bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Phase Priming by IL-3 for Leukotriene C4 Generation in Human Basophils: Difference in Characteristics Between Acute and Late Priming Effects
Authors: Miura Katsushi , MacGlashan Donald W.
Published: April 2014

Previous studies have suggested that enhancement of mediator release from human basophils by IL-3 occurs in at least two phases, and the current studies further characterize the signaling changes that accompany these two phases of the basophil in response to IL-3. The test stimulus for these studies was anaphylatoxin split product of C component (C5a), which does not induce leukotriene C4 release without prior IL-3 treatment. Functionally, IL-3 priming occurs after 5 min, disappears by 2 h, and returns by 18 h. In contrast, the kinetics of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK1/2) phosphorylation, induced by IL-3, do not show the second rise by 18 h. The kinetics of cPLA2 and ERK1/2 phosphorylation following stimulation with C5a are the same for cells that were not treated with IL-3 as for those treated for 18 h, i.e., a lag in phosphorylation of cPLA2 and ERK1/2 lasting 30 s before its eventual rise. Previous studies showed that a 5-min treatment with IL-3 induced little change in the C5a-induced cytosolic calcium response, while 24 h of treatment resulted in a marked and sustained cytosolic calcium elevation during the C5a-induced response. The first phase of the IL-3 priming effect (5–15 min of treatment) was unaffected by cycloheximide, while the second phase (18 h) was inhibited. These data suggest that early IL-3 priming results from preconditioning cPLA2, i.e., causing its phosphorylation, while late priming results from a qualitative change in the cytosolic calcium response.
Dominance of IL-12 Over IL-4 in γδ T Cell Differentiation Leads to Default Production of IFN-γ: Failure to Down-Regulate IL-12 Receptor β2-Chain Expression
Authors: Yin Zhinan , Zhang Dong-Hong , Welte Thomas , Bahtiyar Gul , Jung Sungsoo , Liu Lanzhen , Fu Xin-Yuan , Ray Anuradha , Craft Joe
Published: April 2014

γδ T cells secrete Th1- and Th2-like cytokines that help mediate innate and acquired immunity. We have addressed the mechanism whereby murine γδ T cells acquire the capacity to differentially produce such cytokines. Splenic γδ T cells could be polarized into IFN-γ- or IL-4-secreting cells in vitro; however, in contrast to CD4+ αβ T cells, γδ T cells predominantly produced IFN-γ, even in the presence of IL-4, a finding independent of genetic background. Like CD4+ Th1 cells, IFN-γ-producing cells expressed the IL-12 receptor β2-chain after activation in the presence of IL-12; however, unlike Th2 cells, IL-4-primed γδ T cells also expressed this receptor, even in the absence of IFN-γ and despite the presence of the transcription factor GATA-3. IL-12 also induced IL-4-primed γδ T cells to proliferate and to translocate Stat3/Stat4, indicating signaling through the IL-12 receptor. These molecular events can account for the predominant production of IFN-γ by γδ T cells in the presence of IL-12, despite the availability of IL-4. Early and predominant production of IFN-γ by γδ T cells likely is critical for the roles that these cells play in protection against intracellular pathogens and in tumor immunity.
Vigorous Allograft Rejection in the Absence of Danger
Authors: Bingaman Adam W. , Ha Jongwon , Waitze Seung-Yeun , Durham Megan M. , Cho Hong Rae , Tucker-Burden Carol , Hendrix Rose , Cowan Shannon R. , Pearson Thomas C. , Larsen Christian P.
Published: April 2014

Tolerance to self is a necessary attribute of the immune system. It is thought that most autoreactive T cells are deleted in the thymus during the process of negative selection. However, peripheral tolerance mechanisms also exist to prevent development of autoimmune diseases against peripheral self-Ags. It has been proposed that T cells develop tolerance to peripheral self-Ags encountered in the absence of inflammation or “danger” signals. We have used immunodeficient Rag 1−/− mice to study the response of T cells to neo-self peripheral Ags in the form of well-healed skin and vascularized cardiac allografts. In this paper we report that skin and cardiac allografts without evidence of inflammation are vigorously rejected by transferred T cells or when recipients are reconstituted with T cells at a physiologic rate by nude bone graft transplantation. These results provide new insights into the role of inflammation or “danger” in the initiation of T cell-dependent immune responses. These findings also have profound implications in organ transplantation and suggest that in the absence of central deletional tolerance, peripheral tolerance mechanisms are not sufficient to inhibit alloimmune responses even in the absence of inflammation or danger.
Induction of Diabetes in Nonobese Diabetic Mice by Th2 T Cell Clones from a TCR Transgenic Mouse
Authors: Poulin Michelle , Haskins Kathryn
Published: April 2014

