bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of Cycling T Lymphocytes in Blood and Lymphoid Organs During Immune Responses
Authors: Vasseur Florence , Le Campion Armelle , Pavlovitch Jana H. , Pénit Claude
Published: December 2022

Proliferation of murine T lymphocytes in blood, lymph nodes, and spleen was studied in four in vivo stimulation systems, using BrdU pulse-labeling of DNA-synthesizing cells. The T cell response to the superantigen Staphylococcus enterotoxin B (SEB) was studied in detail. Vβ8+ T cells showed a peak of DNA synthesis 16–24 h after SEB injection, and the percentage of BrdU+ CD4 and CD8 T cells was higher in blood than in lymph nodes and spleen. DNA synthesis was preceded by massive migration of Vβ8+ cells from blood to lymphoid organs, in which the early activation marker CD69 was first up-regulated. SEB-nonspecific Vβ6+ cells showed minimal stimulation but, when cycling, also expressed a high level of CD69. The other systems studied were injection of the IFN-γ inducer polyinosinic:polycytidylic acid, infection by the BM5 variants of murine leukemia virus (the causative agent of murine AIDS), and T cell expansion after transfer of normal bone marrow and lymph node cells into recombinase-activating gene-2-deficient mice. In each case, a peak of T cell proliferation was observed in blood. These data demonstrate the extensive redistribution of cycling T cells in the first few hours after activation. Kinetic studies of blood lymphocyte status appear crucial for understanding primary immune responses because cycling and redistributing T lymphocytes are enriched in the circulating compartment.
Human CD4+ T Cells Mediate Rejection of Porcine Xenografts
Authors: Friedman Tony , Shimizu Akira , Smith Rex Neal , Colvin Robert B. , Seebach Jörg D. , Sachs David H. , Iacomini John
Published: December 2022

It has previously been demonstrated that xenograft rejection in rodents is dependent on CD4+ T cells. However, because of the lack of an appropriate in vivo model, little is known about the cellular basis of human T cell-mediated rejection of xenografts. In this study, we have evaluated the ability of human T cells to mediate rejection of porcine skin grafts in a novel in vivo experimental system using immunodeficient mice as recipients. Recombinase-activating gene-1-deficient mice (R−) lacking mature B and T cells were grafted with porcine skin and received human lymphocytes stimulated in vitro with irradiated porcine PBMC. Skin grafts on mice given either unseparated, activated human lymphocytes, or NK cell-depleted lymphocyte populations were rejected within 18 days after adoptive cell transfer. In contrast, skin grafts on mice given T cell-depleted human lymphocytes or saline showed no gross or histologic evidence of rejection up to 100 days after adoptive transfer. Purified CD4+ T cells were also able to mediate rejection of porcine skin grafts. These data suggest that human CD4+ T cells are sufficient to induce rejection of porcine xenografts. Thus, strategies directed toward CD4+ T cells may effectively prevent cellular rejection of porcine xenografts in humans.
Virus and Autoimmunity: Induction of Autoimmune Disease in Mice by Mouse T Lymphotropic Virus (MTLV) Destroying CD4+ T Cells
Authors: Morse Stephen S. , Sakaguchi Noriko , Sakaguchi Shimon
Published: December 2022

Neonatal infection of the mouse T lymphotropic virus (MTLV), a member of herpes viridae, causes various organ-specific autoimmune diseases, such as autoimmune gastritis, in selected strains of normal mice. The infection selectively depletes CD4+ T cells in the thymus and periphery for 2–3 wk from 1 wk after infection. Thymectomy 3 wk after neonatal MTLV infection enhances the autoimmune responses and produces autoimmune diseases at higher incidences and in a wider spectrum of organs than MTLV infection alone. On the other hand, inoculation of peripheral CD4+ cells from syngeneic noninfected adult mice prevents the autoimmune development. These autoimmune diseases can be adoptively transferred to syngeneic athymic nude mice by CD4+ T cells. The virus is not detected by bioassay in the organs/tissues damaged by the autoimmune responses. Furthermore, similar autoimmune diseases can be induced in normal mice by manipulating the neonatal thymus/T cells (e.g., by neonatal thymectomy) without virus infection. These results taken together indicate that neonatal MTLV infection elicits autoimmune disease by primarily affecting thymocytes/T cells, not self Ags. It may provoke or enhance thymic production of CD4+ pathogenic self-reactive T cells by altering the thymic clonal deletion mechanism, or reduce the production of CD4+ regulatory T cells controlling self-reactive T cells, or both. The possibility is discussed that other T cell-tropic viruses may cause autoimmunity in humans and animals by affecting the T cell immune system, not the self Ags to be targeted by the autoimmunity.
Bcl-2-Induced Changes in E2F Regulatory Complexes Reveal the Potential for Integrated Cell Cycle and Cell Death Functions
Authors: Lind Evan F. , Wayne Jay , Wang Qi-Zhi , Staeva Teodora , Stolzer Amy , Petrie Howard T.
Published: December 2022

