumulation at sites of cutaneous inflammation, we have studied the ability of human leukemic mast cells (HMC-1 cells) to express functionally active IL-8 receptors. Expression of mRNA for both types of IL-8 receptors (CXCR1 and CXCR2) was demonstrated by PCR and of both proteins by flow cytometry. Binding and competition studies with 125I-labeled IL-8 and its homologue melanoma growth stimulating activity (125I-labeled MGSA) revealed two specific binding sites for IL-8, K1 = 1.1 × 1011 M−1 and K2 = 5 × 107 M−1; and for MGSA, K1 = 2.8 × 1010 M−1 and K2 = 5 × 107 M−1. This finding was supported by a dose-dependent rise of cytosolic free calcium concentration ([Ca2+]i) induced by both chemokines and to a lesser extent by the homologue neutrophil-activating peptide-2 (NAP-2). A significant migratory response of human leukemic mast cells (HMC-1) was observed with all three chemokines at a range from 10−8 M to 10−9 M. Moreover, the formation of cellular F-actin was induced in a rapid, dose-dependent fashion, with a maximally 1.7-fold increase at 10−7 M. Using postembedding immunoelectron microscopy, we could show the expression of CXCR1 on the cytoplasmatic membrane of isolated human skin mast cells whereas CXCR2 was located in mast cell-specific granules. These findings demonstrate for the first time the functional expression of both types of IL-8 receptors on human mast cells, suggesting a role for their ligands during mast cell activation and recruitment.
Cutting Edge: Enhanced Anti-HIV-1 Activity and Altered Chemotactic Potency of NH2-Terminally Processed Macrophage-Derived Chemokine (MDC) Imply an Additional MDC Receptor
Authors: Struyf Sofie , Proost Paul , Sozzani Silvano , Mantovani Alberto , Wuyts Anja , De Clercq Erik , Schols Dominique , Van Damme Jo
Published: December 2022

Posttranslational processing of chemokines increases (IL-8) or decreases (monocyte chemotactic protein-1) their chemotactic potency. Macrophage-derived chemokine (MDC) attracts monocytes, dendritic cells, activated lymphocytes, and NK cells and has reportedly anti-HIV-1 activity. Here we report that truncation of MDC by deletion of two NH2-terminal residues resulted in impaired binding to CC chemokine receptor (CCR)4, the only identified MDC receptor sofar. Truncated MDC(3-69) failed to desensitize calcium mobilization by MDC(1-69) or thymus- and activation-regulated chemokine (TARC), another CCR4 ligand. MDC(3-69) lacked HUT-78 T cell chemotactic activity but retained its capacity to attract monocytes and to desensitize chemotaxis. Compared with MDC(1-69), MDC(3-69) had weak but enhanced antiviral activity against M- and T-tropic HIV-1 strains. Furthermore, both MDC forms failed to signal through the orphan receptors Bonzo/STRL33 and BOB/GPR15 and to desensitize RANTES and stromal cell-derived factor (SDF)-1 responses in CCR5-transfected and CXC chemokine receptor (CXCR)4-transfected cells, respectively. These findings suggest that MDC recognizes another, yet unidentified, receptor. We conclude that minimal NH2-terminal truncation of MDC differentially affects its various immunologic functions.
IFN-α Is a Survival Factor for Human Myeloma Cells and Reduces Dexamethasone-Induced Apoptosis
Authors: Ferlin-Bezombes Martine , Jourdan Michel , Liautard Janny , Brochier Jean , Rossi Jean-François , Klein Bernard
Published: December 2022