We have produced a panel of cloned T cell lines from the BDC-2.5 TCR transgenic (Tg) mouse that exhibit a Th2 cytokine phenotype in vitro but are highly diabetogenic in vivo. Unlike an earlier report in which T cells obtained from the Tg mouse were cultured for 1 wk under Th2-promoting conditions and were found to induce disease only in NOD.scid recipients, we found that long-term T cell clones with a fixed Th2 cytokine profile can transfer disease only to young nonobese diabetic (NOD) mice and never to NOD.scid recipients. Furthermore, the mechanism by which diabetes is transferred by a Tg Th2 T cell clone differs from that of the original CD4+ Th1 BDC-2.5 T cell clone made in this laboratory. Whereas the BDC-2.5 clone rapidly causes disease in NOD.scid recipients less than 2 wk old, the Tg Th2 T cell clones can do so only when cotransferred with other diabetogenic T cells, suggesting that the Th2 T cell requires the presence of host T cells for initiation of disease.
CD8+ T Cell-Dependent Elimination of Dendritic Cells In Vivo Limits the Induction of Antitumor Immunity
Authors: Hermans Ian F. , Ritchie David S. , Yang Jianping , Roberts Joanna M. , Ronchese Franca
Published: April 2014

The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable ∼40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in “memory” mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.
Systematic Analysis of the Role of CD19 Cytoplasmic Tyrosines in Enhancement of Activation in Daudi Human B Cells: Clustering of Phospholipase C and Vav and of Grb2 and Sos with Different CD19 Tyrosines
Authors: Brooks Stephen R. , Li Xiaoli , Volanakis Emmanuel J. , Carter Robert H.
Published: April 2014

CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-γ2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9–12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-γ2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.
Description of an Ectothermic TCR Coreceptor, CD8α, in Rainbow Trout
Authors: Hansen John D. , Strassburger Pamela
Published: April 2014

We have cloned the first CD8α gene from an ectothermic source using a degenerate primer for Ig superfamily V domains. Similar to homologues in higher vertebrates, the rainbow trout CD8α gene encodes a 204-aa mature protein composed of two extracellular domains including an Ig superfamily V domain and hinge region. Differing from mammalian CD8α V domains, lower vertebrate (trout and chicken) sequences do not contain the extra cysteine residue (C strand) involved in the abnormal intrachain disulfide bridging within the CD8α V domain of mice and rats. The trout membrane proximal hinge region contains the two essential cysteine residues involved in CD8 dimerization (αα or αβ) and threonine, serine, and proline residues which may be involved in multiple O-linked glycosylation events. Although the transmembrane region is well conserved in all CD8α sequences analyzed to date, the putative trout cytoplasmic region differs and, in fact, lacks the consensus p56lck motif common to other CD8α sequences. We then determined that the trout CD8α genomic structure is similar to that of humans (six exons) but differs from that of mice (five exons). Additionally, Northern blotting and RT-PCR demonstrate that trout CD8α is expressed at high levels within the thymus and at weaker levels in the spleen, kidney, intestine, and peripheral blood leukocytes. Finally, we show that trout CD8α can be expressed on the surface of cells via transfection. Together, our results demonstrate that the basic structure and expression of CD8α has been maintained for more than 400 million years of evolution.
NKT Cells in the Rat: Organ-Specific Distribution of NK T Cells Expressing Distinct Vα14 Chains
Authors: Matsuura Akihiro , Kinebuchi Miyuki , Chen Hong-Zhi , Katabami Shigeo , Shimizu Tadakazu , Hashimoto Yuji , Kikuchi Kokichi , Sato Noriyuki
Published: April 2014