Proliferation and cell death are tightly linked fates during cell and tissue differentiation. In the past few years, it has been shown that Bcl-2 exhibits a potent cell cycle inhibitory effect, in addition to its better known role in the antagonism of cell death. In the present study, we show that the cell cycle effects of Bcl-2 apparently occur at the level of E2F control of gene transcription. Under conditions of normal cell growth, or under conditions that lead to cell death in the absence of Bcl-2, bcl-2 expression results in a reduction of free (active) E2F isoforms and in an increase in the formation of higher-order (inactive) complexes. Bcl-2-induced changes in E2F complex formation are paralleled by an apparent increase in pRb regulatory activity, by the up-regulation of p130 protein expression, and by the formation of E2F/p130 complexes at the expense of those consisting of E2F/p107. Cells lacking bcl-2 expression respond to growth factor withdrawal in the opposite manner, by the liberation of E2F from inactivating complexes and by continued cell cycle leading to cell death. These analyses reveal a mechanism for cell cycle regulation by Bcl-2 that occurs at the level of E2F transcriptional activity. Further, since specific E2F activities are clearly linked to the induction of cell death, these findings may help to consolidate the cell survival and cell cycle effects of Bcl-2 through a common transcriptional mechanism.
In Vitro Induction of the Expression of Multiple IgA Isotype Genes in Rabbit B Cells by TGF-β and IL-2
Authors: Spieker-Polet Helga , Yam Pi-Chen , Arbieva Zarema , Zhai Shi-Kang , Knight Katherine L.
Published: December 2022

The rabbit genome has 13 different Cα genes that are expressed at different levels in mucosal tissues. To analyze the factors involved in the differential expression of these Cα genes, we cloned and sequenced the promoters of the Iα regions that control the expression of sterile mRNA. We found that all Cα genes, including Cα3 and Cα8, which are not expressed, and Cα4, which is expressed at high levels, have similar nucleotide sequences in the Iα region, and all contain the recognition elements for TGF-β in the promoter. B lymphocytes from popliteal lymph nodes or Peyer’s patch activated in vitro could be induced by TGF-β to express sterile IgA transcripts of all IgA isotypes, except Cα2, Cα3, and Cα8. Many single B lymphocytes transcribed sterile mRNA of more than one IgA isotype, which demonstrates that transcription of sterile mRNA alone does not regulate the IgA isotype switch. The addition of IL-2 led to the expression of transcripts of mature IgA of all isotypes, except Cα2, Cα3, and Cα8. The predominantly expressed isotype in these experiments was Cα4. With the use of an IgA4-specific mAb we found that IgA4+ plasma cells are unevenly distributed throughout the small intestine such that many of the IgA+ plasma cells in the duodenum-jejunum produced IgA4, whereas in the lower part of the ileum IgA4-producing cells were almost absent. Because the microbial flora varies throughout the intestine, we suggest that the microbial flora creates different local environments and thus affects either isotype switching or homing of IgA-expressing cells.
Down-Regulation of CXCR4 by Human Herpesvirus 6 (HHV-6) and HHV-7
Authors: Yasukawa Masaki , Hasegawa Atsuhiko , Sakai Ikuya , Ohminami Hideki , Arai Junko , Kaneko Shin , Yakushijin Yoshihiro , Maeyama Kazutaka , Nakashima Hideki , Arakaki Rieko , Fujita Shigeru
Published: December 2022

Recent studies have demonstrated that human herpesvirus 6 (HHV-6) and HHV-7 interact with HIV-1 and alter the expression of various surface molecules and functions of T lymphocytes. The present study was undertaken to clarify whether coreceptors for HIV-1, CXCR4 and CCR5, are necessary for HHV-6 and HHV-7 infection. Although CXCR4 and CCR5 appeared not to be the coreceptors for these viruses, marked down-regulation of CXCR4, but not CCR5, was detected in HHV-6 variant A (HHV-6A)-, HHV-6 variant B (HHV-6B)-, and HHV-7-infected cells. Down-regulation of CXCR4 resulted in impairment of chemotaxis and a decreased level of elevation of the intracellular Ca2+ concentration in response to stromal cell-derived factor-1. Northern blot analysis of mRNAs extracted from HHV-6A-, HHV-6B-, and HHV-7-infected CD4+ T lymphocytes demonstrated a markedly decreased level of CXCR4 gene transcription, but the posttranscriptional stability of CXCR4 mRNA was not significantly altered. These data demonstrate that unlike HIV-1, HHV-6 and HHV-7 infections do not require expression of CXCR4 or CCR5, whereas marked down-regulation of CXCR4 is induced by these viruses, suggesting that HHV-6 and HHV-7 infections may render CD4+ T lymphocytes resistant to T lymphocyte-tropic HIV-1 infection.
Class II MHC/Peptide Complexes Are Released from APC and Are Acquired by T Cell Responders During Specific Antigen Recognition
Authors: Patel Dhaval M. , Arnold Paula Y. , White Gregory A. , Nardella John P. , Mannie Mark D.
Published: December 2022

