bluuebunny/crossref_metadata_2025 huggingface dataset:
Age-Related Dysregulation in CD8 T Cell Homeostasis: Kinetics of a Diversity Loss
Authors: LeMaoult Joël , Messaoudi Ilhem , Manavalan John S. , Potvin Heather , Nikolich-Z̆ugich Dragana , Dyall Ruben , Szabo Paul , Weksler Marc E. , Nikolich-Z̆ugich Janko
Published: April 2014

Relative diversity and representation of peripheral T cells bearing different TCR Vβ families are remarkably tightly regulated between birth and advanced adulthood. By contrast, individual elderly humans and C3H.SW and B10.BR aged mice display drastic disruption in such regulation. It was suggested that the alterations in the murine aged T cell compartment were due to age-related clonal T cell expansions (TCE). Here, we studied the kinetics of homeostatic dysregulation of T cell populations in aged C57BL/6 (B6) mice. Using mAb staining, we show that the percentages of αβ+CD8+ or CD4+ T cells bearing different TCRVβ elements remain virtually constant in mice up to 12 mo of age. In 22-mo-old mice, however, there is a dramatic disturbance of this pattern owing to the emergence of CD8+ TCE. Expanded T cells did not show any obvious bias in Vβ usage and were derived in all cases examined thus far from a single clone. TCE appeared later in life, compared with B cell clonal expansions. However, and in contrast to those detected in humans, TCE were frequently unstable disappearing within 2–4 mo, with other TCE appearing within the same time frame. Additional studies carried on thymic T cells, thymectomized mice, and young T transferred cells into Rag1−/− mice suggest that the clonal expansions occur in the periphery and that their onset is accelerated by decreased thymic output and/or function(s).
Mycophenolate Mofetil Impairs the Maturation and Function of Murine Dendritic Cells
Authors: Mehling Annette , Grabbe Stephan , Voskort Maik , Schwarz Thomas , Luger Thomas A. , Beissert Stefan
Published: April 2014

The immunosuppressive drug, mycophenolate mofetil (MMF), has been successfully introduced in allogeneic transplantation medicine and, more recently, in the treatment of autoimmune skin disorders. MMF inhibits lymphocyte proliferation via a blockade of the enzyme inosine 5′-monophosphate dehydrogenase, an enzyme on which lymphocytes solely depend to generate the purines necessary for DNA/RNA synthesis. To investigate the effects of MMF on cutaneous immune responses, a murine model of contact hypersensitivity (CHS) was used, with oxazolone or trinitrochlorobenzene as a contact allergen. Compared with the respective vehicle, i.p. applied MMF significantly inhibited the elicitation and, surprisingly, the induction of CHS responses. This prompted further studies into the effects of MMF on Ag presentation. Bone marrow-derived dendritic cells (DC) were cultured with GM-CSF and IL-4 in the presence of MMF and were tested for their Ag-presenting capacity. Sensitization and elicitation of CHS and delayed-type hypersensitivity responses by s.c. injected haptenated DC were reduced upon preincubation of DC with MMF. CHS responses were not impaired upon resensitization, indicating that MMF does not induce hapten-specific immunotolerance. In addition, MMF decreased the ability of DC to stimulate allogeneic T cells in MLR assays. Accordingly, flow cytometric analyses revealed a dose-dependent reduction of the expression of CD40, CD80, CD86, I-A, and ICAM-1 on DC with a concurrent reduction of IL-12 production. These data suggest that MMF, in addition to affecting T lymphocytes, directly affects APC, resulting in an impairment of immune responses. They furthermore point to a possible role of inosine 5′-monophosphate dehydrogenase in the maturation of DC.
IL-3 Increases Production of B Lymphoid Progenitors from Human CD34+CD38− Cells
Authors: Crooks Gay M. , Hao Qian-Lin , Petersen Denise , Barsky Lora W. , Bockstoce David
Published: April 2014

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38− cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38− cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34−CD19+ B cells. B cell production was increased whether CD34+CD38− cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Rα expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Rα protein expression was largely restricted to myeloid progenitors. CD34+CD38− cells had low to undetectable levels of IL-3Rα by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Rα protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38− cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.
Antibody Production in Mice Deficient for Complement Receptors 1 and 2 Can Be Induced by IgG/Ag and IgE/Ag, But Not IgM/Ag Complexes
Authors: Applequist Steven E. , Dahlström Jörgen , Jiang Ning , Molina Hector , Heyman Birgitta
Published: April 2014