Rat invariant TCR α-chains and NKT cells were investigated to clarify whether CD1d-mediated recognition by NKT cells is conserved further in evolution. Rats had multiple-copies of TRAV14 genes, which can be categorized into two types according to the diversity accumulated in the CDR2 region. Rats retained invariant TCRα forms with the homogeneous junctional region similar to mouse invariant TRAV14-J281. The proportion of invariant TCR among Vα14+ clones was 12.9% in the thymus and increased in the periphery, 31% in the spleen and 95% in hepatic sinusoidal cells. The invariant TRAV14-J281 was expressed by liver sinusoidal and splenic NKT cells with CD8, CD44high, and TCR Vβ8. Type 1 invariant TCRα was expressed more frequently in hepatic lymphocytes, while type 2 invariant TCRα was expressed predominantly in the spleen. Both types of cells cytolyzed to and were stimulated to proliferate by CD1d-expressing cells in a CD1d-restricted manner. These results suggested that rat NKT cells bearing distinct Vα14 chains are distributed in a tissue-specific pattern. NKT cell populations in rats were more variable than those in mice, indicating that they play novel roles in nature. The implication of the molecular interaction between the structurally diverse invariant TCRα and CD1d/ligand complex in different organs is discussed.
HLA-DR Restricted Peptide Candidates for Bee Venom Immunotherapy
Authors: Texier Catherine , Pouvelle Sandra , Busson Marc , Hervé Mireille , Charron Dominique , Ménez André , Maillère Bernard
Published: April 2014

T cell epitopes containing peptides have been recently proposed as an alternative to conventional immunotherapy of allergic diseases because they are expected to be better tolerated than allergen extracts. A principal limitation to their clinical use is that they present an important diversity, which primarily results from the polymorphism of HLA class II molecules. In Caucasian populations, however, seven alleles of the most expressed molecules (namely DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominate. Peptides from allergens that would efficiently bind to them should be potential candidates for specific immunotherapy. In this paper, we have determined the peptides present in the major bee venom allergen by investigating the capacity of synthetic peptides that encompass its whole sequence to bind to each allele. Several efficient binders have been identified and are either allele-specific or common to several HLA-DR molecules. Interestingly enough, the 81–97 sequence is universal in the sense that it binds to all studied molecules. This sequence is surrounded by several active regions, which make the 76–106 sequence particularly rich of binding determinants and a good candidate for specific immunotherapy. Statistical analyses of the binding data also provide an overview of the preponderant HLA-DR alleles specificity.
Unprecedented Polymorphism of Mhc-DRB Region Configurations in Rhesus Macaques
Authors: Doxiadis Gaby G. M. , Otting Nel , de Groot Natasja G. , Noort Riet , Bontrop Ronald E.
Published: April 2014

The rhesus macaque is an important model in preclinical transplantation research and for the study of chronic and infectious diseases, and so extensive knowledge of its MHC (MhcMamu) is needed. Nucleotide sequencing of exon 2 allowed the detection of 68 Mamu-DRB alleles. Although most alleles belong to loci/lineages that have human equivalents, identical Mhc-DRB alleles are not shared between humans and rhesus macaques. The number of -DRB genes present per haplotype can vary from two to seven in the rhesus macaque, whereas it ranges from one to four in humans. Within a panel of 210 rhesus macaques, 24 Mamu-DRB region configurations can be distinguished differing in the number and composition of loci. None of the Mamu-DRB region configurations has been described for any other species, and only one of them displays major allelic variation giving rise to a total of 33 Mamu-DRB haplotypes. In the human population, only five HLA-DRB region configurations were defined, which in contrast to the rhesus macaque exhibit extensive allelic polymorphism. In comparison with humans, the unprecedented polymorphism of the Mamu-DRB region configurations may reflect an alternative strategy of this primate species to cope with pathogens. Because of the Mamu-DRB diversity, nonhuman primate colonies used for immunological research should be thoroughly typed to facilitate proper interpretation of results. This approach will minimize as well the number of animals necessary to conduct experiments.
TGF-β2 Reduces Demyelination, Virus Antigen Expression, and Macrophage Recruitment in a Viral Model of Multiple Sclerosis
Authors: Drescher Kristen M. , Murray Paul D. , Lin Xiaoqi , Carlino Joseph A. , Rodriguez Moses
Published: April 2014