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-γ. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A− T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.
Primary Peripheral Blood Eosinophils Rapidly Degrade Transfected Granulocyte-Macrophage Colony-Stimulating Factor mRNA
Authors: Esnault Stéphane , Malter James S.
Published: December 2022

Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMV-driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t1/2 in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t1/2 of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded, suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM-CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow detailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.
Lad, an Adapter Protein Interacting with the SH2 Domain of p56lck, Is Required for T Cell Activation
Authors: Choi Young Bong , Kim Chan Ki , Yun Yungdae
Published: December 2022

T cell-specific Src family tyrosine kinase, p56lck, plays crucial roles in T cell differentiation, activation, and proliferation. These multiple functions of p56lck are believed to be conducted through the protein-protein interactions with various cellular signaling proteins. To clarify the mechanisms through which p56lck contributes to T cell signaling, we identified the proteins binding to the Src homology 2 (SH2) domain of p56lck through a tyrosine phosphorylation-dependent yeast two-hybrid screening. Subsequent characterization of positive clones revealed the presence of a protein of 366 aa named Lad (Lck-associated adapter protein), which is a potential murine homologue of previously reported TSAd, a T cell-specific adapter protein. Lad contains several protein-protein interaction domains including a zinc-finger motif, an SH2 domain, a proline-rich SH3 binding motif, and several phosphotyrosine sites. Furthermore, Lad was tyrosine phosphorylated and associated with p56lckin vivo and redistributed from cytoplasm to the plasma membrane in a T cell activation-dependent manner. Moreover in T cells, IL-2 promoter activity was enhanced upon coexpression of Lad but was inhibited by the coexpression of antisense Lad RNA. These characteristics of Lad suggest that Lad play an essential role as an adapter protein in p56lck-mediated T cell signaling.
CD4+ T Cell Responses to CD40-Deficient APCs: Defects in Proliferation and Negative Selection Apply Only with B Cells as APCs
Authors: Ozaki Minette E. , Coren Barbara A. , Huynh Tracy N. , Redondo Deborah J. , Kikutani Hitoshi , Webb Susan R.
Published: December 2022

During T-APC interactions in vivo, interfering with CD40-CD154 interactions leads to reduced T cell priming, defects in effector function, and, in some cases, T cell tolerance. As shown here, however, presentation of conventional peptide Ags by CD40-deficient spleen APC in vitro leads to normal CD4+ T cell proliferative responses. By contrast, responses to the same peptides presented by purified B cells were markedly reduced in the absence of CD40. Thus, the requirement for CD40-CD154 interactions appears to be strongly influenced by the type of APC involved. Analysis of responses to endogenous superantigens, which are known to be strongly dependent on B cells for presentation, indicated that CD4+ responses to strong Ags are less dependent on CD40 than are responses to weak Ags. Similar findings applied to negative selection in the thymus. Thus, deletion of potentially autoreactive cells depended on CD40 expression when B APC were involved, and this requirement was most pronounced when negative selection was directed to weak Ags.
Autoimmune Myocarditis Does Not Require B Cells for Antigen Presentation
Authors: Malkiel Susan , Factor Stephen , Diamond Betty
Published: December 2022

T cells constitute the pathogenic effector cell population in autoimmune myocarditis in BALB/c mice. Using mice rendered deficient for B cells by a targeted disruption to the IgM transmembrane domain or by treatment with anti-IgM Ab from birth, we asked whether B cells are a critical APC in the induction of autoimmune myocarditis. B cell-deficient mice immunized with cardiac myosin develop myocarditis comparable in incidence and severity to that in wild-type mice, suggesting that autoreactive T cells that cause myocarditis in BALB/c mice are activated by macrophages or dendritic cells. Since it does not appear that presentation of cryptic epitopes is critical for the breakdown of self tolerance, potentially pathogenic T cells recognizing dominant myosin epitopes must have escaped tolerization. Either anatomic sequestration of cardiac myosin peptide-MHC complexes or subthreshold presentation of cardiac myosin peptides by conventional APC can explain the survival of these autoreactive T cells.
Structure/Function Relationship of Activating Ly-49D and Inhibitory Ly-49G2 NK Receptors
Authors: Ortaldo John R. , Winkler-Pickett Robin , Willette-Brown Jami , Wange Ronald L. , Anderson Stephen K. , Palumbo Gregory J. , Mason Llewellyn H. , McVicar Daniel W.
Published: December 2022