Deficiencies in C factors C2, C3, or C4 as well as lack of C receptors 1 and 2 (CR1/2) lead to impaired Ab production. Classical pathway activation plays a major role, as mice deficient in factor B, a key factor in the alternative pathway, have normal Ab production. Abs in complex with their specific Ag are known to feedback regulate the Ab response, and enhanced responses are initiated by IgM, IgE, and IgG. IgM acts via the C system, whereas IgE and IgG can operate independently of C via Fc receptors. Here we have investigated whether these isotypes are able to enhance Ab responses in mice lacking CR1/2. SRBC-specific IgM, administered with SRBC, does not enhance Ab responses in these animals. In contrast, 2,4,6-trinitrophenyl-specific IgE and IgG2a, administered with BSA-2,4,6-trinitrophenyl, induce potent Ab responses in CR1/2-deficient mice. Additionally, BSA administered with CFA or alum induced strong Ab responses in the absence of CR1/2. These results indicate that CR1/2 is needed to promote IgM-mediated induction of primary Ab responses. The data also show that the need for CR1/2 can be circumvented by Abs typical of a secondary immune response forming complexes with Ag or by conventional adjuvants, presumably mimicking physiological inflammatory reactions.
Contemporary Analysis of MHC-Related Immunodominance Hierarchies in the CD8+ T Cell Response to Influenza A Viruses
Authors: Belz Gabrielle T. , Stevenson Philip G. , Doherty Peter C.
Published: April 2014

Early studies of influenza virus-specific CD8+ T cell-mediated immunity indicated that the level of CTL activity associated with H2Db is greatly diminished in mice that also express H2Kk. Such MHC-related immunodominance hierarchies are of some interest, as they could lead to variable outcomes for peptide-based vaccination protocols in human populations. The influence of H2Kk on the H2Db-restricted response profile has thus been looked at again using a contemporary, quantitative, IFN-γ-based flow cytometric assay. The depressive effect of H2Kk was very apparent for the influenza DbPA224 epitope and was also reproduced when CTL activity was measured for H2Db-expressing targets pulsed with the immunodominant NP366 peptide. The secondary CD8+IFN-γ+ DbNP366-specific response was much greater in parental H2b than in H2k×bF1 mice, but the sizes of the CD8+ sets specific for KkNP50 and DbNP366 were essentially equivalent in the F1 animals. Thus, although the immunodominance profile associated with DbNP366 is lost when H2Kk is also present, the response is still substantial. A further, MHC-related effect was also identified for the KkNS1152 epitope, which was consistently associated with a greater CD8+IFN-γ+ response in H2KkDb recombinant than in (H2KkDk × H2KbDb)F1 mice. The diminished DbPA224 response in H2k×bF1 mice was characterized by loss of a prominent Vβ7 TCR responder phenotype, supporting the idea that TCR deletion during ontogeny shapes the available repertoire. The overall conclusion is that these MHC-related immunodominance hierarchies are more subtle than the early CTL assays suggested and, although inherently unpredictable, are unlikely to cause a problem for peptide-based vaccine strategies.
An Essential Role for the IL-7 Receptor During Intrathymic Expansion of the Positively Selected Neonatal T Cell Repertoire
Authors: Hare Katherine J. , Jenkinson Eric J. , Anderson Graham
Published: April 2014

Intrathymic T cell development is a multistage process involving discrete phases of proliferation as well as differentiation. From studies on IL-7 or IL-7Rα-deficient mice, it is clear that the IL-7 receptor (IL-7R) plays a critical role during the initial stages of intrathymic CD4−8− precursor development. In contrast, the role of IL-7R in later stages of thymocyte development are unclear. Here, we have used various approaches to investigate directly the role of the IL-7R in thymocyte positive selection and the recently described phase of postselection proliferation. First, we show that positive selection involves selective up-regulation of IL-7Rα- and IL-7Rγ-chains, with the majority of CD4+ and CD8+ cells being IL-7R+. Second, MHC class II+ thymic epithelium—which drives postselection proliferation—expresses IL-7 mRNA. Finally, analysis of positive selection and postselection proliferation in thymocytes from IL-7Rα−/− neonates shows that positive selection occurs normally, whereas postselection expansion is drastically reduced. Thus, our data provide the first evidence that, as well as playing a role during early phases of thymic development, IL-7R mediates intrathymic expansion of positively selected thymocytes, which may aid in establishment of the neonatal peripheral T cell pool.
T Cell Effector Function and Anergy Avoidance Are Quantitatively Linked to Cell Division
Authors: Wells Andrew D. , Walsh Matthew C. , Sankaran David , Turka Laurence A.
Published: April 2014