TGF-β2 is a potent immunoregulatory mediator that influences B cell, T cell, and macrophage function. To test whether this cytokine alters pathology in a model of virus-induced demyelinating disease, we treated SJL/J mice with TGF-β2 either before or after infection with Theiler’s murine encephalomyelitis virus. Treatment continued three times weekly through day 35 postinfection. TGF-β2 administration resulted in significantly smaller lesions and fewer virus Ag-positive cells in the spinal cords of infected SJL/J mice. Mice treated with TGF-β2 had similar levels of virus-specific IgG as infected, control-treated mice. TGF-β2 administration significantly increased the level of non-virus-specific activated CTLs, but had no effect on virus-specific CTLs. TUNEL revealed a decrease in the number of apoptotic nuclei in the spinal cord white matter of mice treated in vivo with TGF-β2. Immunostaining with an Ab to F4/80 revealed that TGF-β2-treated mice had significantly fewer F4/80-positive cells in the white matter of the spinal cord as compared with infected control-treated mice. These data suggest that TGF-β2 may control virus-induced demyelination via an immunomodulatory mechanism that reduces macrophage infiltration.
Blockade of CD14 Increases Shigella-Mediated Invasion and Tissue Destruction
Authors: Wennerås Christine , Ave Patrick , Huerre Michel , Arondel Josette , Ulevitch Richard J. , Mathison John C. , Sansonetti Philippe
Published: April 2014

Shigella is a diarrheal pathogen that causes disease through invasion of the large intestinal mucosa. The endotoxin of the invading bacterium may play a key role in the disease process by causing inflammation and tissue injury during infection. Earlier studies have shown that various animal species lacking functional CD14 were protected against endotoxin-mediated shock. Rabbits experimentally infected with Shigella were used to test the hypothesis that blockade of endotoxin-induced cell activation with anti-CD14 mAb would diminish inflammation and thus disease severity. Unexpectedly, we observed that the intestinal mucosa of anti-CD14-treated animals exhibited a 50-fold increase in bacterial invasion and more severe tissue injury compared with controls. Despite higher bacterial loads in treated animals, the numbers of polymorphonuclear leukocytes that were recruited to the infection site were similar to those in controls. Furthermore, the phagocytic cells of CD14-blocked animals produced IL-1 and TNF-α. Moreover, in vitro blockade of CD14 did not impede bactericidal activity. Thus, anti-CD14 treatment interfered with host defense mechanisms involved with removal/eradication of Shigella.
p38 Mitogen-Activated Protein Kinase and c-Jun-NH2-Terminal Kinase Regulate RANTES Production by Influenza Virus-Infected Human Bronchial Epithelial Cells
Authors: Kujime Kousei , Hashimoto Shu , Gon Yasuhiro , Shimizu Kazufumi , Horie Takashi
Published: April 2014

Airway epithelial cells which are the initial site of influenza virus (IV) infection are suggested to participate in airway inflammatory response by expressing various cytokines including RANTES; however, the intracellular signal that regulates RANTES expression has not been determined. In the present study, we examined the role of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and c-Jun-NH2-terminal kinase (JNK) in RANTES production by IV-infected human bronchial epithelial cells. The results showed that IV infection induced increases in p38 MAP kinase, and Erk and JNK phosphorylation and activity. SB 203580, PD 98059, and CEP-1347 attenuated IV-infection induced p38 MAP kinase activity, Erk activity, and JNK activity, respectively. SB 203580 and CEP-1347 attenuated RANTES production by 45.3% and 45.2%, respectively, but a combination of these inhibitors additively attenuated by 69.1%. In contrast, PD 98059 did not attenuate. Anti-IL-1α mAb, anti-IL-1β mAb, anti-TNF-α mAb, anti-IL-8 mAb, anti-IFN-β mAb, anti-RANTES mAb, and a combination of these mAbs did not affect IV infection-induced increases in p38 MAP kinase, Erk, and JNK phosphorylation, indicating that each cytokine neutralized by corresponding Ab was not involved in IV infection-induced phosphorylation of MAP kinases. N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. These results indicate that p38 MAP kinase and JNK, at least in part, regulate RANTES production by bronchial epithelial cells.
Superfibronectin, a Multimeric Form of Fibronectin, Increases HIV Infection of Primary CD4+ T Lymphocytes
Authors: Tellier Marinka C. , Greco Giampaolo , Klotman Mary , Mosoian Arevik , Cara Andrea , Arap Wadih , Ruoslahti Erkki , Pasqualini Renata , Schnapp Lynn M.
Published: April 2014