Murine NK cells express Ly-49 family receptors capable of either inhibiting or activating lytic function. The overlapping patterns of expression of the various receptors have complicated their precise biochemical characterization. Here we describe the use of the Jurkat T cell line as the model for the study of Ly-49s. We demonstrate that Ly-49D is capable of delivering activation signals to Jurkat T cells even in the absence of the recently described Ly-49D-associated chain, DAP-12. Ly-49D signaling in Jurkat leads to tyrosine phosphorylation of TCRζ and requires Syk/Zap70 family kinases and arginine 54 of Ly-49D, suggesting that Ly-49D signals via association with TCRζ. Coexpression studies in 293-T cells confirmed the ability of Ly-49D to associate with TCRζ. In addition, we have used this model to study the functional interactions between an inhibitory Ly-49 (Ly-49G2) and an activating Ly-49 (Ly-49D). Ly-49G2 blocks activation mediated by Ly-49D in an immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent manner. In contrast, Ly-49G2 was incapable of inhibiting activation by the TCR even though human killer cell inhibitory receptor (KIR) (KIR3DL2(GL183)) effectively inhibits TCR. Both the ability of Ly-49G2 to block Ly-49D activation and the failure of Ly-49G2 to inhibit TCR signaling were confirmed in primary murine NK cells and NK/T cells, respectively. These data demonstrate the dominant effects of the inhibitory receptors over those that activate and suggest an inability of the Ly-49 type II inhibitory receptors to efficiently inhibit type I transmembrane receptor signaling in T cells and NK cells.
Constitutive Expression and Role of the TNF Family Ligands in Apoptotic Killing of Tumor Cells by Human NK Cells
Authors: Kashii Yoshiro , Giorda Roberto , Herberman Ronald B. , Whiteside Theresa L. , Vujanovic Nikola L.
Published: December 2022

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-α, LT-β, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-α1β2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-α1β2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.
Amino Acids Specifying MHC Class Preference in TCR Vα2 Regions
Authors: Correia-Neves Margarida , Waltzinger Caroline , Wurtz Jean-Marie , Benoist Christophe , Mathis Diane
Published: December 2022

Some TCR variable regions are preferentially expressed in CD4+ or CD8+ T cells, reflecting a predilection for interacting with MHC class II or class I molecules. The molecular basis for MHC class bias has been studied previously, in particular for Vα3 family members, pointing to a dominant role for two amino acid positions in complementary-determining regions (CDRs) 1 and 2. We have evaluated the generality of these findings by examining the MHC class bias of Vα2 family members, an attractive system because it shows more variability within the CDR1 and -2, exhibits variation in the framework regions, and includes a member for which the crystal structure has been determined. We find that preferential recognition of MHC class I or II molecules does not always depend on residues at the same positions of CDR1 and -2; rules for one family may be reversed in another. Instead, there are multiple influences exerted by various CDR1/2 positions as well as the CDR3s of both the TCR α- and TCR β-chains.
Active Matrix Metalloproteinases Are Present in Cartilage During Immune Complex-Mediated Arthritis: A Pivotal Role for Stromelysin-1 in Cartilage Destruction
Authors: van Meurs Joyce , van Lent Peter , Holthuysen Astrid , Lambrou Dimitri , Bayne Ellen , Singer Irwin , van den Berg Wim
Published: December 2022

The involvement of immune complexes during experimental arthritis in induction of metalloproteinases (MMP)-induced neoepitopes in aggrecan in cartilage, as well as the role of stromelysin-1 (SLN-1) in the induction of this neoepitope, was investigated. Passive immune complex arthritis was induced, and generation of the MMP-specific cleavage product (VDIPEN) was studied by immunolocalization. The role of SLN-1 was studied with use of SLN-1-deficient (SLN-1KO) mice. VDIPEN expression was studied in vitro by exposing the cartilage to IL-1 and subsequent activation of latent MMPs. Immune complex arthritis was characterized by an acute inflammation, with influx of mainly polymorphonuclear cells into the joint cavity. Expression of VDIPEN neoepitopes was consistently found in areas extensively depleted from proteoglycans. SLN-1KO mice did not show expression of the VDIPEN neoepitope, although inflammation and proteoglycan depletion was comparable to wild-type mice. In addition, erosions of cartilage were absent in SLN-1KO mice, but were present in wild-type mice, suggesting an important role for SLN-1 in cartilage destruction. In vitro studies showed that SLN-1 is also pivotally involved in IL-1-induced MMP activity. Stimulated polymorphonuclear neutrophils were able to activate latent MMPs present in the cartilage. Neutrophil elastase was also capable of activating IL-1-induced latent MMPs, which identifies elastase as a possible activator for latent VDIPEN-inducing MMPs. This study suggests that IC are important in the activation of latent MMPs in cartilage, possibly through polymorphonuclear neutrophil activation on the cartilage edge. SLN-1 is a pivotal enzyme in overall MMP-activity in cartilage during immune complex-mediated arthritis.
Regulation of Human Eosinophil Migration Across Lung Epithelial Monolayers by Distinct Calcium Signaling Mechanisms in the Two Cell Types
Authors: Liu Lixin , Ridefelt Peter , Håkansson Lena , Venge Per
Published: December 2022

