bluuebunny/crossref_metadata_2025 huggingface dataset:
NK1.1+ T Cells in the Liver Arise in the Thymus and Are Selected by Interactions with Class I Molecules on CD4+CD8+ Cells
Authors: Coles Mark C. , Raulet David H.
Published: April 2014

NK1.1+ T cells represent a specialized T cell subset specific for CD1d, a nonclassical MHC class I-restricting element. They are believed to function as regulatory T cells. NK1.1+ T cell development depends on interactions with CD1d molecules presented by hematopoietic cells rather than thymic epithelial cells. NK1.1+ T cells are found in the thymus as well as in peripheral organs such as the liver, spleen, and bone marrow. The site of development of peripheral NK1.1+ T cells is controversial, as is the nature of the CD1d-expressing cell that selects them. With the use of nude mice, thymectomized mice reconstituted with fetal liver cells, and thymus-grafted mice, we provide direct evidence that NK1.1+ T cells in the liver are thymus dependent and can arise in the thymus from fetal liver precursor cells. We show that the class I+ (CD1d+) cell type necessary to select NK1.1+ T cells can originate from TCRα−/− precursors but not from TCRβ−/− precursors, indicating that the selecting cell is a CD4+CD8+ thymocyte. 5-Bromo-2′-deoxyuridine-labeling experiments suggest that the thymic NK1.1+ T cell population arises from proliferating precursor cells, but is a mostly sessile population that turns over very slowly. Since liver NK1.1+ T cells incorporate 5-bromo-2′-deoxyuridine more rapidly than thymic NK1.1+ T cells, it appears that liver NK1.1+ T cells either represent a subset of thymic NK1.1+ T cells or are induced to proliferate after having left the thymus. The results indicate that NK1.1+ T cells, like conventional T cells, arise in the thymus where they are selected by interactions with restricting molecules.
NOD/LtSz-Rag1nullMice: An Immunodeficient and Radioresistant Model for Engraftment of Human Hematolymphoid Cells, HIV Infection, and Adoptive Transfer of NOD Mouse Diabetogenic T Cells
Authors: Shultz Leonard D. , Lang Pamela A. , Christianson Sherri W. , Gott Bruce , Lyons Bonnie , Umeda Syuji , Leiter Edward , Hesselton RuthAnn , Wagar Eric J. , Leif Jean H. , Kollet Órit , Lapidot Tsvee , Greiner Dale L.
Published: April 2014

Development of a small animal model for the in vivo study of human immunity and infectious disease remains an important goal, particularly for investigations of HIV vaccine development. NOD/Lt mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation readily support engraftment with high levels of human hematolymphoid cells. However, NOD/LtSz-scid mice are highly radiosensitive, have short life spans, and a small number develop functional lymphocytes with age. To overcome these limitations, we have backcrossed the null allele of the recombination-activating gene (Rag1) for 10 generations onto the NOD/LtSz strain background. Mice deficient in RAG1 activity are unable to initiate V(D)J recombination in Ig and TCR genes and lack functional T and B lymphocytes. NOD/LtSz-Rag1null mice have an increased mean life span compared with NOD/LtSz-scid mice due to a later onset of lymphoma development, are radioresistant, and lack serum Ig throughout life. NOD/LtSz-Rag1null mice were devoid of mature T or B cells. Cytotoxic assays demonstrated low NK cell activity. NOD/LtSz-Rag1null mice supported high levels of engraftment with human lymphoid cells and human hemopoietic stem cells. The engrafted human T cells were readily infected with HIV. Finally, NOD/LtSz-Rag1null recipients of adoptively transferred spleen cells from diabetic NOD/Lt+/+ mice rapidly developed diabetes. These data demonstrate the advantages of NOD/LtSz-Rag1null mice as a radiation and lymphoma-resistant model for long-term analyses of engrafted human hematolymphoid cells or diabetogenic NOD lymphoid cells.
Lymphotoxin-α-Dependent Spleen Microenvironment Supports the Generation of Memory B Cells and Is Required for Their Subsequent Antigen-Induced Activation
Authors: Fu Yang-Xin , Huang Guangming , Wang Yang , Chaplin David D.
Published: April 2014

Lymphotoxin α-deficient (LTα−/−) mice show dramatically reduced IgG responses after either primary or secondary immunizations with sheep red blood cells (SRBC). When splenocytes from SRBC-primed wild-type donor mice were infused into irradiated naive wild-type recipient mice, they generated a robust memory IgG response, but not when infused into LTα−/− recipients, indicating that the microenvironment that develops in LTα−/− mice is incompetent to support the activation of this memory response. When irradiated wild-type mice were reconstituted with splenocytes from primed LTα−/− donors and then challenged with the same immunizing Ag, no memory response was observed, indicating further that memory cells could not be generated in the LTα−/− environment. To address which lymphocyte subsets were impaired in the LTα−/− mice, we performed reconstitution experiments using a hapten/carrier system and T cells and B cells from different primed donors. There was no detectable defect in either the generation or expression of memory T cells from LTα−/− donors. In contrast, B cells were not primed for memory in the microenvironment of LTα−/− mice. Additionally, primed wild-type memory B cells could not express a memory IgG response in the LTα−/− microenvironment. Thus, splenic white pulp structure, which depends on the expression of LTα for its development and maintenance, is needed to support the generation of memory B cells and to permit existing memory B cells to express an isotype switched memory Ig response following antigenic challenge.
IL-12-Independent Th1-Type Immune Responses to Respiratory Viral Infection: Requirement of IL-18 for IFN-γ Release in the Lung But Not for the Differentiation of Viral-Reactive Th1-Type Lymphocytes
Authors: Xing Zhou , Zganiacz Anna , Wang Jun , Divangahi Maziar , Nawaz Fauzia
Published: April 2014