IFN-α is used as a maintenance therapy in patients with multiple myeloma, but its benefit is a matter of controversy. In vitro studies show that IFN-α can both stimulate and inhibit myeloma cell proliferation. We have tested the effect of IFN-α on the survival of myeloma cell lines and primary plasma cells. IFN-α significantly reduced the apoptosis induced by removal of IL-6 in four IL-6-dependent myeloma cell lines. It also reduced the level of apoptosis induced by dexamethasone in these cell lines as well as in purified primary myeloma cells from seven patients. IFN-α promoted the survival of myeloma cells, which, following removal of IL-6, were blocked in G1 and died. However, unlike IL-6, IFN-α-treated cells remained mainly blocked in the G1 phase of the cycle. While the effects of IL-6 are mediated through stimulation of its gp130 receptor subunit, the IFN-α-induced survival of myeloma cells was independent of gp130 transducer activation (as demonstrated using a neutralizing anti-gp130 Ab). However, the signal transduction cascades activated by these two cytokines share at least some common elements, since stimulation with either IFN-α or IL-6 resulted in STAT3 phosphorylation. These results indicate that IFN-α promotes the survival, but not the proliferation, of myeloma cells, preventing the apoptosis induced by removal of IL-6 or addition of dexamethasone. This survival factor activity may explain the conflicting reports on the effects of IFN-α on myeloma cell proliferation.
Two Regions in the CD80 Cytoplasmic Tail Regulate CD80 Redistribution and T Cell Costimulation
Authors: Doty Raymond T. , Clark Edward A.
Published: December 2022

CD28 is a major T cell costimulatory molecule, delivering signals distinct from those of the CD3/TCR complex, which regulate cytokine and cytokine receptor expression, cell proliferation, and cell viability. CD28 needs to be cross-linked to initiate signals, yet both of its ligands, CD80 and CD86, are expressed as monomers. Previously, we determined the cytoplasmic tail of CD80 is required for CD28-mediated costimulation and subcellular relocalization of CD80 in lymphocytes. In this study, we report that Reh B cell transfectants expressing CD80 with mutations in the cytoplasmic tail region either at 275–278 (RRNE→AAAA, CD80/4A) or serine 284 (S→A, CD80/SA) can bind ligand similar to transfectants expressing wild-type CD80, yet are unable to costimulate T cell proliferation. These mutant CD80 molecules are expressed on the surface of the Reh cells in small clusters or foci indistinguishable from those of wild-type CD80 molecules. However, mutant CD80 molecules unlike wild-type CD80 cannot be readily induced by ligand into caps. Thus, small clusters of CD80 found on APC are insufficient to initiate CD28-mediated signals, and the formation of CD80 caps appears to be a critical factor regulating the initiation of T cell costimulation. A 30-kDa phosphoprotein that associates with the cytoplasmic tail of CD80 in activated cells may play a role in CD80 redistribution and thus CD28-mediated costimulation. These results indicate two distinct regions of the CD80 cytoplasmic tail regulate its costimulatory function, and both regions are required for CD80 function.
The BB1 Monoclonal Antibody Recognizes Both Cell Surface CD74 (MHC Class II-Associated Invariant Chain) as Well as B7-1 (CD80), Resolving the Question Regarding a Third CD28/CTLA-4 Counterreceptor
Authors: Freeman Gordon J. , Cardoso Angelo A. , Boussiotis Vassiliki A. , Anumanthan Anukanth , Groves Richard W. , Kupper Thomas S. , Clark Edward A. , Nadler Lee M.
Published: December 2022

The identification of all CD28/CTLA-4 counterreceptors is critical to our understanding of this pivotal pathway of T cell activation. Clouding our understanding has been the reported discrepancies in expression and function of the B7-1 (CD80) molecule based upon the use of the BB1 vs other anti-B7-1 mAbs. To resolve this issue, we have cloned a BB1-binding molecule from the BB1+B7-1− NALM-6 pre-B cell line. Here, we demonstrate that this BB1-binding molecule is identical to the cell surface form of CD74 (MHC class II-associated invariant chain). CD74-transfected cells bound the BB1 mAb but not other anti-CD80 mAbs, CD28-Ig, or CTLA4Ig. Absorption and blocking experiments confirmed the reactivity of BB1 mAb with CD74. A region of weak homology was identified between CD74 and the region of B7-1 encoding the BB1 epitope. Therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Many of the puzzling observations in the literature concerning the expression of human B7-1 are resolved by an understanding that BB1 staining is the summation of CD74 plus B7-1 expression. This observation requires the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.
Superclustering of B Cell Receptor and FcγRIIB1 Activates Src Homology 2-Containing Protein Tyrosine Phosphatase-1
Authors: Sato Katsuaki , Ochi Atsuo
Published: December 2022