The ability of viruses and bacteria to interact with the extracellular matrix plays an important role in their infectivity and pathogenicity. Fibronectin is a major component of the extracellular matrix in lymph node tissue, the main site of HIV deposition and replication during the chronic phase of infection. Therefore, we asked whether matrix fibronectin (FN) could affect the ability of HIV to infect lymphocytes. To study the role of matrix FN on HIV infection, we used superfibronectin (sFN), a multimeric form of FN that closely resembles in vivo matrix FN. In this study we show that HIV-1IIIB efficiently binds to multimeric fibronectin (sFN) and that HIV infection of primary CD4+ lymphocytes is enhanced by >1 order of magnitude in the presence of sFN. This increase appears to be due to increased adhesion of viral particles to the cell surface in the presence of sFN, followed by internalization of virus. Enzymatic removal of cell surface proteoglycans inhibited the adhesion of HIV-1IIIB/sFN complexes to lymphocytes. In contrast, Abs to integrins had no effect on binding of HIV-1IIIB/sFN complexes to lymphocytes. The III1-C peptide alone also bound HIV-1IIIB efficiently and enhanced HIV infection, although not as effectively as sFN. HIV-1IIIB gp120 envelope protein binds to the III1-C region of sFN and may be important in the interaction of virus with matrix FN. We conclude that HIV-1IIIB specifically interacts with the III1-C region within matrix FN, and that this interaction may play a role in facilitating HIV infection in vivo, particularly in lymph node tissue.
A Poxvirus Protein That Binds to and Inactivates IL-18, and Inhibits NK Cell Response
Authors: Born Teresa L. , Morrison Lynda A. , Esteban David J. , VandenBos Tim , Thebeau Lydia G. , Chen Nanhai , Spriggs Melanie K. , Sims John E. , Buller R. Mark L.
Published: April 2014

IL-18 induces IFN-γ and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R. The dissociation constant of p13 for murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectromelia evades the host immune response.
CD14 Employs Hydrophilic Regions to “Capture” Lipopolysaccharides
Authors: Cunningham Mark D. , Shapiro Robert A. , Seachord Carrie , Ratcliffe Kent , Cassiano Linda , Darveau Richard P.
Published: April 2014

CD14 participates in the host innate inflammatory response to bacterial LPS obtained from Escherichia coli and other Gram-negative bacteria. Evidence from several laboratories suggests that different regions of the amino-terminal portion of the molecule may be involved in LPS binding. In this report a series of single-residue serine replacement and charge reversal mutations were generated to further elucidate the mechanism by which this protein may bind a multitude of different LPS ligands. Single-residue CD14 mutation proteins were examined for their ability to bind LPS obtained from E. coli, Porphyromonas gingivalis, and Helicobacter pylori and facilitate the activation of E-selectin from human endothelial cells. In addition, the single-residue CD14 mutation proteins were employed to perform monoclonal epitope-mapping studies with three LPS-blocking Abs that bound tertiary epitopes. Evidence that several different hydrophilic regions of the amino-terminal region of CD14 are involved in LPS binding was obtained. Epitope-mapping studies revealed that these hydrophilic regions are located on one side of the protein surface. These studies suggest that CD14 employs a charged surface in a manor similar to the macrophage scavenger receptor to “capture” LPS ligands and “present” them to other components of the innate host defense system.
Role of CCAAT/Enhancer-Binding Protein Site in Transcription of Human Neutrophil Peptide-1 and -3 Defensin Genes
Authors: Tsutsumi-Ishii Yuko , Hasebe Takeshi , Nagaoka Isao
Published: April 2014

The human neutrophil defensins (human neutrophil peptides (HNPs)), major components of azurophilic granules, contribute to innate and acquired host immunities through their potent antimicrobial activities and ability to activate T cells. Despite being encoded by nearly identical genes, HNP-1 is more abundant in the granules than HNP-3. We investigated the regulation of HNP-1 and HNP-3 expression at the transcriptional level using a promyelocytic HL-60 cell line. Luciferase analysis showed that transcriptional levels of HNP-1 and HNP-3 promoters were equivalent and that an ∼200-bp region identical between promoters was sufficient for transcriptional activity. Furthermore, overlapping CCAAT/enhancer-binding protein (C/EBP) and c-Myb sites in the region were found to be required for efficient transcription. Gel mobility shift assay demonstrated that C/EBPα predominantly bound to the C/EBP/c-Myb sites using HL-60 nuclear extracts. No specific binding to C/EBP/c-Myb sites was observed in nuclear extracts from mature neutrophils, which expressed neither C/EBPα protein nor HNP mRNAs. Taken together, these findings suggest that the difference in the amounts of HNP-1 and HNP-3 peptides in neutrophils is caused by posttranscriptional regulation and that C/EBPα plays an important role in the transcription of HNP genes in immature myeloid cells.
Chemokines Fail to Up-Regulate β1 Integrin-Dependent Adhesion in Human Th2 T Lymphocytes
Authors: Clissi Barbara , D’Ambrosio Daniele , Geginat Jens , Colantonio Lucia , Morrot Alexander , Freshney Norman W. , Downward Julian , Sinigaglia Francesco , Pardi Ruggero
Published: April 2014

Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of P- and E-selectin ligands and selected chemokine receptors. Here we investigated the expression and function of β1 integrins in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands laminin 1 and fibronectin in response to chemokines such as RANTES and stromal cell-derived factor-1, and this process is paralleled by the activation of the Rac1 GTPase and by a rapid burst of actin polymerization. Selective inhibitors of phosphoinositide-3 kinase prevent efficiently all of the above processes, whereas the protein kinase C inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting Rac1 activation and actin polymerization. Notably, chemokine-induced adhesion to β1 integrin ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving phosphoinositide-3 kinase, Rac1, and actin turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced β1 integrin-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.
Leukocyte Arrest During Cytokine-Dependent Inflammation In Vivo
Authors: Kunkel Eric J. , Dunne Jessica L. , Ley Klaus
Published: April 2014

Leukocyte rolling along the walls of inflamed venules precedes their adhesion during inflammation. Rolling leukocytes are thought to arrest by engaging β2 integrins following cellular activation. In vitro studies suggest that chemoattractants may instantaneously activate and arrest rolling leukocytes. However, how leukocytes stop rolling and become adherent in inflamed venules in vivo has remained rather mysterious. In this paper we use a novel method of tracking individual leukocytes through the microcirculation to show that rolling neutrophils become progressively activated while rolling down the venular tree. On average, leukocytes in wild-type mice roll for 86 s (and cover 270 μm) before becoming adherent with an efficiency around 90%. These rolling leukocytes exhibit a gradual β2 integrin-dependent decrease in rolling velocity that correlates with an increase in intracellular free calcium concentration before arrest. Similar tracking analyses in gene-targeted mice demonstrate that the arrest of rolling leukocytes is very rare when β2 integrins are absent or blocked by a mAb. Arrest is ∼50% less efficient in the absence of E-selectin. These data suggest a model of leukocyte recruitment in which β2 integrins play a critical role in stabilizing leukocyte rolling during a protracted cellular activation period before arrest and firm adhesion.
IFN-γ-Dependent and -Independent Mechanisms in Adverse Effects Caused by Concomitant Administration of IL-18 and IL-12
Authors: Nakamura Shuji , Otani Takeshi , Ijiri Yoshihiro , Motoda Ryuichi , Kurimoto Masashi , Orita Kunzo
Published: April 2014

IL-18 is a new type of inflammatory cytokine similar to but distinct from IL-12 and IL-1β. One intriguing property of IL-18 is synergism with IL-12 in many respects. In this study we examined the in vivo synergistic effects of IL-18/IL-12 in mice and found lethal toxicity accompanying an elevated IFN-γ level in the serum. Since treatment with IL-18 alone did not have any apparent toxicity, and treatment with IL-12 alone showed only limited toxicity in our system, the synergy between the two cytokines was all the more remarkable. The major symptoms of the toxicity were weight loss, diarrhea, hemorrhagic colitis, splenomegaly, fatty liver, and atrophic thymus, most of which are similarly found in endotoxin-induced septic shock. However, in contrast to septic shock, TNF-α was not induced. The involvement of IFN-γ in the toxicity was further studied in detail. Treatment of athymic nude mice with anti-asialo-GM1 did not reduce the toxicity, whereas anti-IFN-γ treatment of wild-type mice alleviated it. When IFN-γ-deficient mice were treated with IL-18/IL-12, the majority of them showed mortality and toxicity with severe pulmonary edema. These results indicate that IL-18/IL-12 treatment induces severe adverse effects through not only IFN-γ-dependent mechanisms but also IFN-γ-independent processes.
α4 Integrin-Dependent Leukocyte Recruitment Does Not Require VCAM-1 in a Chronic Model of Inflammation
Authors: Johnston Brent , Chee Alex , Issekutz Thomas B. , Ugarova Tatiana , Fox-Robichaud Alison , Hickey Michael J. , Kubes Paul
Published: April 2014

Rats immunized with Mycobacterium butyricum in Freund’s adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an α4 integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that α4 integrin-dependent leukocyte rolling and adhesion was inhibited with a β1 integrin, but not a β7 integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for α4β1 integrin in the vasculature. However, α4β1 integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature. To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient α4 integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95–100%. Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via α4 integrin. Another ligand for α4β1 integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that α4β1 integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.
The Coupling of 5-Oxo-Eicosanoid Receptors to Heterotrimeric G Proteins
Authors: O’Flaherty Joseph T. , Taylor Jennifer S. , Kuroki Mitsuyuki
Published: April 2014