We demonstrated that IL-12 was induced during primary or secondary pulmonary adenoviral infection in wild-type (wt) mice. However, cellular responses were not compromised in the lungs of IL-12−/− mice. The level of IFN-γ in the lung was similar in wt and IL-12−/− mice during pulmonary viral infection. Upon Ag stimulation in vitro, lymphocytes from draining lymph nodes or spleen of infected IL-12−/− mice released large amounts of IFN-γ, but not IL-4, which were comparable to those released by wt lymphocytes. Furthermore, a predominantly IgG2a response to adenoviral infection was unimpaired in IL-12−/− mice. These significant anti-adenoviral Th1-type responses in IL-12−/− mice led to an efficient clearance of virus-infected cells in the lung. Whether IL-18 was involved in IL-12-independent anti-adenoviral immune responses was investigated. Abrogation of endogenous IL-18 by an Ab resulted in diminished IFN-γ release and lymphocytic infiltrate in the lung during adenoviral infection. Nevertheless, the development of lymphocytes of the Th1 phenotype was unimpaired in the absence of both IL-12 and IL-18. In contrast to their intact ability to mount Th1-type responses to viral infection, IL-12−/− mice suffered impaired Th1-type immune responses to pulmonary mycobacterial infection. Our findings suggest that IL-12, although induced, is not required for Th1-type responses to respiratory viral infection, in contrast to mycobacterial infection. IL-18 is required for the optimal release of IFN-γ in the lung during viral infection, but is not required for the generation of virus-reactive Th1-type lymphocytes. Th1 differentiation during respiratory adenoviral infection may involve molecules different from IL-12 or IL-18.
Schistosome Infection of Transgenic Mice Defines Distinct and Contrasting Pathogenic Roles for IL-4 and IL-13: IL-13 Is a Profibrotic Agent
Authors: Fallon Padraic G. , Richardson Emma J. , McKenzie Grahame J. , McKenzie Andrew N. J.
Published: April 2014

Experimental Schistosoma mansoni infections of mice lead to a dynamic type 2 cytokine-mediated pathological process. We have used IL-4-deficient, IL-13-deficient, and IL-4/13-deficient mice to dissect the role of these cytokines in the development of immune response and pathology following S. mansoni infection. We demonstrate that while both of these cytokines are necessary to develop a robust Th2 cell-driven, eosinophil-rich granuloma response, they also perform disparate functions that identify novel sites for therapeutic intervention. IL-13-deficient mice demonstrated significantly enhanced survival following infection, which correlated with reduced hepatic fibrosis. In contrast, increased mortality was manifest in IL-4-deficient and IL-4/13-deficient mice, and this correlated with hepatocyte damage and intestinal pathology. Therefore, we demonstrate that during a dynamic type 2 cytokine disease process IL-13 is detrimental to survival following infection, whereas IL-4 is beneficial.
Lipopolysaccharide Inhibits HIV-1 Infection of Monocyte- Derived Macrophages Through Direct and Sustained Down-Regulation of CC Chemokine Receptor 5
Authors: Franchin Giovanni , Zybarth Gabriele , Dai Wei Wei , Dubrovsky Larisa , Reiling Norbert , Schmidtmayerova Helena , Bukrinsky Michael , Sherry Barbara
Published: April 2014

It is now well established that HIV-1 requires interactions with both CD4 and a chemokine receptor on the host cell surface for efficient infection. The expression of the CCR5 chemokine receptor in human macrophages facilitates HIV-1 entry into these cells, which are considered important in HIV pathogenesis not only as viral reservoirs but also as modulators of altered inflammatory function in HIV disease and AIDS. LPS, a principal constituent of Gram-negative bacterial cell walls, is a potent stimulator of macrophages and has been shown to inhibit HIV infection in this population. We now present evidence that one mechanism by which LPS mediates its inhibitory effect on HIV-1 infection is through a direct and unusually sustained down-regulation of cell-surface CCR5 expression. This LPS-mediated down-regulation of CCR5 expression was independent of de novo protein synthesis and differed from the rapid turnover of these chemokine receptors observed in response to two natural ligands, macrophage-inflammatory protein-1α and -1β. LPS did not act by down-regulating CCR5 mRNA (mRNA levels actually increased slightly after LPS treatment) or by enhancing the degradation of internalized receptor. Rather, the observed failure of LPS-treated macrophages to rapidly restore CCR5 expression at the cell-surface appeared to result from altered recycling of chemokine receptors. Taken together, our results suggest a novel pathway of CCR5 recycling in LPS-stimulated human macrophages that might be targeted to control HIV-1 infection.
Decreased Resistance of B Cell-Deficient Mice to Infection with Toxoplasma gondii Despite Unimpaired Expression of IFN-γ, TNF-α, and Inducible Nitric Oxide Synthase
Authors: Kang Hoil , Remington Jack S. , Suzuki Yasuhiro
Published: April 2014