In asthmatic patients, eosinophils massively infiltrate the lung tissues and migrate through lung epithelium into the airways. The regulatory mechanisms involved are obscure. We studied the role of calcium in the migration of human eosinophils across monolayers of human lung epithelial H292 cell line cells induced by combined chemotactic solutions of platelet-activating factor and C5a. The transepithelial migration of eosinophils was attenuated by depletion of the external Ca2+ in the migration system, whereas the eosinophil migration itself was unaffected as evidenced by measuring eosinophil chemotaxis in the Boyden chamber in the absence of epithelial cells. Buffering of intracellular Ca2+ in eosinophils with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibited both eosinophil transepithelial migration and eosinophil chemotaxis in the Boyden chamber, suggesting the importance of intracellular Ca2+ in eosinophil transmigration. Although loading of BAPTA/AM or addition of thapsigargin to the epithelial cells effectively changed their cytoplasmic free Ca2+ concentrations, neither of these treatments affected transepithelial migration of eosinophils. Interestingly, addition of La3+ (0.2 mM) to epithelial cells suppressed eosinophil transmigration whereas addition of La3+ to eosinophils did not. Taken together, these results show the importance of Ca2+ in eosinophil migration across lung epithelium and support a distinctive regulatory role of intracellular and extracellular Ca2+ for the two cell types involved in this process; i.e., the transmigration of human eosinophils across a monolayer of lung epithelial cells is regulated by the intracellular Ca2+ in eosinophils, whereas the ability of the lung epithelial cell monolayer to allow eosinophil passage is dependent on the extracellular Ca2+.
IFN-γ-Inducible Protein-10 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via Inhibition of Angiogenesis
Authors: Keane Michael P. , Belperio John A. , Arenberg Douglas A. , Burdick Marie D. , Xu Zuo J. , Xue Ying Y. , Strieter Robert M.
Published: December 2022

Few studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis (BPF). For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during BPF are dependent in part on a relative deficiency of the angiostatic CXC chemokine, IFN-γ-inducible protein-10 (IP-10). To test this hypothesis, we measured IP-10 by specific ELISA in whole lung homogenates in either bleomycin-treated or control mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a decrease in the presence of IP-10 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Systemic administration of IP-10 significantly reduced BPF without any alteration in lung lymphocyte or NK cell populations. This was also paralleled by a reduction in angiogenesis. Furthermore, IP-10 had no direct effect on isolated pulmonary fibroblasts. These results demonstrate that the angiostatic CXC chemokine, IP-10, inhibits fibroplasia and deposition of extracellular matrix by regulating angiogenesis.
Anti-TGF-β Treatment Prevents Skin and Lung Fibrosis in Murine Sclerodermatous Graft-Versus-Host Disease: A Model for Human Scleroderma
Authors: McCormick Laura L. , Zhang Yan , Tootell Elena , Gilliam Anita C.
Published: December 2022

Scleroderma, a debilitating acquired connective tissue disease, is characterized by fibrosis, particularly of the skin and lungs. Monocyte-produced TGF-β1, a potent stimulus for collagen synthesis, is thought to drive the fibrosis. Here, we thoroughly characterize a murine sclerodermatous graft-vs-host disease (Scl GVHD) model for scleroderma that reproduces important features of scleroderma including skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA, which is preceded by monocyte infiltration and the up-regulation of cutaneous TGF-β1 mRNA. Most importantly, we can prevent fibrosis in both the skin and lungs of mice with Scl GVHD by inhibiting TGF-β with neutralizing Abs. The murine Scl GVHD model provides the unique opportunity to study basic immunologic mechanisms that drive fibrosing diseases and GVHD itself and will be useful for testing new therapies for these diseases.
Bone Marrow-Derived Cells Are Responsible for the Development of Autoimmune Arthritis in Human T Cell Leukemia Virus Type I-Transgenic Mice and Those of Normal Mice Can Suppress the Disease
Authors: Saijo Shinobu , Kotani Motoko , Habu Kiyoshi , Ishitsuka Chiho , Yamamoto Hiroaki , Sekiguchi Toyozo , Iwakura Yoichiro
Published: December 2022

Previously, we reported that human T cell leukemia virus type I env-pX region-introduced transgenic (pX-Tg) mice developed an inflammatory polyarthropathy associated with a development of autoimmunity. To elucidate roles of autoimmunity in the development of arthritis, the immune cells were reciprocally replaced between pX-Tg mice and non-transgenic (Tg) mice. When bone marrow (BM) cells and spleen cells from pX-Tg mice were transferred into irradiated non-Tg mice, arthritis developed in these mice. In contrast, arthritis in pX-Tg mice was completely suppressed by non-Tg BM and spleen cells. Similar results were obtained with BM cells only. After the transplantation, T cells, B cells, and macrophages were replaced completely, whereas cells in the joints were replaced partially. In those mice, serum Ig and rheumatoid factor levels correlated with the disease development, and inflammatory cytokine expression was elevated in the arthritic joints. Furthermore, involvement of T cells in the joint lesion was suggested, because the incidence was greatly reduced in athymic nu/nu mice although small proportion of the mice still developed arthritis. These observations suggest that BM stem cells are abnormal, causing autoimmunity in pX-Tg mice, and this autoimmunity plays an important, but not absolute, role in the development of arthritis in this Tg mouse.
Determination of Residues Involved in Ligand Binding and Signal Transmission in the Human IFN-α Receptor 2
Authors: Chuntharapai A. , Gibbs V. , Lu J. , Ow A. , Marsters S. , Ashkenazi A. , De Vos A. , Kim K. Jin
Published: December 2022