We have shown previously that T cells activated by optimal TCR and CD28 ligation exhibit marked proliferative heterogeneity, and ∼40% of these activated cells fail entirely to participate in clonal expansion. To address how prior cell division influences the subsequent function of primary T cells at the single cell level, primary CD4+ T cells were subjected to polyclonal stimulation, sorted based on the number of cell divisions they had undergone, and restimulated by ligation of TCR/CD28. We find that individual CD4+ T cells exhibit distinct secondary response patterns that depend upon their prior division history, such that cells that undergo more rounds of division show incrementally greater IL-2 production and proliferation in response to restimulation. CD4+ T cells that fail to divide after activation exist in a profoundly hyporesponsive state that is refractory to both TCR/CD28-mediated and IL-2R-mediated proliferative signals. We find that this anergic state is associated with defects in both TCR-coupled activation of the p42/44 mitogen-activated protein kinase (extracellular signal-related kinase 1/2) and IL-2-mediated down-regulation of the cell cycle inhibitor p27kip1. However, these defects are selective, as TCR-mediated intracellular calcium flux and IL-2R-coupled STAT5 activation remain intact in these cells. Therefore, the process of cell division or cell cycle progression plays an integral role in anergy avoidance in primary T cells, and may represent a driving force in the formation of the effector/memory T cell pool.
Selective Inhibition of IgG-Mediated Phagocytosis in Gelsolin-Deficient Murine Neutrophils
Authors: Serrander Lena , Skarman Petra , Rasmussen Birgitta , Witke Walter , Lew Daniel P. , Krause Karl-Heinz , Stendahl Olle , Nüße Oliver
Published: April 2014

Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton. We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn−) mice. The phagocytosis of complement opsonized yeast was only minimally affected. In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn− neutrophils. Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis. Furthermore, attachment of IgG-opsonized yeast to Gsn− neutrophils was reduced (∼50%) but not to the same extent as ingestion (∼73%). This was not due to reduced surface expression of the Fcγ-receptor or its lateral mobility. This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis. We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase. All these functions were normal in Gsn− neutrophils. Thus, the role of gelsolin is specific for IgG-mediated phagocytosis. Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis. The latter requires a more dynamic reorganization of the cytoskeleton.
Selective Malformation of the Splenic White Pulp Border in L1-Deficient Mice
Authors: Wang Shih-Lien , Kutsche Michael , DiSciullo Gino , Schachner Melitta , Bogen Steven A.
Published: April 2014

Lymphocytes enter the splenic white pulp by crossing the poorly characterized boundary of the marginal sinus. In this study, we describe the importance of L1, an adhesion molecule of the Ig superfamily, for marginal sinus integrity. We find that germline insertional mutation of L1 is associated with a selective malformation of the splenic marginal sinus. Other splenic structures remain intact. Immunofluorescence analysis of the extracellular framework of the spleen, using an Ab to laminin, reveals that L1 knockout mice have an irregularly shaped, discontinuous white pulp margin. Electron microscopic analysis shows that it is associated with bizarrely shaped marginal sinus lining cells at the periphery of the white pulp. These abnormalities correlate with the localization of L1 in normal mice in that L1 is normally expressed on marginal sinus lining cells at the white pulp border. These L1-immunopositive lining cells coexpress high levels of mucosal addressin cell adhesion molecule-1 and vimentin, indicating that they are of fibroblastic lineage and express a well-characterized addressin. Our findings are the first to implicate L1 in splenic lymphoid architectural development. Moreover, these findings help define the poorly characterized sinusoidal boundary across which mononuclear cells cross to enter the splenic white pulp.
MHC Class II Transactivator Inhibits IL-4 Gene Transcription by Competing with NF-AT to Bind the Coactivator CREB Binding Protein (CBP)/p300
Authors: Sisk Tyler J. , Gourley Tania , Roys Stacey , Chang Cheong-Hee
Published: April 2014