FcγRIIB1 (CD32) is a receptor that binds the Fc domain of Ag-complexed IgG. Coaggregation of B cell receptor (BCR) and FcγRIIB1 generates a dominant negative signal that inhibits B cell activation. In Ag-specific Id-positive B cells, the co-cross-linking of BCR and FcγRIIB1 by anti-Id Ab resulted in the association of both Src homology 2-containing protein tyrosine phosphatase (SHP-1) and Src homology 2-containing inositol phosphatase (SHIP) with the FcγRIIB1; however, only SHIP activity was detected. “Superclustering” of the BCR and FcγRIIB1 complex induced by stimulation with anti-Id Ab plus polyvalent Ag synergistically activated SHP-1. The degree of co-cross-linking between BCR and FcγRIIB1 may determine the activation status of SHP-1 and SHIP.
The Role of B7-1 and B7-2 Costimulation for the Generation of CTL Responses In Vivo
Authors: Sigal Luis J. , Reiser Hans , Rock Kenneth L.
Published: December 2022

The role of B7-1 and B7-2 costimulatory molecules in the generation of Ag-specific CD8+ CTLs is not well understood. In this paper, we analyze the role of both B7-1 and B7-2 in the generation of CTLs to nonliving, exogenous Ag and to live virus. To analyze the role of B7 costimulation in the induction of CTLs, we blocked B7-1 and/or B7-2 in vivo by injecting C57BL/6 mice with anti-B7-1 and/or anti-B7-2 mAbs; the mice were subsequently immunized with either chicken OVA that had been cross-linked to beads as a model of exogenous Ags or with wild-type and recombinant vaccinia virus expressing different forms of chicken OVA as models of viral Ags. Our results indicate that B7 costimulation is necessary in the generation of CTLs for all of these Ags. Since the B7 molecules could be costimulating CD8+ and/or CD4+ T cells in wild-type animals, we also examined the role of costimulation in the generation of CTLs to exogenous and viral Ag in MHC class II-deficient mice lacking most CD4+ T cells. In these animals, a combination of both mAbs also blocked all CTL responses, indicating that the Th cell-independent activation of CTLs is dependent upon the B7-costimulatory signals supplied to the CD8+ cell. These findings contribute to the understanding of the role of costimulation for the generation of CTLs. We also discuss the implications of these findings on the role of professional APCs in the initiation of CTL responses.
Multiple Lupus Susceptibility Loci Map to Chromosome 1 in BXSB Mice
Authors: Hogarth Maxine B. , Slingsby Jason H. , Allen Penelope J. , Thompson E. Mary , Chandler Phillip , Davies Kevin A. , Simpson Elizabeth , Morley Bernard J. , Walport Mark J.
Published: December 2022

BXSB mice spontaneously develop a lupus-like syndrome that is accelerated by the Yaa gene (Y-linked autoimmune accelerator). We studied the phenotype of disease in (B10 × BXSB)F1 and (BXSB × (B10 × BXSB)F1) backcross mice and genotyped 224 backcross animals to allow a microsatellite-based genome-wide linkage analysis to be conducted. In the backcross population, three intervals on chromosome 1 showed significant linkage to disease, suggesting that multiple loci contribute to the production of autoimmune disease. D1Mit5 at 32.8 cM was linked to development of nephritis (χ2 = 15.68, p = 7.5 × 10−5), as was D1Mit12 at 63.1 cM (χ2 = 20.17, p = 7.1 × 10−6). D1Mit403 at 100 cM was linked to anti-dsDNA Ab production (χ2 = 17.28, p = 3.2 × 10−5). Suggestive linkages to antinuclear Abs and nephritis were identified on chromosome 3, to splenomegaly on chromosome 4, and to anti-ssDNA Ab production on chromosome 10. Chromosome 4 and the telomeric region of chromosome 1 have previously been linked to disease in other mouse models of systemic lupus erythematosus; however, the centromeric regions of chromosome 1 and chromosomes 3 and 10 are unique to BXSB. This implies that, though some loci may be common to a number of mouse models of lupus, different clusters of disease genes confer disease susceptibility in different strains of mice.
Studies Using Antigen-Presenting Cells Lacking Expression of Both B7-1 (CD80) and B7-2 (CD86) Show Distinct Requirements for B7 Molecules During Priming Versus Restimulation of Th2 But Not Th1 Cytokine Production
Authors: Schweitzer A. Nicola , Sharpe Arlene H.
Published: December 2022

The differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases. B7 costimulation has been shown to affect the production of both Th1 and Th2 cytokines, depending on the system studied. There is, consequently, great interest in manipulating the B7 costimulatory signal for therapeutic purposes. To optimally manipulate this key immunoregulatory pathway, the contribution of B7 costimulation to cytokine production requires further clarification. We have compared the B7 requirement for cytokine production by naive vs previously activated T cells using DO11.10 TCR transgenic CD4+ T cells and splenic APCs from mice lacking B7 expression. Our data indicate that induction of IL-4 production and Th2 differentiation by naive T cells is highly dependent on B7 molecules, whereas IL-4 production by previously activated T cells is B7 independent. The predominant contribution of B7-mediated signals to Th1 cytokine production by both naive and primed T cells is upon IL-2 production (and expansion) rather than IFN-γ (effector cytokine) production. Thus, our studies demonstrate that the antigenic experience of a T cell at the time of B7 blockade may determine whether blockade predominantly affects T cell expansion, differentiation, or effector cytokine production. These differential effects of B7 costimulation on IL-2 vs IFN-γ production and on IL-4 production by naive vs primed T cells have important implications for understanding how B7:CD28/CTLA4 blockade can be effectively used to manipulate cytokine production in vivo.
Chimeric Receptors Providing Both Primary and Costimulatory Signaling in T Cells from a Single Gene Product
Authors: Finney Helene M. , Lawson Alastair D. G. , Bebbington Christopher R. , Weir A. Neil C.
Published: December 2022

Single chain Fv chimeric receptors, or T-bodies, are described with intracellular sequences comprising the costimulatory signaling domain of CD28 in series with the ζ-chain from the TCR complex. Using an engineered human single chain Fv derived from P67, an mAb with specificity for human CD33, and a spacer comprising an Ab hinge region with either Fcγ or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to mediate both primary signaling and costimulation from one extracellular binding event. Constructs with the CD28 signaling domain proximal and the ζ-chain distal to the membrane were found to express more efficiently in Jurkat than constructs with the opposite orientation and were capable of mediating up to 20 times more IL-2 production on stimulation with solid phase Ag when compared with transfectants expressing chimeric receptors with ζ-chain intracellular signaling domains only. IL-2 production was specific to Ag challenge and was completely inhibited by incubation with free Ab of the same specificity as the extracellular binding site of the construct, but not by an isotype-matched control Ab. The CD28 intracellular domain of these fusion proteins was shown to be capable of binding the p85 subunit of phosphatidylinositol 3′-kinase. These constructs represent the first of a new generation of single gene multidomain chimeric receptors capable of mediating both primary and costimulatory signaling specifically from a single extracellular recognition event.
Caspase Dependence of Target Cell Damage Induced by Cytotoxic Lymphocytes
Authors: Sarin Apurva , Haddad Elias K. , Henkart Pierre A.
Published: December 2022

Since the CTL secreted granule protease granzyme B can activate multiple target caspases, it has been proposed that this pathway is responsible for CTL-induced cytolysis of Fas-negative targets. However, target lysis via the granule exocytosis pathway is completely resistant to caspase inhibitors. To test the possibility that granzymes trigger a postcaspase cytoplasmic apoptotic pathway leading to lysis, we have examined the caspase dependence of several cytoplasmic changes associated with apoptotic death. Rapid prelytic phosphatidylserine externalization was induced in Jurkat target cells by both the Fas ligand (FasL)/Fas and the granule exocytosis effector pathways. This was specifically blocked by peptide ketone caspase inhibitors when induced by the former, but not by the latter, pathway. A rapid prelytic loss of target mitochondrial ψ was also induced by both CTL effector pathways, and this was also specifically blocked by caspase inhibitors when induced by the FasL/Fas, but not by the granule exocytosis, pathway. Similarly, target membrane blebbing induced by CTL via the FasL/Fas, but not via the granule exocytosis, effector pathway was specifically blocked by caspase inhibitors. In contrast to the above nonnuclear damage, CTL-induced target staining by the lipid probe FM1–43 reflecting plasma membrane endocytosis was blocked by caspase inhibitors. Thus, when caspase activation is blocked, the granule exocytosis pathway triggers several parameters of target apoptotic damage in addition to lysis, suggesting that granzymes directly trigger a postcaspase cytoplasmic apoptotic death pathway.
Commitment of Individual Th1-Like Lymphocytes to Expression of IFN-γ Versus IL-4 and IL-10: Selective Induction of IL-10 by Sequential Stimulation of Naive Th Cells with IL-12 and IL-4
Authors: Assenmacher Mario , Löhning Max , Scheffold Alexander , Richter Anne , Miltenyi Stefan , Schmitz Jürgen , Radbruch Andreas
Published: December 2022