The role of B cells in resistance against Toxoplasma gondii was studied using B cell-deficient (μMT) mice. Following peroral infection with 10 cysts of the ME49 strain, all μMT mice survived the acute stage of the infection but died between 3 and 4 wk after infection. In contrast, all control mice were alive at 8 wk after infection. At the stage during which μMT animals succumbed to the infection, parasite replication and pathology were most evident in their brains; small numbers of tachyzoites were also detectable in their lungs. Significantly greater numbers of T. gondii cysts and areas of inflammation associated with tachyzoites were observed in brains of μMT than in control mice. Large areas of necrosis associated with numerous tachyzoites were observed only in brains of μMT mice. Anti-T. gondii IgG Abs were detected only in sera of control mice, whereas similar levels of IFN-γ were detected in sera of both strains of mice. Amounts of mRNA for IFN-γ, IL-10, and inducible NO synthase in the brain did not differ between infected μMT and control mice. Expression of mRNA for TNF-α was increased in brains of μMT mice. Administration of polyclonal rabbit anti-T. gondii IgG Ab prevented early mortality and pathology associated with tachyzoites in the brain in the infected μMT mice. These results indicate that B cells play an important role, most likely through their production of specific Abs, in resistance to persistent active (tachyzoite) infection with T. gondii in mice, especially in the brain and lung.
Th Cell-Deficient Mice Control Influenza Virus Infection More Effectively Than Th- and B Cell-Deficient Mice: Evidence for a Th-Independent Contribution by B Cells to Virus Clearance
Authors: Mozdzanowska Krystyna , Maiese Krista , Gerhard Walter
Published: April 2014

The notion that MHC class I- restricted CD8+ T (Tc) cells are capable of resolving autonomously infections with influenza virus is based largely on studies testing virus strains of low pathogenicity in CD4+ T (Th) cell-deficient/depleted mice. To test whether this holds also for pathogenic strains and to exclude possible contributions by B cells, we analyzed PR8 infection in Th cell-depleted B cell-deficient (μMT) mice. These mice, termed μMT (−CD4), showed 80% mortality after infection with a small dose of PR8, which resulted in insignificant mortality in intact or Th cell-depleted BALB/c mice. Infection of μMT(−CD4) mice with a virus of low pathogenicity was resolved without mortality, but, compared with intact BALB/c mice, with delay of ∼5 and ∼20 days from lung and nose, respectively. The low mortality of Th cell-depleted BALB/c mice suggested that B cells contributed to recovery in a Th-independent manner. This was verified by showing that transfer of 8–10 million T cell-depleted naive spleen cells into μMT(−CD4) mice 1 day before infection reduced mortality to 0%. The mechanism by which B cells improved recovery was investigated. We found no evidence that they operated by improving the lung-associated Tc response. Treatment of infected μMT(−CD4) mice with normal mouse serum spiked with hemagglutinin-specific IgM did not reduce mortality. Taken together, the data show that 1) the Tc response is capable of resolving autonomously (in conjunction with innate defenses) influenza virus infections, although with substantial delay compared with intact mice, and 2) B cells can contribute to recovery by a Th-independent mechanism.
Neutralization of IL-18 Reduces Neutrophil Tissue Accumulation and Protects Mice Against Lethal Escherichia coli and Salmonella typhimurium Endotoxemia
Authors: Netea Mihai G. , Fantuzzi Giamila , Kullberg Bart Jan , Stuyt Rogier J. L. , Pulido Edward J. , McIntyre Robert C. , Joosten Leo A. B. , Van der Meer Jos W. M. , Dinarello Charles A.
Published: April 2014

In addition to stimulating IFN-γ synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1β and the chemokines IL-8 and macrophage inflammatory protein-1α. We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice. IL-1β converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1β, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium. In contrast, both wild-type and IL-1β−/− mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-γ but not IL-1β in ICE−/− mice is responsible for this resistance. However, IFN-γ-deficient mice were not resistant to S. typhimurium LPS, suggesting an IFN-γ-independent role for IL-18. Anti-IL-18 Abs protected mice against a lethal injection of either LPS. Anti-IL-18 treatment also reduced neutrophil accumulation in liver and lungs. The increased survival was accompanied by decreased levels of IFN-γ and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E. coli LPS, whereas IFN-γ and TNF concentrations were decreased in treated mice challenged with S. typhimurium. In conclusion, neutralization of IL-18 during lethal endotoxemia protects mice against lethal effects of LPS. This protection is partly mediated through inhibition of IFN-γ production, but mechanisms involving decreased neutrophil-mediated tissue damage due to the reduction of either chemokines (E. coli LPS) or TNF (S. typhimurium LPS) synthesis by anti-IL-18 treatment may also be involved.
Neutrophil Activation by Bacterial Lipoprotein Versus Lipopolysaccharide: Differential Requirements for Serum and CD14
Authors: Soler-Rodriguez Ana Maria , Zhang Hongwei , Lichenstein Henri S. , Qureshi Nilofer , Niesel David W. , Crowe Sheila E. , Peterson Johnny W. , Klimpel Gary R.
Published: April 2014