The MHC class II transactivator (CIITA) activates the expression of multiple genes involved in Ag presentation, but inhibits Th2-type cytokine production, including IL-4, during Th1 cell differentiation. Th1 cells derived from CIITA-deficient mice produce both Th1- and Th2-type cytokines, and the introduction of CIITA to Th2 cells down-regulates Th2-type cytokine gene transcription. Here we show that the IL-4 promoter is regulated by multiple protein-protein interactions among CIITA, NF-AT, and coactivator CBP/p300. The introduction of CBP/p300 and NF-AT enhances the IL-4 promoter activity, and this activation was repressed by CIITA. Furthermore, our data show that CIITA competes with NF-AT to bind CBP/p300 and that this competition dramatically influences transcriptional activation of the IL-4 promoter. We identified two domains of CIITA that interact with two distinct domains of CBP/p300 that are also recognized by NF-AT. CIITA mutants that retain the ability to interact with CBP/p300 are sufficient to inhibit NF-AT-mediated IL-4 gene expression.
Identification and Functional Characterization of a New Gene Encoding the Mouse Terminal Complement Inhibitor CD59
Authors: Qian Yue-Ming , Qin Xuebin , Miwa Takashi , Sun Xiujun , Halperin Jose A. , Song Wen-Chao
Published: April 2014

CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.
The IgA/IgM Receptor Expressed on a Murine B Cell Lymphoma Is Poly-Ig Receptor
Authors: Phillips-Quagliata Julia M. , Patel Samir , Han Jing-Kang , Arakelov Sergei , Rao T. Dharma , Shulman Marc J. , Fazel Shafie , Corley Ronald B. , Everett Margaret , Klein Michel H. , Underdown Brian J. , Corthésy Blaise
Published: April 2014

T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116–120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Cα2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells.
Efficient Internalization of IL-2 Depends on the Distal Portion of the Cytoplasmic Tail of the IL-2R Common γ-Chain and a Lymphoid Cell Environment
Authors: Yu Aixin , Olosz Ferenc , Choi Chris Y. , Malek Thomas R.
Published: April 2014

The common γ-chain (γc), a subunit of the IL-2R, is essential for high affinity ligand binding and signal transduction due to Jak3 association to γc. Another consequence of IL-2/IL-2R interaction is rapid receptor-mediated endocytosis of the receptor-ligand complex. In the present study, we establish that this rapid endocytosis of IL-2 in a T cell tumor line is dependent upon the cytoplasmic tail of γc. Deletion mutants of the cytoplasmic tail mapped this activity to 9 aa of γc, 45–54 aa distal to the transmembrane region. In contrast, ligand-independent constitutive endocytosis of γc occurred more slowly and was dependent upon a PEST sequence in a more membrane-proximal region of the cytoplasmic tail of γc. Thus, this receptor subunit may use distinct sorting signals for its constitutive regulation and ligand-induced endocytosis. Rapid endocytosis of IL-2 was inhibited by the tyrosine kinase inhibitor genistein, implicating a role for a signal transduction pathway in IL-2 internalization. However, one T cell line bearing a mutant γc exhibited impaired endocytosis of IL-2, despite normal IL-2-induced Jak/STAT activation. Furthermore, inefficient endocytosis of IL-2 was noted after transfection of the COS7 epithelial cell line with the IL-2R, and further reconstitution of these cells with Jak/STAT proteins did not enhance this internalization. Collectively, these latter findings indicate that rapid endocytosis of IL-2 is dependent upon cellular signaling in lymphoid cell environment that is not solely a consequence of the presence of the Jak/STAT pathway.
Effect of Integrin β2 Subunit Truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) Assembly, Surface Expression, and Function
Authors: Tan Suet-Mien , Hyland Robert H. , Al-Shamkhani Aymen , Douglass Wendy A. , Shaw Jacqueline M. , Law S. K. Alex
Published: April 2014

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the β2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized β2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated β2 variants, we showed that the subregion Q23-D300 of the β2 subunit is sufficient to combine with the αL and αM subunits intracellularly. However, only the β2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the β2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0.5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the β2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.
Molecular Cloning and Characterization of a Novel CXC Chemokine Macrophage Inflammatory Protein-2γ Chemoattractant for Human Neutrophils and Dendritic Cells
Authors: Cao Xuetao , Zhang Weiping , Wan Tao , He Long , Chen Taoyong , Yuan Zhenglong , Ma Shihua , Yu Yizhi , Chen Guoyou
Published: April 2014