Commitment of Th lymphocytes to the Th1 phenotype, as characterized by the expression of the major proinflammatory cytokine IFN-γ, may be critically involved in the establishment of chronic inflammation and inflammatory autoimmune disease. To date, it has been shown that in IL-12-stimulated murine Th cell lines containing a major fraction of Th1 cells, Th2 cells can be induced by IL-4 until about 2 wk after initial activation, but not later. Here we analyze, based on the magnetic isolation of viable Th1 cells according to their specific expression of IFN-γ, the cytokine commitment of individual Th1 cells. After activation of naive Th cells with Ag and IL-12 for up to 5 wk, isolated IFN-γ-producing cells were restimulated with Ag and IL-4. Within the first 3 to 4 wk of IL-12 stimulation, some IFN-γ+ cells stopped expression of IFN-γ when restimulated with IL-4. However, within only 1 to 2 wk of IL-12 stimulation, few IFN-γ+ cells could be converted to produce IL-4. Others continued to express IFN-γ and thus were already committed to a proinflammatory, Th1-like phenotype. Surprisingly, within 3 wk of IL-12 stimulation, many of the IFN-γ-producing cells responded to IL-4 restimulation by expression of IL-10, but neither IFN-γ nor IL-4, i.e., by conversion to a suppressive, anti-inflammatory phenotype.
The Th2 Cytokine IL-4 Is Not Required for the Progression of Antibody-Dependent Autoimmune Myasthenia Gravis
Authors: Balasa Balaji , Deng Caishu , Lee Jae , Christadoss Premkumar , Sarvetnick Nora
Published: December 2022

Experimental autoimmune myasthenia gravis (EAMG), a disorder of the neuromuscular junction, is mediated by autoantibodies against muscle nicotinic acetylcholine receptor (AChR). The roles of IFN-γ (Th1) and IL-4 (Th2) cytokines in the initiation and progression of this disease are not fully understood. Recently, we have demonstrated that IFN-γ is necessary for the initiation of tAChR-induced EAMG in mice. However, the role of IL-4 in the progression of clinical EAMG remained undetermined. In this study we have addressed the contribution of IL-4 in the disease progression in IL-4−/− C57BL/6j mice whose IL-4 gene has been disrupted. Following immunization with Torpedo (t) AChR, the IL-4−/− mice readily developed signs of muscle weakness and succumbed to clinical EAMG with kinetics similar to the susceptibility of IL-4+/+ mice. The tAChR-primed lymph node cells from IL-4−/− mice vigorously proliferated to tAChR and to its dominant α146–162 sequence associated with disease pathogenesis. However, these T cells secreted higher levels of IFN-γ and IL-2, suggesting the development of a Th1 default pathway in these mice. Nevertheless, the IL-4 mutation had no effect on the recruitment of CD4+ Vβ6+ T cells specific to the dominant tAChR α146–162 sequence in vivo. Immune sera from IL-4−/− mice showed a dramatic increase in mouse AChR-specific IgG2a levels followed by a concomitant decrease in IgG1 levels, but these mice did not exhibit an accelerated disease. In conclusion, we have demonstrated for the first time that IL-4 is not required either for the generation of a pathogenic anti-AChR humoral immune response or for progression of clinical EAMG in mice.
α-Melanocyte-Stimulating Hormone Inhibits the Nuclear Transcription Factor NF-κB Activation Induced by Various Inflammatory Agents
Authors: Manna Sunil K. , Aggarwal Bharat B.
Published: December 2022