Neutrophil activation plays an important role in the inflammatory response to Gram-negative bacterial infections. LPS has been shown to be a major mediator of neutrophil activation which is accompanied by an early down-regulation of L-selectin and up-regulation of CD1lb/CD18. In this study, we investigated whether lipoprotein (LP), the most abundant protein in the outer membrane of bacteria from the family Enterobacteriaceae, can activate neutrophils and whether this activation is mediated by mechanisms that differ from those used by LPS or Escherichia coli diphosphoryl lipid A (EcDPLA). Neutrophil activation was assessed by measuring down-regulation of L-selectin and up-regulation of CD11b/CD18. When comparing molar concentrations of LP vs EcDPLA, LP was more potent (four times) at activating neutrophils. In contrast to LPS/EcDPLA, LP activation of neutrophils was serum independent. However, LP activation of neutrophils was enhanced by the addition of soluble CD14 and/or LPS-binding protein. In the presence of serum, LP activation of neutrophils was inhibited by different mAbs to CD14. This inhibition was significantly reduced or absent when performed in the absence of serum. Diphosphoryl lipid A from Rhodobacter spheroides (RaDPLA) completely inhibited LPS/EcDPLA activation of neutrophils but only slightly inhibited LP activation of neutrophils. These results suggest that LP activation of human neutrophils can be mediated by a mechanism that is different from LPS activation and that LP is a potentially important component in the development of diseases caused by Gram-negative bacteria of the family Enterobacteriaceae.
Functional Genomic Analysis in Arthritis-Affected Cartilage: Yin-Yang Regulation of Inflammatory Mediators by α5β1 and αVβ3 Integrins
Authors: Attur Mukundan G. , Dave Mandar N. , Clancy Robert M. , Patel Indravadan R. , Abramson Steven B. , Amin Ashok R.
Published: April 2014

Osteoarthritis-affected cartilage exhibits enhanced expression of fibronectin (FN) and osteopontin (OPN) mRNA in differential display and bioinformatics screen. Functional genomic analysis shows that the engagement of the integrin receptors α5β1 and αvβ3 of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of α5β1 using activating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory mediators such as NO and PGE2 as well as the cytokines, IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by α5β1 integrin ligation is mediated via induction and autocrine production of IL-1β, because type II soluble IL-1 decoy receptor inhibits their production. In contrast, αvβ3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1β, NO, and PGE2 (triggered by α5β1, IL-1β, IL-18, or IL-1β, TNF-α, plus LPS) in a dominant negative fashion by osteoarthritis-affected cartilage and activated bovine chondrocytes. These data demonstrate a cross-talk in signaling mechanisms among integrins and show that integrin-mediated “outside in” and “inside out” signaling very likely influences cartilage homeostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis.
Pivotal Role of CCR1-Positive Leukocytes in Bleomycin- Induced Lung Fibrosis in Mice
Authors: Tokuda Atsuko , Itakura Meiji , Onai Nobuyuki , Kimura Hiroshi , Kuriyama Takayuki , Matsushima Kouji
Published: April 2014

We have investigated the involvement of chemokine receptor CCR1-positive cells in bleomycin-induced lung injury, a model of pulmonary fibrosis. After bleomycin challenge in C57BL/6J mice, the expression of CCR1 mRNA increased and peaked at day 7, which paralleled to the expression of its ligands, macrophage-inflammatory protein-1α and RANTES. Immunohistochemical study showed that CCR1-positive cells accumulated in the interstitial inflammatory site. Furthermore, the treatment of anti-CCR1 Ab significantly reduced the accumulation of inflammatory cells and collagen deposition, resulting in dramatic improvement of survival. These results suggest that CCR1-positive cells play significant roles in the pathogenesis of pulmonary fibrosis subsequent to bleomycin-induced lung injury, and that CCR1 could be a novel molecular target for intervention therapy against pulmonary fibrosis.
Time-Dependent Loss of Mac-1 from Infiltrating Neutrophils in the Reperfused Myocardium
Authors: Youker Keith A. , Beirne Joshua , Lee Justin , Michael Lloyd H. , Smith C. Wayne , Entman Mark L.
Published: April 2014

Numerous studies have shown that polymorphonuclear neutrophils (PMNs) infiltrate the myocardium immediately after reperfusion of infarcted tissue. Studies with mAbs in vivo and cellular studies in vitro suggest that PMN-induced injury of the cardiac myocyte involve Mac-1 adhesion to myocyte ICAM-1. In this study we demonstrate that PMNs that have infiltrated the ischemic area begin to lose Mac-1 within the first 3 h. By the fifth hour of reperfusion, minimal CD11b staining is seen on PMNs using immunostaining, whereas CD11a remained unchanged. Immunoreactivity of postreperfusion cardiac lymph with R15.7 (anti-CD18) or MY904 (anti-CD11b) was positive in all animals but not for CD11a (R7.1), indicating a specific loss of Mac-1. Immunoprecipitation with either R15.7 or MY904 resulted in identical peptides (a doublet at 190 kDa and a band at 80 kDa), suggesting that both α and β subunits of Mac-1 heterodimer were released. Immunoprecipitation of control PMN lysates revealed bands of 198 kDa and 91 kDa slightly greater than those from the released Mac-1. An in vitro model of homotypic aggregation showed a similar loss of Mac-1 from PMNs; immunoprecipitates of the supernatant demonstrated peptide bands identical with those found in postischemic cardiac lymph. The appearance of soluble Mac-1 in vitro was prevented by anti-CD18 mAb, R15.7, and also by protease inhibition by PMSF. Thus, in vivo and in vitro, activated PMNs lose Mac-1 in a process that may be dependent upon adhesion and subsequent proteolysis.
Accumulation of Clonally Related B Lymphocytes in the Cerebrospinal Fluid of Multiple Sclerosis Patients
Authors: Colombo Monica , Dono Mariella , Gazzola Paola , Roncella Silvio , Valetto Angelo , Chiorazzi Nicholas , Mancardi Giovanni L. , Ferrarini Manlio
Published: April 2014