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2αβ, hence is designated as MIP-2γ. Mouse MIP-2γ was identified by electrocloning and is highly homologous to human MIP-2γ. Northern blotting revealed that MIP-2γ was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2γ was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2γ expression by monocyte-derived DC. Recombinant MIP-2γ from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2γ is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2γ does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.
Enhanced Pulmonary Allergic Responses toAspergillusin CCR2−/− Mice
Authors: Blease Kate , Mehrad Borna , Standiford Theodore J. , Lukacs Nicholas W. , Gosling Jennifa , Boring Landin , Charo Israel F. , Kunkel Steven L. , Hogaboam Cory M.
Published: April 2014

Allergic responses to Aspergillus species exacerbate asthma and cystic fibrosis. The natural defense against live Aspergillus fumigatus spores or conidia depends on the recruitment and activation of mononuclear and polymorphonuclear leukocytes, events that are dependent on chemotactic cytokines. In this study, we explored the relative contribution of the monocyte chemoattractant protein-1 receptor, CCR2, in the pulmonary response to A. fumigatus conidia. Following sensitization to soluble A. fumigatus Ags, mice lacking CCR2 due to targeted deletion were markedly more susceptible to the injurious effects of an intrapulmonary challenge with live conidia compared with mice that expressed CCR2 or CCR2+/+. CCR2−/− mice exhibited a major defect in the recruitment of polymorphonuclear cells, but these mice also had significantly more eosinophils and lymphocytes in bronchoalveolar lavage samples. CCR2−/− mice also had significant increases in serum levels of total IgE and whole lung levels of IL-5, IL-13, eotaxin, and RANTES compared with CCR2+/+ mice. Airway inflammation, hyper-responsiveness to spasmogens, and subepithelial fibrosis were significantly enhanced in CCR2−/− mice compared with CCR2+/+ mice after the conidia challenge. Thus, these findings demonstrate that CCR2 plays an important role in the immune response against A. fumigatus, thereby limiting the allergic airway inflammatory and remodeling responses to this fungus.
Identification of STAT4-Dependent and Independent Mechanisms of Resistance toToxoplasma gondii
Authors: Cai Guifang , Radzanowski Thad , Villegas Eric N. , Kastelein Robert , Hunter Christopher A.
Published: April 2014

The capacity of IL-12 to stimulate T and NK cell production of IFN-γ is required for resistance to Toxoplasma gondii. To identify the transcription factors involved in this mechanism of resistance, mice deficient in STAT4, a protein involved in IL-12 signaling, were infected with T. gondii and their immune responses were analyzed. STAT4−/− mice were unable to control parasite replication and died during the acute phase of infection, whereas wild-type mice controlled parasite replication and survived this challenge. The susceptibility of STAT4−/− mice to toxoplasmosis correlated with a defect in their ability to produce IFN-γ in response to infection, whereas administration of IFN-γ to these mice inhibited parasite replication and delayed time to death. Interestingly, analysis of infected STAT4−/− mice revealed that these mice did produce low levels of IFN-γ during infection, and the ability of splenocytes from infected or uninfected STAT4−/− mice to produce IFN-γ was enhanced by the addition of IL-2 plus IL-18. Moreover, administration of IL-2 plus IL-18 to STAT4−/− mice resulted in elevated serum levels of IFN-γ associated with a decreased parasite burden and delayed time to death. In vivo depletion studies demonstrated that the ability of IL-2 plus IL-18 to mediate STAT4-independent resistance to T. gondii is dependent on NK cell production of IFN-γ. Together, these studies identify STAT4 as an important transcription factor required for development of the innate NK and adaptive T cell responses necessary for resistance to T. gondii. However, other signaling pathways can be used to bypass STAT4-dependent production of IFN-γ and enhance innate resistance to T. gondii.
An Immunomodulatory Function for Neutrophils During the Induction of a CD4+ Th2 Response in BALB/c Mice Infected withLeishmania major
Authors: Tacchini-Cottier Fabienne , Zweifel C. , Belkaid Y. , Mukankundiye C. , Vasei M. , Launois P. , Milon G. , Louis J. A.
Published: April 2014