The human IFN-α receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-α2/α1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-α2/α1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-α8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-α2, but remained responsive to hIFN-β. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-α2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.
Mechanisms of Lung Neutrophil Activation After Hemorrhage or Endotoxemia: Roles of Reactive Oxygen Intermediates, NF-κB, and Cyclic AMP Response Element Binding Protein
Authors: Shenkar Robert , Abraham Edward
Published: December 2022

Acute inflammatory lung injury occurs frequently in the setting of severe infection or blood loss. Accumulation of activated neutrophils in the lungs and increased pulmonary proinflammatory cytokine levels are major characteristics of acute lung injury. In the present experiments, we examined mechanisms leading to neutrophil accumulation and activation in the lungs after endotoxemia or hemorrhage. Levels of IL-1β, TNF-α, and macrophage inflammatory protein-2 mRNA were increased in lung neutrophils from endotoxemic or hemorrhaged mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic, hemorrhaged, or control mice. The transcriptional regulatory factors NF-κB and cAMP response element binding protein were activated in lung but not blood neutrophils after hemorrhage or endotoxemia. Xanthine oxidase inhibition, achieved by feeding allopurinol or tungsten-containing diets, did not affect neutrophil trafficking to the lungs after hemorrhage or endotoxemia. Xanthine oxidase inhibition did prevent hemorrhage- but not endotoxemia- induced increases in proinflammatory cytokine expression among lung neutrophils. Hemorrhage- or endotoxemia-associated activation of NF-κB in lung neutrophils was not affected by inhibition of xanthine oxidase. cAMP response element binding protein activation was increased after hemorrhage, but not endotoxemia, in mice fed xanthine oxidase-inhibiting diets. Our results indicate that xanthine oxidase modulates cAMP response element binding protein activation and proinflammatory cytokine expression in lung neutrophils after hemorrhage, but not endotoxemia. These findings suggest that the mechanisms leading to acute inflammatory lung injury after hemorrhage differ from those associated with endotoxemia.
Chemotaxis of Rat Mast Cells Toward Adenine Nucleotides
Authors: McCloskey Michael A. , Fan Yihong , Luther Stacie
Published: December 2022

Rat mucosal mast cells express P2 purinoceptors, occupation of which mobilizes cytosolic Ca2+ and activates a potassium conductance. The primary function of this P2 system in mast cell biology remains unknown. Here, we show that extracellular ADP causes morphological changes in rat bone marrow-cultured mast cells (BMMC) typical of those occurring in cells stimulated by chemotaxins, and that the nucleotides ADP, ATP, and UTP are effective chemoattractants for rat BMMC. ADP was also a chemotaxin for murine J774 monocytes. The nucleotide selectivity and pertussis toxin sensitivity of the rat BMMC migratory response suggest the involvement of P2U receptors. Poorly hydrolyzable derivatives of ADP and ATP were effective chemotaxins, obviating a role for adenosine receptors. Buffering of external Ca2+ at 100 nM or reduction of the electrical gradient driving Ca2+ entry (by elevating external K+) blocked ADP-driven chemotaxis, suggesting a role for Ca2+ influx in this process. Anaphylatoxin C5a was a potent chemotaxin (EC50 ≈0.5 nM) for J774 monocytes, but it was inactive on rat BMMC in the presence or absence of laminin. Ca2+ removal or elevated [K+] had modest effects on C5a-driven chemotaxis of J774 cells, implicating markedly different requirements for Ca2+ signaling in C5a- vs ADP-mediated chemotaxis. This is supported by the observation that depletion of Ca2+ stores with thapsigargin completely blocked migration induced by ADP but not C5a. These findings suggest that adenine nucleotides liberated from parasite-infested tissue could participate in the recruitment of mast cells by intestinal mucosa.
Cutting Edge: HIV-1 Tat Protein Differentially Modulates the B Cell Response of Naive, Memory, and Germinal Center B Cells
Authors: Lefevre Eric A. , Krzysiek Roman , Loret Erwann P. , Galanaud Pierre , Richard Yolande
Published: December 2022

Critical steps of B cell differentiation occur within lymphoid organs that are also major sites of HIV-1 replication. Because Tat can be released by infected cells, we investigated whether extracellular HIV-1 Tat modulates cell proliferation of B cells at critical stages of their differentiation. Here we show that extracellular Tat inhibited the proliferation of B cell receptor-triggered naive and memory B cells by >80% but had no effect on their CD40 mAb and IL-4-mediated proliferation. In striking contrast, Tat doubled the germinal center B cell proliferation induced by CD40 mAb and IL-4. These effects were dose dependent and required the addition of Tat at the initiation of the culture, suggesting that Tat acts on early stages of cell cycle progression. By its effects on B cell subsets, Tat might directly affect the normal B cell differentiation process in HIV-positive patients and favor the occurrence of AIDS-associated B cell lymphomas.
Th1 Cytokines and NK Cells Participate in the Development of Murine Syngeneic Graft-Versus-Host Disease
Authors: Flanagan Diana Lowery , Jennings C. Darrell , Bryson J. Scott
Published: December 2022