The accumulation of B lymphocyte clones in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and patients with other neurological disorders was investigated using PCR technologies. Oligoclonal B cell accumulations were detected in 10 of 10 MS patients, but only in 3 of 10 of the patients with other neurological disorders. Analyses of the Ig V(D)J sequences on the CSF from MS patients disclosed that VH3 and VH4 genes were extensively mutated compared with germline sequences. Moreover, a substantial proportion of the molecular clones analyzed shared the same third CDR of the H chain variable region gene (HCDR3) and the same VH genes, albeit with different numbers and locations of point mutations, thus indicating an ongoing process of intraclonal diversification. A larger number of clonally related VH sequences could be obtained by using a VH3 gene-specific PCR so that genealogical trees depicting the process of diversification could be drawn. Analyses of the Ig V(D)J from the CSF of a patient with viral meningitis and oligoclonal B cell accumulations revealed that VH3 genes were extensively mutated. However, no intraclonal diversification could be observed even using VH3 gene-specific PCR methodologies. Clone-specific PCR and sequencing was used to detect the V(D)J found in the CSF of one MS patient in the PBL of the same patient. Only 1/3 of the V(D)J sequences investigated could be demonstrated in the PBL, indicating that the V(D)J genes utilized by B cells in the CSF are much less represented in the PBL. Collectively, the data suggest that in MS there is a compartmentalized clonal expansion.
Rebound from Nitric Oxide Inhibition Triggers Enhanced Monocyte Activation and Chemotaxis
Authors: Magazine Harold I. , Chang Jungshan , Goumon Yannick , Stefano George B.
Published: April 2014

Exposure of human peripheral blood monocytes to the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) resulted in a rapid shift in cellular conformation of spontaneously activated cells from ameboid to round. The population of activated cells, ∼7.1 ± 1.2%, was reduced 7-fold to 1.1 ± 0.4% following 0.5 h exposure to SNAP. Observation of monocytes for 6 h demonstrated a gradual release from NO inhibition initiating at 2.5 h following SNAP treatment and a period of hyperactivity that was maximal at ∼5 h following SNAP exposure. During the rebound from the NO inhibition phase, there was a significant increase in the population of activated monocytes and an increased responsiveness to chemotactic agents such as IL-1, IL-8, and fMLP relative to that of cells treated with the chemotactic agents alone. Conformational changes induced by SNAP were associated with a reduction in F-actin and loss of filopodial extension. The loss and recovery of F-actin staining paralleled changes in cell activity, suggesting that NO may alter cellular activity by modulation of cytoskeletal actin. These data taken together suggest that inhibition of monocyte activity by NO results in an excitatory phase observed subsequent to release from NO inhibition and increased sensitivity to chemotactic agents. We propose that this rebound from NO inhibition may provide increased immunosurveillance to rectify immunological problems that have been encountered during the period of inhibition.
Linkage of the CCR5Δ32 Mutation with a Functional Polymorphism of CD45RA
Authors: Liao Hua-Xin , Montefiori David C. , Patel Dhavalkumar D. , Lee David M. , Scott William K. , Pericak-Vance Margaret , Haynes Barton F.
Published: April 2014

A 32-bp deletion in CCR5 (CCR5Δ32) confers to PBMC resistance to HIV-1 isolates that use CCR5 as a coreceptor. To study this mutation in T cell development, we have screened 571 human thymus tissues for the mutation. We identified 72 thymuses (12.6%) that were heterozygous and 2 (0.35%) that were homozygous for the CCR5Δ32 mutation. We found that thymocyte development was normal in both CCR5Δ32 heterozygous and homozygous thymuses. In 3% of thymuses we identified a functional polymorphism of CD45RA, in which cortical and medullary thymocytes failed to down-regulate the 200- and 220-kDa CD45RA isoforms during T cell development. Moreover, we found an association of this CD45 functional polymorphism in thymuses with the CCR5Δ32 mutation (p = 0.00258). In vitro HIV-1 infection assays with CCR5-using primary isolates demonstrated that thymocytes with the heterozygous CCR5Δ32 mutation produced less p24 than did CCR5 wild-type thymocytes. However, the functional CD45RA polymorphism did not alter the susceptibility of thymocytes to HIV-1 infection. Taken together, these data demonstrate association of the CCR5Δ32 mutation with a polymorphism in an as yet unknown gene that is responsible for the ability to down-regulate the expression of high m.w. CD45RA isoforms. Although the presence of the CCR5Δ32 mutation down-regulates HIV-1 infection of thymocytes, the functional CD45RA polymorphism does not alter the susceptibility of thymocytes to HIV-1 infection in vitro.
CD1d-Specific NK1.1+ T Cells with a Transgenic Variant TCR
Authors: Sköld Markus , Faizunnessa Nurun N. , Wang Chyung-Ru , Cardell Susanna
Published: April 2014