The possible immunomodulatory role of polymorphonuclear leukocytes (PMN) in CD4+ T lymphocyte differentiation in mice was examined by studying the effect of transient depletion of PMN during the early phase after Leishmania major delivery. A single injection of the PMN-depleting NIMP-R14 mAb 6 h before infection with L. major prevented the early burst of IL-4 mRNA transcription otherwise occurring in the draining lymph node of susceptible BALB/c mice. Since this early burst of IL-4 mRNA transcripts had previously been shown to instruct Th2 differentiation in mice from this strain, we examined the effect of PMN depletion on Th subset differentiation at later time points after infection. The transient depletion of PMN in BALB/c mice was sufficient to inhibit Th2 cell development otherwise occurring after L. major infection. Decreased Th2 responses were paralleled with partial resolution of the footpad lesions induced by L. major. Furthermore, draining lymph node-derived CD4+ T cells from PMN-depleted mice remained responsive to IL-12 after L. major infection, unlike those of infected BALB/c mice receiving control Ab. PMN depletion had no effect when the NIMP-R14 mAb was injected 24 h postinfection. The protective effect of PMN depletion was shown to be IL-12 dependent, as concomitant neutralization of IL-12 reversed the protective effect of PMN depletion. These results suggest a role for an early wave of PMN in the development of the Th2 response characteristic of mice susceptible to infection with L. major.
The Anti-Tumor Activity of IL-12: Mechanisms of Innate Immunity That Are Model and Dose Dependent
Authors: Smyth Mark J. , Taniguchi Masaru , Street Shayna E. A.
Published: April 2014

IL-12 has been demonstrated to have potent anti-tumor activities in a variety of mouse tumor models, but the relative roles of NK, NKT, and T cells and their effector mechanisms in these responses have not been fully addressed. Using a spectrum of gene-targeted or Ab-treated mice we have shown that for any particular tumor model the effector mechanisms downstream of IL-12 often mimic the natural immune response to that tumor. For example, metastasis of the MHC class I-deficient lymphoma, EL4-S3, was strictly controlled by NK cells using perforin either naturally or following therapy with high-dose IL-12. Intriguingly, in B16F10 and RM-1 tumor models both NK and NKT cells contribute to natural protection from tumor metastasis. In these models, a lower dose of IL-12 or delayed administration of IL-12 dictated a greater relative role of NKT cells in immune protection from tumor metastasis. Overall, both NK and NKT cells can contribute to natural and IL-12-induced immunity against tumors, and the relative role of each population is tumor and therapy dependent.
The Role of a Mitochondrial Pathway in the Induction of Apoptosis by Chemicals Extracted from Diesel Exhaust Particles
Authors: Hiura Timothy S. , Li Ning , Kaplan Roman , Horwitz Marcus , Seagrave Jean-Clare , Nel Andre E.
Published: April 2014

We are interested in the cytotoxic and proinflammatory effects of particulate pollutants in the respiratory tract. We demonstrate that methanol extracts made from diesel exhaust particles (DEP) induce apoptosis and reactive oxygen species (ROS) in pulmonary alveolar macrophages and RAW 264.7 cells. The toxicity of these organic extracts mimics the cytotoxicity of the intact particles and could be suppressed by the synthetic sulfhydryl compounds, N-acetylcysteine and bucillamine. Because DEP-induced apoptosis follows cytochrome c release, we studied the effect of DEP chemicals on mitochondrially regulated death mechanisms. Crude DEP extracts induced ROS production and perturbed mitochondrial function before and at the onset of apoptosis. This mitochondrial perturbation follows an orderly sequence of events, which commence with a change in mitochondrial membrane potential, followed by cytochrome c release, development of membrane asymmetry (annexin V staining), and propidium iodide uptake. Structural damage to the mitochondrial inner membrane, evidenced by a decrease in cardiolipin mass, leads to O⨪2 generation and uncoupling of oxidative phosphorylation (decreased intracellular ATP levels). N-Acetylcysteine reversed these mitochondrial effects and ROS production. Overexpression of the mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress apoptosis. Taken together, these results suggest that DEP chemicals induce apoptosis in macrophages via a toxic effect on mitochondria.
Activation of Human Leukocytes Reduces Surface P-Selectin Glycoprotein Ligand-1 (PSGL-1, CD162) and Adhesion to P-Selectin In Vitro
Authors: Davenpeck Kelly L. , Brummet Mary E. , Hudson Sherry A. , Mayer Ruth J. , Bochner Bruce S.
Published: April 2014