α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide found mainly in the brain, pituitary, and circulation. It inhibits most forms of inflammation by a mechanism that is not known. As most types of inflammation require activation of NF-κB, we investigated the effect of α-MSH on the activation of this transcription factor by a wide variety of inflammatory stimuli. Electrophoretic mobility shift assay showed that α-MSH completely abolished TNF-mediated NF-κB activation in a dose- and time-dependent manner. It also suppressed NF-κB activation induced by LPS, okadaic acid, and ceramide. The effect was specific, as the activation of the transcription factor activating protein-1 by TNF was unaffected. Western blot analysis revealed that TNF-dependent degradation of the inhibitory subunit of NF-κB, IκBα, and nuclear translocation of the p65 subunit of NF-κB were also inhibited. This correlated with suppression of NF-κB-dependent reporter gene expression induced by TNF. The inhibitory effect of α-MSH appeared to be mediated through generation of cAMP, as inhibitors of adenylate cyclase and of protein kinase A reversed its inhibitory effect. Similarly, addition of membrane-permeable dibutyryl cAMP, like α-MSH, suppressed TNF-induced NF-κB activation. Overall, our results suggest that α-MSH suppresses NF-κB activated by various inflammatory agents and that this mechanism probably contributes to its anti-inflammatory effects.
Overexpression of BSAP/Pax-5 Inhibits Switching to IgA and Enhances Switching to IgE in the I.29μ B Cell Line
Authors: Qiu Gang , Stavnezer Janet
Published: December 2022

B cell-specific activator protein (BSAP)/Pax-5 is a paired domain DNA-binding protein expressed in the developing nervous system, testis, and in all B lineage cells, except terminally differentiated plasma cells. BSAP regulates transcription of several genes expressed in B cells and also the activity of the 3′ IgH enhancer. As it has binding sites within or 5′ to the switch regions of nearly all Ig heavy chain C region genes and also is known to increase transcription of the germline ε RNA, BSAP has been hypothesized to be involved in regulation of Ab class switch recombination. To directly examine the effects of BSAP on isotype switching, we use a tetracycline-regulated expression system to overexpress BSAP in the surface IgM+ I.29μ B cell line, a mouse cell line that can be induced to undergo class switch recombination. We find that overexpression of BSAP inhibits switching to IgA in I.29μ cells stimulated with LPS + TGF-β1 + nicotinamide, but enhances switching to IgE in cells stimulated with LPS + IL-4 + nicotinamide. Parallel to its effects on switching, overexpression of BSAP inhibits germline α RNA expression and the transcriptional activity of the germline α promoter, while enhancing activity of the germline ε promoter. Proliferation of I.29μ cells is not affected in this system. The possible mechanisms and significance of the effect of BSAP on isotype switching are discussed.
Triple Role of Platelet-Activating Factor in Eosinophil Migration Across Monolayers of Lung Epithelial Cells: Eosinophil Chemoattractant and Priming Agent and Epithelial Cell Activator
Authors: Liu Lixin , Zuurbier Astrid E. M. , Mul Frederik P. J. , Verhoeven Arthur J. , Lutter René , Knol Edward F. , Roos Dirk
Published: December 2022

Infiltration of eosinophils into the lung lumen is a hallmark of allergic asthmatic inflammation. To reach the lung lumen, eosinophils must migrate across the vascular endothelium, through the interstitial matrix, and across the lung epithelium. The regulation of this process is obscure. In this study, we investigated the migration of human eosinophils across confluent monolayers of either human lung H292 epithelial cells or primary human bronchial epithelial cells. Established eosinophil chemoattractants (IL-8, RANTES, platelet-activating factor (PAF), leukotriene B4, and complement fragment 5a (C5a)) or activation of the epithelial cells with IL-1β induced little eosinophil transmigration (<7% in 2 h). In contrast, addition of PAF in combination with C5a induced extensive (>20%) transepithelial migration of unprimed and IL-5-primed eosinophils. Eosinophil migration assessed in a Boyden chamber assay, i.e., without an epithelial monolayer, was only slightly increased upon addition of PAF and C5a. Preincubation of eosinophils with the PAF receptor antagonist WEB 2086 only inhibited migration of unprimed eosinophils toward PAF and C5a, whereas preincubation of epithelial cells with WEB 2086 abolished migration of both IL-5-primed and unprimed eosinophils. This latter result indicated the presence of PAF receptors on epithelial cells. Indeed, addition of PAF to epithelial cells induced an increase in cytosolic free Ca2+, which was blocked by the PAF receptor antagonists WEB 2086 and TCV-309. Our results show that PAF induces permissive changes in epithelial cells, and that PAF acts as a chemoattractant and priming agent for the eosinophils.
Evidence for the Involvement of Fas Ligand and Perforin in the Induction of Vascular Leak Syndrome
Authors: Rafi Asimah Q. , Zeytun Ahmet , Bradley Michael J. , Sponenberg D. Phillip , Grayson Randolph L. , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: December 2022