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Vα14-Jα281 rearrangement and a limited set of TCR Vβ segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Vα3.2/Vβ9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-γ, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the “non-Vα 14” diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.
Cutting Edge: Introduction of an Endopeptidase Cleavage Motif into a Determinant Flanking Region of Hen Egg Lysozyme Results in Enhanced T Cell Determinant Display
Authors: Schneider Susanne C. , Ohmen Jeff , Fosdick Lisa , Gladstone Brian , Guo Jane , Ametani Akio , Sercarz Eli E. , Deng Hongkui
Published: April 2014

The choice of which determinants of a whole Ag will be presented on cell surface MHC class II molecules after uptake and processing by APC is the result of the interplay between structural characteristics of the Ag and the processing machinery of the APC. In this study, we demonstrate that introduction of a dibasic motif adjacent to a subdominant determinant enhances the presentation of this determinant from the whole molecule. This is the first report showing that a single amino acid substitution in a whole Ag, designed to introduce an endopeptidase recognition site, enhances display of class II-restricted determinants, most likely by creating a peptide chain cleavage in the antigenic molecule. Our findings have important implications for the understanding of immunodominance and for vaccine design.
The Polarization Defect of Wiskott-Aldrich Syndrome Macrophages Is Linked to Dislocalization of the Arp2/3 Complex
Authors: Linder Stefan , Higgs Henry , Hüfner Katharina , Schwarz Klaus , Pannicke Ulrich , Aepfelbacher Martin
Published: April 2014

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally characterized by the clinical triad eczema, thrombocytopenia, and severe immunodeficieny, with recurrent bacterial and viral infections, indicating a profound immune cell defect. Such altered immune cells include monocytes, macrophages, and dendritic cells, which were reported to display disturbed cell polarization or chemotaxis. WAS is caused by mutations in the WAS protein (WASp), which is thought to organize the actin cytoskeleton through the Arp2/3 complex. Here we show that the Arp2/3 complex is an integral part of podosomes, actin-rich adhesion structures of macrophages, and that WAS macrophages fail to organize the Arp2/3 complex into podosomes. We also demonstrate that microinjection of a C-terminal acidic stretch of WASp into normal macrophages displaces Arp2/3 from podosomes and, in combination with chemoattractant stimulation of cells, induces a phenotype resembling the polarization-defective phenotype of stimulated WAS macrophages. These findings point to an important role of the Arp2/3 complex in polarization and migration of immune cells.
Microchimerism, Donor Dendritic Cells, and Alloimmune Reactivity in Recipients of Flt3 Ligand-Mobilized Hemopoietic Cells: Modulation by Tacrolimus
Authors: Morelli Adrian E. , Antonysamy Mary A. , Takayama Takuya , Hackstein Holger , Chen Zongyou , Qian Shiguang , Zurowski Nancy B. , Thomson Angus W.
Published: April 2014

Flt3 ligand (FL) is a potent hemopoietic growth factor that strikingly enhances stem cells and dendritic cells (DC) in vivo. We examined the impact of infusing FL-mobilized bone marrow (BM) cells on microchimerism and anti-donor reactivity in normal and tacrolimus-immunosuppressed, noncytoablated allogeneic recipients. BM from B10 (H2b) mice given FL (10 μg/day; days 0–8; FL-BM) contained a 7-fold higher incidence of potentially tolerogenic immature CD11c+ DC (CD40low, CD80low, CD86low, MHC IIlow) that induced alloantigen-specific T cell hyporesponsiveness in vitro. C3H (H2k) mice received 50 × 106 normal or FL-BM cells (day 0) and tacrolimus (2 mg/kg/day; days 0–12). On day 15, enhanced numbers of donor (IAb+) cells were detected in the thymi and spleens of FL-BM recipients. Tacrolimus markedly enhanced microchimerism, which declined as a function of time. Ex vivo splenocyte proliferative and CTL responses and Th1 cytokine (IFN-γ) production in response to donor alloantigens were augmented by FL-BM infusion, but reduced by tacrolimus. Systemic infusion of purified FL-BM immature DC, equivalent in number to that in corresponding whole BM, confirmed their capacity to sensitize, rather than tolerize, recipient T cells in vivo. In vitro, tacrolimus suppressed GM-CSF-stimulated growth of myeloid DC from normal BM much more effectively than from FL-BM without affecting MHC class II or costimulatory molecule expression. Infusion of normal B10 BM cells at the time of transplant prolonged C3H heart allograft survival, whereas FL-BM cells did not. A therapeutic effect of tacrolimus on graft survival was observed in combination with normal, but not FL-BM cells. These findings suggest the need for alternative immunosuppressive strategies to calcineurin inhibition to enable the engraftment, survival, and immunomodulatory function of FL-enhanced, immature donor DC.
T Cell Tolerance Based on Avidity Thresholds Rather Than Complete Deletion Allows Maintenance of Maximal Repertoire Diversity
Authors: Sandberg Johan K. , Franksson Lars , Sundbäck Jonas , Michaelsson Jakob , Petersson Max , Achour Adnane , Wallin Robert P. A. , Sherman Nicholas E. , Bergman Tomas , Jörnvall Hans , Hunt Donald F. , Kiessling Rolf , Kärre Klas
Published: April 2014