Syngeneic graft-vs-host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow cells followed by a short course of therapy with the immunosuppressive agent cyclosporine A. Following cessation of cyclosporine A therapy, animals develop clinical symptoms of SGVHD: weight loss, runting, and diarrhea. While it has been suggested that T cells are responsible for the induction and effector phases of SGVHD, the role of nonspecific effector cells and cytokine mediators has yet to be examined in the disease process. Mice with SGVHD had increased levels of message for IL-12p40, IFN-γ, and TNF-α in the target organs of SGVHD as compared with transplant controls and asymptomatic cyclosporine A-treated mice. Concomitant with the increase in Th1 cytokines was an enhanced cellular infiltrate in the target organs of SGVHD mice as determined by histological analysis. To directly examine the role of IL-12 in the development of SGVHD, in vivo neutralization of IL-12 was performed. Treatment of mice with Abs to IL-12 inhibited SGVHD-mediated tissue pathology and mortality. Because IL-12 has been shown to activate both T cells and NK cells to secrete IFN-γ and to become more cytolytic, studies were initiated to ascertain which lymphocyte populations play a role in the development of murine SGVHD. Depletion of NK cells inhibited clinical symptoms of SGVHD. In contrast, T cell depletion did not alter the disease process. Therefore, these findings collectively demonstrate a role for IL-12 and NK cells in the effector phase of murine SGVHD.
Functional Similarity and Differences Between Selection-Independent CD4−CD8− αβ T Cells and Positively Selected CD8 T Cells Expressing the Same TCR and the Induction of Anergy in CD4−CD8− αβ T Cells in Antigen-Expressing Mice
Authors: Caveno Jordan , Zhang Yiqun , Motyka Bruce , Teh Soo-Jeet , Teh Hung-Sia
Published: December 2022

In TCR-αβ transgenic mice, CD4−CD8− TCR-αβ+ (αβ DN) cells arise in the absence of positively selecting MHC molecules and are resistant to clonal deletion in Ag-expressing mice. In this study the activation requirements and functional properties of αβ double-negative (DN) cells were compared with those of positively selected CD8+ cells expressing equivalent levels of the same MHC class I-restricted transgenic TCR. We found that positively selected CD8+ cells required a lower density of the antigenic ligand for optimal proliferative responses compared with αβ DN cells derived from nonpositively selecting mice. However, when the CD8 coreceptor on CD8+ cells was blocked with an anti-CD8 mAb, both αβ DN and CD8+ cells exhibited the same dose-response curve to the antigenic ligand and the same dependence on CD28/B7 costimulation. Positively selected CD8+ cells also differed from αβ DN cells in that they differentiated into more efficient killers and IL-2 producers after Ag stimulation, even after CD8 blockade. However, Ag-activated αβ DN and CD8+ cells were equally efficient in producing IFN-γ, suggesting that this functional property is independent of positive selection. We also found that αβ DN cells recovered from the lymph nodes of Ag-expressing mice were functionally anergic. This anergic state was associated with defective proliferation and IL-2 production in response to Ag stimulation. These observations indicate that αβ DN cells can be anergized in vivo by physiological levels of the antigenic ligand.
Requirements for Signal Delivery Through CD44: Analysis Using CD44-Fas Chimeric Proteins
Authors: Ishiwatari-Hayasaka Haruko , Fujimoto Takashi , Osawa Tomoko , Hirama Toshiyasu , Toyama-Sorimachi Noriko , Miyasaka Masayuki
Published: December 2022

CD44 is a transmembrane glycoprotein involved in various cell adhesion events, including lymphocyte migration, early hemopoiesis, and tumor metastasis. To examine the requirements of CD44 for signal delivery through the extracellular domain, we constructed a chimeric CD44 protein fused to the intracellular domain of Fas on its C-terminus. In cells expressing the CD44-Fas fusion protein, apoptosis could be induced by treatment with certain anti-CD44 mAbs alone, especially those recognizing the epitope group d, which has been previously shown to play a role in ligand binding, indicating that ligation of a specific region of the CD44 extracellular domain results in signal delivery. Of note was that appropriate ligation of the epitope h also resulted in the generation of apoptotic signal, although this region was not thought to be involved in ligand binding. In contrast, the so-called blocking anti-CD44 mAbs (epitope group f) that can abrogate the binding of hyaluronate (HA) failed to induce apoptosis even after further cross-linking with the secondary Ab, indicating that a mere mAb-induced oligomerization of the chimeric proteins is insufficient for signal generation. However, these blocking mAbs were instead capable of inhibiting apoptosis induced by nonblocking mAb (epitope group h). Furthermore, a chimeric protein bearing a mutation in the HA binding domain and hence lacking the ability to recognize HA was incapable of mediating the mAb-induced apoptosis, suggesting that the functional integrity of the HA binding domain is crucial to the signal generation in CD44.
Direct Suppression of TCR-Mediated Activation of Extracellular Signal-Regulated Kinase by Leukocyte Protein Tyrosine Phosphatase, a Tyrosine-Specific Phosphatase
Authors: Oh-hora Masatsugu , Ogata Masato , Mori Yoshiko , Adachi Masaaki , Imai Kohzoh , Kosugi Atsushi , Hamaoka Toshiyuki
Published: December 2022