Endothelial cell injury resulting in vascular leak syndrome (VLS) is one of the most widely noted phenomenons in a variety of clinical diseases. In the current study we used IL-2-induced VLS as a model to investigate the role of cytolytic lymphocytes in the cytotoxicity of endothelial cells. Administration of IL-2 (75,000 U/mouse, three times a day for 3 days) into BL/6 wild-type mice triggered significant VLS in the lungs, liver, and spleen. Interestingly, perforin-knockout (KO) mice exhibited a marked decrease in IL-2-induced VLS in all three organs tested. Also, Fas ligand-defective (gld) mice and Fas-deficient (lpr) mice exhibited decreased VLS in the liver and spleen, but not in the lungs. The decreased VLS seen in perforin-KO, gld, and lpr mice was not due to any defect in lymphocyte migration or homing to various organs because histopathologic studies in these mice demonstrated significant and often greater perivascular infiltration of lymphocytes compared with the IL-2-treated wild-type mice. Ultrastructural studies of the lungs demonstrated significant damage to the endothelial cells in IL-2-treated wild-type mice and decreased damage in perforin-KO mice. IL-2 administration caused up-regulation of CD44 in all strains of mice tested and triggered increased LAK activity against an endothelial cell line in wild-type and gld mice, but not in perforin-KO mice. The current study demonstrates for the first time that perforin and Fas ligand may actively participate in endothelial cell injury and induction of VLS in a variety of organs.
CD40-Mediated Signal Transduction in Human Airway Smooth Muscle
Authors: Lazaar Aili L. , Amrani Yassine , Hsu Jason , Panettieri Reynold A. , Fanslow William C. , Albelda Steven M. , Puré Ellen
Published: December 2022

CD40 is a member of the TNF receptor family that was initially described on the surface of B cells. Recently, CD40 has also been described on mesenchymal cells, such as endothelial cells and fibroblasts, where engagement by its ligand CD40 ligand can lead to up-regulation of costimulatory and cell adhesion molecules, as well as secretion of proinflammatory cytokines. Since airway inflammation potentially involves cell-cell interactions of T cells and eosinophils (which express CD40 ligand) with airway smooth muscle (ASM) cells, we postulated that ASM may express CD40 and that engagement of ASM CD40 may modulate smooth muscle cell function. We demonstrate that CD40 is expressed on cultured human ASM and that expression can be increased by treatment with TNF-α or IFN-γ. Cross-linking CD40 on ASM resulted in enhanced IL-6 secretion and an increase in intracellular calcium concentrations, which were dependent on calcium influx. We show that CD40-mediated signaling events include protein tyrosine phosphorylation and activation of NF-κB. Pretreatment of ASM with the tyrosine kinase inhibitors genistein or herbimycin inhibited the rapid mobilization of calcium induced via CD40, suggesting that calcium mobilization was coupled to activation of protein tyrosine kinases. In addition, inhibition of calcium influx inhibited both CD40-mediated NF-κB activation and enhancement of IL-6 secretion. These results delineate a potentially important CD40-mediated signal-transduction pathway in ASM, involving protein tyrosine kinase-dependent calcium mobilization, NF-κB activation, and IL-6 production. Together, these results suggest a mechanism whereby T cell/smooth muscle cell interactions may potentiate airway inflammation.
Th2-Induced Eotaxin Expression and Eosinophilia Coexist with Th1 Responses at the Effector Stage of Lung Inflammation
Authors: Li Li , Xia Yiyang , Nguyen Andrea , Feng Lili , Lo David
Published: December 2022