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1α (EF1α). EF1α occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1α peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1α peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1α isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.
An IFN-γ-Inducible Transcription Factor, IFN Consensus Sequence Binding Protein (ICSBP), Stimulates IL-12 p40 Expression in Macrophages
Authors: Wang I-Ming , Contursi Cristina , Masumi Atsuko , Ma Xiaojing , Trinchieri Giorgio , Ozato Keiko
Published: April 2014

IL-12 is a cytokine that links innate and adaptive immunity. Its subunit p40 is induced in macrophages following IFN-γ/LPS stimulation. Here we studied the role for IFN consensus sequence binding protein (ICSBP), an IFN-γ/LPS-inducible transcription factor of the IFN regulatory factor (IRF) family in IL-12 p40 transcription. Macrophage-like cells established from ICSBP−/− mice did not induce IL-12 p40 transcripts, nor stimulated IL-12 p40 promoter activity after IFN-γ/LPS stimulation, although induction of other inducible genes was normal in these cells. Transfection of ICSBP led to a marked induction of both human and mouse IL-12 p40 promoter activities in ICSBP+/+ and ICSBP−/− cells, even in the absence of IFN-γ/LPS stimulation. Whereas IRF-1 alone was without effect, synergistic enhancement of promoter activity was observed following cotransfection of ICSBP and IRF-1. Deletion analysis of the human promoter indicated that the Ets site, known to be important for activation by IFN-γ/LPS, also plays a role in the ICSBP activation of IL-12 p40. A DNA affinity binding assay revealed that endogenous ICSBP is recruited to the Ets site through protein-protein interaction. Last, transfection of ISCBP alone led to induction of the endogenous IL-12 p40 mRNA in the absence of IFN-γ and LPS. Taken together, our results show that ICSBP induced by IFN-γ/LPS, acts as a principal activator of IL-12p40 transcription in macrophages.
Ly-6I, a New Member of the Murine Ly-6 Superfamily with a Distinct Pattern of Expression
Authors: Pflugh David L. , Maher Stephen E. , Bothwell Alfred L. M.
Published: April 2014

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.
Ascaris suum, an Intestinal Parasite, Produces Morphine
Authors: Goumon Yannick , Casares Federico , Pryor Stephen , Ferguson Lee , Brownawell Bruce , Cadet Patrick , Rialas Christos M. , Welters Ingeborg D. M. , Sonetti Dario , Stefano George B.
Published: April 2014

The parasitic worm Ascaris suum contains the opiate alkaloid morphine as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of this material is 1168 ± 278 ng/g worm wet weight. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. To determine whether the morphine was active, we exposed human monocytes to the material, and they immediately released nitric oxide in a naloxone-reversible manner. The anatomic distribution of morphine immunoreactivity reveals that the material is in the subcuticle layers and in the animals’ nerve chords. Furthermore, as determined by RT-PCR, Ascaris does not express the transcript of the neuronal μ receptor. Failure to demonstrate the expression of this opioid receptor, as well as the morphine-like tissue localization in Ascaris, suggests that the endogenous morphine is intended for secretion into the microenvironment.
IL-4-Independent Inhibition of IL-12 Responsiveness DuringLeishmania amazonensisInfection
Authors: Jones Douglas E. , Buxbaum Laurence U. , Scott Phillip
Published: April 2014

Leishmania amazonensis induces a nonhealing infection in C3H mice, whereas infection with Leishmania major is self-healing. We found that C3H mice infected with L. amazonensis exhibited decreased IL-12 production, which could account for the susceptibility to this organism. However, exogenous IL-12 administration failed to induce a healing immune response. The failure of L. amazonensis-infected C3H mice to respond to IL-12 was associated with a specific defect in IL-12 receptor β2 (IL-12Rβ2) mRNA expression by CD4+ T cells. Furthermore, decreased IL-12Rβ2 mRNA expression correlated with a decrease in the IL-12-signaling capacity of the lymph node (LN) cells. IL-4 did not contribute to susceptibility or down-regulation of the IL-12Rβ2 subunit, because IL-4−/− mice remained susceptible to L. amazonensis infection, even after IL-12 administration, and CD4+ cells from infected IL-4−/− mice also had reduced expression of IL-12Rβ2 mRNA. These results demonstrate that regulation of the IL-12 receptor, independent of IL-4, is a point of control for the immune response to leishmaniasis. In contrast to experimental L. major infections, where host genetics control susceptibility, these studies demonstrate that the lack of IL-12 responsiveness may be dictated by the pathogen, rather than the host.
Genetic Mapping of Two Murine Loci that Influence the Development of IL-4-Producing Thy-1dull γδ Thymocytes
Authors: Azuara Véronique , Pereira Pablo
Published: April 2014