5-Oxo-eicosatetraenoic acid (5-oxoETE) stimulated human neutrophil (PMN) and eosinophil chemotaxis, PMN hexose uptake, and PMN membrane GTP/GDP exchange. Pertussis toxin (PT), a blocker of heterotrimeric G proteins (GP), completely inhibited these responses, but proved far less effective on the same responses when elicited by leukotriene B4, C5a, FMLP, platelet-activating factor, IL-8, or RANTES chemotactic factors. 5-OxoETE also specifically bound to the membrane preparations that conducted GTP/GDP exchange. This binding was down-regulated by GTPγS, but not ADPγS, and displaced by 5-oxoETE analogues, but not by leukotriene B4, lipoxin A4, or lipoxin B4. Finally, PMN expressed PT-sensitive GP αι2 and PT-resistant GP αq/11- and α13-chains; eosinophils expressed only αi2 and αq/11. We conclude that 5-oxoETE activates granulocytes through a unique receptor that couples preferentially to PT-sensitive GP. The strict dependency of this putative receptor on PT-sensitive GP may underlie the limited actions of 5-oxoETE, compared with other CF, and help clarify the complex relations between receptors, GP, cell signals, and cell responses.
Expression and Activation of NF-κB in the Antrum of the Human Stomach
Authors: van den Brink Gijs R. , ten Kate Fiebo J. , Ponsioen Cyriel Y. , Rive Maaike M. , Tytgat Guido N. , van Deventer Sander J. H. , Peppelenbosch Maikel P.
Published: April 2014

Both in vitro studies and experiments in mice suggest a key role for transcription factor NF-κB as a mediator of mucosal inflammation. Experiments in vitro show that NF-κB activation may be a critical event in the production of proinflammatory molecules in Helicobacter pylori-associated gastritis. This study examines the expression and activity of NF-κB in situ in antral biopsies of 69 consecutive patients with immunohistochemical techniques. In the uninflamed stomach, NF-κB was highly expressed and active in a subset of epithelial cells, which were identified as predominantly G cells. In accordance with this activity, G cells were shown to express high levels of the NF-κB target cytokine TNF-α, a well-documented stimulator of gastrin production. In patients with H. pylori-associated gastritis, NF-κB activity was markedly enhanced. Activation occurred preferentially in the epithelial cells. The number of cells showing activated NF-κB correlated with the activity of gastritis, a measure of neutrophil influx, whereas no correlation was found with the chronicity of inflammation, a measure of the presence of mononuclear inflammatory cells. This correlation is direct evidence of the importance of NF-κB-dependent signal transduction for neutrophil influx in H. pylori-associated gastritis.
CD4+ T Cell and Eosinophil Adhesion Is Mediated by Specific ICAM-3 Ligation and Results in Eosinophil Activation
Authors: Douglas Ivor S. , Leff Alan R. , Sperling Anne I.
Published: April 2014

T cells and eosinophils, which are found in close proximity in asthmatic lungs, express many surface receptors that are counterligands. These data suggest that direct interactions between these cell types could play an important role in regulating airway inflammation in asthma. We examined the effect of selective adhesion between counterligands on human eosinophils and CD4+ T cells to determine 1) the existence of specific adhesive interactions and 2) if augmented specific adhesion to CD4+ T cells also caused augmented secretion of leukotriene C4 (LTC4) from eosinophils. A new method for binding of human CD4+ T cells to microwell plates was developed, which allowed for specific quantitative assessment of eosinophil adhesion to individual CD4+ T cells in culture. Adhesion of CD4+ T cells to eosinophils was minimal in unstimulated cells but increased after activation of T cells by PMA. Augmented adhesion was regulated substantially through binding of ICAM-3 and only minimally by ICAM-1. We further evaluated whether this specific adhesion up-regulated stimulated secretion of LTC4 from eosinophils. Adhesion with CD4+ T cells augmented eosinophil secretion of LTC4 caused by FMLP plus cytochalasin. Blockade of ICAM-3, as well as ICAM-1, inhibited completely the augmented secretion of eosinophil LTC4. We demonstrate that eosinophils and CD4+ T cells are capable of ligand-specific adhesion that is mediated predominantly by ICAM-3 ligation and that this binding causes augmented eosinophil secretion.
Cutaneous Injection of Human Subjects with Macrophage Inflammatory Protein-1α Induces Significant Recruitment of Neutrophils and Monocytes
Authors: Lee Sang Chin , Brummet Mary E. , Shahabuddin Syed , Woodworth Thasia G. , Georas Steve N. , Leiferman Kristin M. , Gilman Steven C. , Stellato Cristiana , Gladue Ron P. , Schleimer Robert P. , Beck Lisa A.
Published: April 2014