The T cell-mediated lung inflammation that is associated with allergic asthma is characterized mainly by massive eosinophil infiltration, which induces airway injury and the subsequent late-phase reactivity. Because Th2 cells are often isolated from asthmatic subjects, these cells are postulated to play a role in asthma pathogenesis. We report that adoptively transferred, influenza hemagglutinin-specific Th1 and Th2 cells induced different patterns of chemokines leading to different types of cellular infiltration. Th2 cells were sufficient to induce dramatic Ag-dependent lung eosinophilia and eotaxin expression; by contrast, Th1 transfer primarily induced neutrophil recruitment with little eotaxin production. To determine whether Th1 cells show inhibitory effects on Th2 cell-mediated responses, Th1 and Th2 cells were cotransferred. Hemagglutinin-specific Th1 cells did not inhibit Ag-induced lung eosinophilia, nor did they inhibit eotaxin expression. Furthermore, influenza virus infection of the lung in mice receiving hemagglutinin-specific Th2 cells also induced eotaxin expression and eosinophilia that could not be inhibited by the cotransfer of Th1 cells. Our results show that Th2-mediated allergic lung inflammation coexists with the Th1-mediated responses that are stimulated by diverse forms of Ags.
IL-12, But Not IFN-γ, Plays a Major Role in Sustaining the Chronic Phase of Colitis in IL-10-Deficient Mice
Authors: Davidson Natalie J. , Hudak Susan A. , Lesley Robin E. , Menon Satish , Leach Michael W. , Rennick Donna M.
Published: December 2022

IL-10-deficient (IL-10−/−) mice develop chronic enterocolitis mediated by CD4+ Th1 cells producing IFN-γ. Because IL-12 can promote Th1 development and IFN-γ production, the ability of neutralizing anti-IL-12 mAb to modulate colitis in IL-10−/− mice was investigated. Anti-IL-12 mAb treatment completely prevented disease development in young IL-10−/− mice. Treatment of adult mice resulted in significant amelioration of established disease accompanied by reduced numbers of mesenteric lymph node and colonic CD4+ T cells and of mesenteric lymph node T cells spontaneously producing IFN-γ. In contrast, anti-IFN-γ mAb had minimal effect on disease reversal, despite a significant preventative effect in young mice. These findings suggested that IL-12 sustains colitis by supporting the expansion of differentiated Th1 cells that mediate disease independently of their IFN-γ production. This conclusion was supported by the finding that anti-IL-12 mAb greatly diminished the ability of a limited number of CD4+ T cells expressing high levels of CD45RB from diseased IL-10−/− mice to expand and cause colitis in recombination-activating gene-2−/− recipients, while anti-IFN-γ mAb had no effect. Furthermore, IL-12 could support pathogenic IL-10−/− T cells stimulated in vitro in the absence of IL-2. While these studies show that IL-12 plays an important role in sustaining activated Th1 cells during the chronic phase of disease, the inability of anti-IL-12 mAb to abolish established colitis or completely prevent disease transfer by Th1 cells suggests that additional factors contribute to disease maintenance.
Expression of Chemokine Receptors CXCR4 and CCR5 in HIV-1-Infected and Uninfected Individuals
Authors: Ostrowski Mario A. , Justement Shawn J. , Catanzaro Andrew , Hallahan Claire A. , Ehler Linda A. , Mizell Stephanie B. , Kumar Princy N. , Mican Jo Ann , Chun Tae-Wook , Fauci Anthony S.
Published: December 2022

The chemokine receptors CXCR4 and CCR5 have been identified as major coreceptors for HIV-1 entry into CD4+ T cells. The majority of primary HIV-1 isolates in early disease use CCR5 as a coreceptor, whereas during disease progression with the emergence of syncytium-inducing viruses, CXCR4 is also used. We performed a cross-sectional study in which we evaluated the expression of two HIV-1 coreceptors, CCR5 and CXCR4, in whole blood samples taken from HIV-1-infected and uninfected individuals. We demonstrate that CXCR4 on CD4+ and CD8+ T cells, and CD14+ monocytes is significantly down-regulated, and CCR5 expression on CD4+ T cells is up-regulated in HIV-infected individuals compared with uninfected controls. Coreceptor expression correlated with the level of cellular activation in vivo in both HIV-infected and uninfected individuals, with CXCR4 being expressed predominantly on quiescent (HLA-DR−) T cells and CCR5 being expressed predominantly on activated (HLA-DR+) T cells. Lower expression of CXCR4 and higher expression of CCR5 on CD4+ T cells correlated with advancing disease. In addition, a tendency for greater activation of CXCR4+CD4+ T cells in patients with advanced disease was observed. Patients who harbored syncytium-inducing viruses, however, could not be distinguished from those who harbored nonsyncytium-inducing viruses based on the level of CD4+ T cell activation or chemokine receptor expression.