IL-4-producing γδ cells belong to a novel subset of γδ lymphocytes that expresses a very restricted repertoire of TCRs. To gain a deeper insight into the development and in vivo functions of these cells, we have analyzed the genetic control of their representation in the thymus. Using an intercross between C57BL/6 and DBA/2 mice we found two loci on chromosomes 13 and 17—named LadT1 and LadT2, respectively—with marked influence in their development. The LadT2 locus does not appear to be the MHC locus. The region identified on mouse chromosome 13 contains the structural genes for TCRγ as well as the IL-9 gene, which has been suggested as a candidate gene influencing the complex pathogenesis of asthma.
Divergent Effects of Platelet-Endothelial Cell Adhesion Molecule-1 and β3 Integrin Blockade on Leukocyte Transmigration In Vivo
Authors: Thompson Richard D. , Wakelin Matthew W. , Larbi Karen Y. , Dewar Ann , Asimakopoulos George , Horton Michael A. , Nakada Marian T. , Nourshargh Sussan
Published: April 2014

The final stage in the migration of leukocytes to sites of inflammation involves movement of leukocytes through the endothelial cell layer and the perivascular basement membrane. Both platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) and the integrin αvβ3 have been implicated in this process, and in vitro studies have identified αvβ3 as a heterotypic ligand for PECAM-1. In the present study we have addressed the roles of these molecules by investigating and comparing the effects of PECAM-1 and αvβ3 blockade on leukocyte migration in vivo. For this purpose we have examined the effects of neutralizing Abs directed against PECAM-1 (domain 1-specific, mAb 37) and β3 integrins (mAbs 7E3 and F11) on leukocyte responses in the mesenteric microcirculation of anesthetized rats using intravital microscopy. The anti-PECAM-1 mAb suppressed leukocyte extravasation, but not leukocyte rolling or firm adhesion, elicited by IL-1β in a dose-dependent manner (e.g., 67% inhibition at 10 mg/kg 37 Fab), but had no effect on FMLP-induced leukocyte responses. Analysis by electron microscopy suggested that this suppression was due to an inhibition of neutrophil migration through the endothelial cell barrier. By contrast, both anti-β3 integrin mAbs, 7E3 F(ab′)2 (5 mg/kg) and F11 F(ab′)2 (5 mg/kg), selectively reduced leukocyte extravasation induced by FMLP (38 and 46%, respectively), but neither mAb had an effect on IL-1β-induced leukocyte responses. These findings indicate roles for both PECAM-1 and β3 integrins in leukocyte extravasation, but do not support the concept that these molecules act as counter-receptors in mediating leukocyte transmigration.
Lethal Granuloma Disintegration in Mycobacteria-Infected TNFRp55−/− Mice Is Dependent on T Cells and IL-12
Authors: Ehlers Stefan , Kutsch Stefanie , Ehlers Eva M. , Benini Jochen , Pfeffer Klaus
Published: April 2014

Genetically susceptible, TNFRp55 gene-deficient (TNFRp55−/−) mice succumb to infection with Mycobacterium avium. Before their death, M. avium-infected TNFRp55−/− mice develop granulomatous lesions that, in contrast to granulomas in wild-type syngeneic mice, undergo acute disintegration. To determine the factors involved in these events, we depleted T cell subsets or neutralized the inflammatory cytokines IFN-γ, IL-12, or TNF in TNFRp55−/− mice infected i.v. with M. avium. Infected TNFRp55−/− mice treated with a control mAb became moribund between days 26 and 34 postinfection, showing widespread inflammatory cell apoptosis within disintegrating granulomas. In contrast, TNFRp55−/− mice depleted of either CD4+ or CD8+ cells after granuloma initiation stayed healthy until at least day 38 postinfection and showed no signs of granuloma destruction. Neutralization of IL-12, but not of IFN-γ or TNF, also protected M. avium-infected TNFRp55−/− mice from granuloma decomposition and from premature death. Treatment with dexamethasone or with a specific inhibitor of inducible NO synthase did not prevent granuloma dissolution or death of TNFRp55−/− mice. In conclusion, granuloma disintegration in TNFRp55−/− mice is a lethal event that is dependent on IL-12 and that is mediated by an excess of T cells.
Cutting Edge: Antilisterial Activity of CD8+ T Cells Derived from TNF-Deficient and TNF/Perforin Double-Deficient Mice
Authors: White Douglas W. , Badovinac Vladimir P. , Kollias George , Harty John T.
Published: April 2014

The mechanisms by which CD8+ T cells mediate immunity against bacterial pathogens remain largely unknown. Perforin-dependent cytolysis plays a role, but is not required for CD8+ T cell-mediated immunity against Listeria monocytogenes. TNF is essential for CD8+ T cell immunity to L. monocytogenes, but the cellular source of TNF is undefined. TNF-deficient and TNF/perforin double-deficient mice were used to generate CD8+ T cells specific for an L. monocytogenes-derived Ag. Wild-type and TNF-deficient CD8+ T cells mediated antilisterial immunity in wild-type but not TNF-deficient host mice, revealing that CD8+ T cell-derived TNF is not required for CD8+ T cell-mediated antilisterial immunity, but demonstrating a role for TNF derived from other cell types. TNF/perforin double-deficient CD8+ T cells mediated antilisterial immunity in the liver, but not in the spleen, of wild-type recipient mice, suggesting that perforin-independent immunity in the spleen requires CD8+ T cell-derived TNF.