Leukocyte protein tyrosine phosphatase (LC-PTP)/hemopoietic PTP is a human cytoplasmic PTP that is predominantly expressed in the hemopoietic cells. Recently, it was reported that hemopoietic PTP inhibited TCR-mediated signal transduction. However, the precise mechanism of the inhibition was not identified. Here we report that extracellular signal-regulated kinase (ERK) is the direct target of LC-PTP. LC-PTP dephosphorylated ERK2 in vitro. Expression of wild-type LC-PTP in 293T cells suppressed the phosphorylation of ERK2 by a mutant MEK1, which was constitutively active regardless of upstream activation signals. No suppression of the phosphorylation was observed by LC-PTPCS, a catalytically inactive mutant. In Jurkat cells, LC-PTP suppressed the ERK and p38 mitogen-activated protein kinase cascades. LC-PTP and LC-PTPCS made complexes with ERK1, ERK2, and p38α, but not with the gain-of-function sevenmaker ERK2 mutant (D321N). A small deletion (aa 1–46) in the N-terminal portion of LC-PTP or Arg to Ala substitutions at aa 41 and 42 resulted in the loss of ERK binding activity. These LC-PTP mutants revealed little inhibition of the ERK cascade activated by TCR cross-linking. On the other hand, the wild-type LC-PTP did not suppress the phosphorylation of sevenmaker ERK2 mutant. Thus, the complex formation of LC-PTP with ERK is the essential mechanism for the suppression. Taken collectively, these results indicate that LC-PTP suppresses mitogen-activated protein kinase directly in vivo.
CD45 Negatively Regulates Lyn Activity by Dephosphorylating Both Positive and Negative Regulatory Tyrosine Residues in Immature B Cells
Authors: Katagiri Tatsuo , Ogimoto Mami , Hasegawa Kiminori , Arimura Yutaka , Mitomo Katsuyuki , Okada Masato , Clark Marcus R. , Mizuno Kazuya , Yakura Hidetaka
Published: December 2022

Using CD45-deficient clones from the immature B cell line, WEHI-231, we previously demonstrated that CD45 selectively dephosphorylates the Src-family protein tyrosine kinase Lyn and inhibits its kinase activity. To further define the mechanisms of CD45 action on Lyn, we metabolically labeled Lyn from CD45-positive and -negative WEHI-231 cells and analyzed cyanogen bromide fragments by SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is tyrosine phosphorylated with little serine or threonine phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa were phosphorylated in the absence of B cell Ag receptor (BCR) ligation. The 8.2-kDa band corresponded to a fragment containing the positive regulatory site (Tyr397), as assessed by its size and its phosphorylation in an in vitro kinase assay. The 4.1-kDa band was phosphorylated by COOH-terminal Src kinase, suggesting that it contains the COOH-terminal negative regulatory site (Tyr508). CD45 was also shown to dephosphorylate autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only the negative but also the positive regulatory tyrosine residues of Lyn. Furthermore, coimmunoprecipitations using anti-Igα Ab demonstrated that Lyn associated with the resting BCR was constitutively phosphorylated and activated in CD45-negative cells. In the parental cells, both regulatory sites were phosphorylated on BCR ligation. Taken collectively, these results suggest that CD45 keeps both BCR-associated and total cytoplasmic pools of Lyn in an inactive state, and a mechanism by which Lyn is activated by relative reduction of CD45 effect may be operative on BCR ligation.
Regulation of Human Intestinal Intraepithelial Lymphocyte Cytolytic Function by Biliary Glycoprotein (CD66a)
Authors: Morales Victor M. , Christ Andreas , Watt Suzanne M. , Kim Hyun S. , Johnson Kevin W. , Utku Nalan , Texieira Ana M. , Mizoguchi Atsushi , Mizoguchi Emiko , Russell Gary J. , Russell Sara E. , Bhan Atul K. , Freeman Gordon J. , Blumberg Richard S.
Published: December 2022

Human small intestinal intraepithelial lymphocytes (iIEL) are a unique population of CD8αβ+ TCR-αβ+ but CD28− T lymphocytes that may function in intestinal epithelial cell immunosurveillance. In an attempt to define novel cell surface molecules involved in iIEL function, we raised several mAbs against activated iIELs derived from the small intestine that recognized an Ag on activated, but not resting, iIELs. Using expression cloning and binding studies with Fc fusion proteins and transfectants, the cognate Ag of these mAbs was identified as the N domain of biliary glycoprotein (CD66a), a carcinoembryonic Ag-related molecule that contains an immune receptor tyrosine-based inhibitory motif. Functionally, these mAbs inhibited the anti-CD3-directed and lymphokine-activated killer activity of the P815 cell line by iIELs derived from the human small intestine. These studies indicate that the expression of biliary glycoprotein on activated human iIELs and, potentially, other mucosal T lymphocytes is involved in the down-regulation of cytolytic function.