Macrophage inflammatory protein (MIP-1α), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1α have been well documented, the in vivo biological activities of MIP-1α in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1α and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10–24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1α stimulation. These studies substantiate the biological effects of MIP-1α on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1α to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.
Genetic Analysis of the Influence of Pertussis Toxin on Experimental Allergic Encephalomyelitis Susceptibility: An Environmental Agent Can Override Genetic Checkpoints
Authors: Blankenhorn Elizabeth P. , Butterfield Russell J. , Rigby Robert , Cort Laura , Giambrone Dana , McDermott Paul , McEntee Kay , Solowski Nancy , Meeker Nathan D. , Zachary James F. , Doerge Rebecca W. , Teuscher Cory
Published: April 2014

Pertussis toxin (PTX) is a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental allergic encephalomyelitis (EAE). To delineate the genetics of PTX effect in EAE, we mapped EAE-modifying (eae-m) loci in cohorts of backcross mice immunized with and without PTX. In this study, we analyzed the genetic basis of EAE susceptibility and severity and the intermediate phenotypes of mononuclear cell infiltration, suppuration, and demyelination. In animals immunized with PTX, one major locus, eae9, controls disease susceptibility and severity. Eae9 also regulates the extent of mononuclear cell infiltration of the spinal cord in male mice. Without PTX, five eae-m loci were noted, including three new loci in intervals on chromosomes 8 (eae14), 10 (eae17), and 18 (eae18). Taken together, these results suggest that eae9 controls the effects of PTX in EAE susceptibility, and is capable of overriding the other genetic checkpoints in the pathogenesis of this disease.
CCR5-Reactive Antibodies in Seronegative Partners of HIV-Seropositive Individuals Down-Modulate Surface CCR5 In Vivo and Neutralize the Infectivity of R5 Strains of HIV-1 In Vitro
Authors: Lopalco Lucia , Barassi Claudia , Pastori Claudia , Longhi Renato , Burastero Samuele E. , Tambussi Giuseppe , Mazzotta Francesco , Lazzarin Adriano , Clerici Mario , Siccardi Antonio G.
Published: April 2014

Exposure to HIV does not necessarily results in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1β (Mip1β) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1β binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89–102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1β-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1β in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.
Blockade of CD28-B7, But Not CD40-CD154, Prevents Costimulation of Allogeneic Porcine and Xenogeneic Human Anti-Porcine T Cell Responses
Authors: Lee Richard S. , Yamada Kazuhiko , Womer Karl L. , Pillsbury Edmund P. , Allison Kenneth S. , Marolewski Ariane E. , Geng Dong , Thall Aron D. , Arn J. Scott , Sachs David H. , Sayegh Mohamed H. , Madsen Joren C.
Published: April 2014

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 μg/ml) was considerably more inhibitory than anti-CD154 mAb (100 μg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-γ by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-γ and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.
Cutting Edge: Activation of HIV-1 Transcription by the MHC Class II Transactivator
Authors: Saifuddin Mohammed , Roebuck Kenneth A. , Chang Cheong-hee , Ting Jenny P. Y. , Spear Gregory T.
Published: April 2014

Both macrophages and activated CD4+ T cells can be productively infected by HIV-1, and both cell types express MHC class II molecules. Expression of MHC class II proteins in these cells is regulated by a specific transcriptional coactivator, the class II transactivator (CIITA). In this study, we report for the first time that CIITA expression profoundly influences HIV-1 replication. Stable expression of CIITA in Jurkat cells markedly increased 1) HIV-1 replication as assessed by the p24 Ag production and 2) luciferase expression after transfection with full-length provirus or long terminal repeat constructs. Similarly, transient expression of CIITA increased provirus expression as well as long terminal repeat promoter activity in 293 and HeLa-T4 cells. In contrast, mutant forms of CIITA did not increase HIV-1 expression. This study shows that expression of CIITA increases HIV-1 replication through a transcriptional mechanism.