P-selectin glycoprotein ligand-1 (PSGL-1), the primary ligand for P-selectin, is constitutively expressed on the surface of circulating leukocytes. The objective of this study was to examine the effect of leukocyte activation on PSGL-1 expression and PSGL-1-mediated leukocyte adhesion to P-selectin. PSGL-1 expression was examined via indirect immunofluorescence and flow cytometry before and after leukocyte stimulation with platelet activating factor (PAF) and PMA. Human neutrophils, monocytes, and eosinophils were all demonstrated to have significant surface expression of PSGL-1 at baseline, which decreased within minutes of exposure to PAF or PMA. PSGL-1 was detected in the supernatants of PAF-activated neutrophils by immunoprecipitation. Along with the expression data, this suggests removal of PSGL-1 from the cell surface. Soluble PSGL-1 was also detected in human bronchoalveolar lavage fluids. Down-regulation of PSGL-1 was inhibited by EDTA. However, inhibitors of L-selectin shedding and other sheddase inhibitors did not affect PSGL-1 release, suggesting that PSGL-1 may be shed by an as yet unidentified sheddase or removed by some other mechanism. Functionally, PSGL-1 down-regulation was associated with decreased neutrophil adhesion to immobilized P-selectin under both static and flow conditions, with the most profound effects seen under flow conditions. Together, these data indicate that PSGL-1 can be removed from the surface of activated leukocytes, and that this decrease in PSGL-1 expression has profound effects on leukocyte binding to P-selectin, especially under conditions of flow.
Secretory Phospholipase A2 Receptor-Mediated Activation of Cytosolic Phospholipase A2 in Murine Bone Marrow-Derived Mast Cells
Authors: Fonteh Alfred N. , Atsumi Gen-ichi , LaPorte Tiffany , Chilton Floyd H.
Published: April 2014

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-α-d-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.
Proinflammatory Effects of IL-10 During Human Endotoxemia
Authors: Lauw Fanny N. , Pajkrt Dasja , Hack C. Erik , Kurimoto Masashi , van Deventer Sander J. H. , van der Poll Tom
Published: April 2014

IL-10 is considered a potent antiinflammatory cytokine that strongly inhibits the production of proinflammatory cytokines. Recent studies have suggested that IL-10 also has immunostimulatory properties on CD4+, CD8+ T cells, and/or NK cells, resulting in increased IFN-γ production. To determine the effect of IL-10 on IFN-γ production and related inflammatory responses in humans, 16 healthy subjects received a bolus i.v. injection of LPS (4 ng/kg) in combination with either placebo or recombinant human IL-10 (25 μg/kg), administered just before or 1 h after LPS. IL-10 treatment, particularly when administered after LPS, enhanced LPS-induced IFN-γ release, as well as the release of the IFN-γ-dependent chemokines IFN-γ-inducible protein-10 and monokine induced by IFN-γ, while inhibiting or not influencing the production of IFN-γ-inducing cytokines. In addition, IL-10 treatment enhanced activation of CTLs and NK cells after LPS injection, as reflected by increased levels of soluble granzymes. These data indicate that high-dose IL-10 treatment in patients with inflammatory disorders can be associated with undesired proinflammatory effects.
Differential Requirement for Classic and Novel PKC Isoforms in Respiratory Burst and Phagocytosis in RAW 264.7 Cells
Authors: Larsen Elaine C. , DiGennaro Jeannine A. , Saito Naoaki , Mehta Sapna , Loegering Daniel J. , Mazurkiewicz Joseph E. , Lennartz Michelle R.
Published: April 2014

The binding of Ab (IgG)-opsonized particles by FcγRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs α, βI, δ, ε, and ζ. Subcellular fractionation demonstrated that PKCs α, δ, and ε translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs δ and ε increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC α or ε confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC ε colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC α mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs δ and/or ε are necessary for phagocytosis.
Cellular and Molecular Mechanism for Kilham Rat Virus-Induced Autoimmune Diabetes in DR-BB Rats
Authors: Chung Young-Hwa , Jun Hee Sook , Son Mike , Bao Min , Bae Hak Yeon , Kang Yup , Yoon Ji-Won
Published: April 2014

Kilham rat virus (KRV) causes autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats; however, the mechanism by which KRV induces autoimmune diabetes without the direct infection of β cells is not well understood. We first asked whether molecular mimicry, such as a common epitope between a KRV-specific peptide and a β cell autoantigen, is involved in the initiation of KRV-induced autoimmune diabetes in DR-BB rats. We found that KRV peptide-specific T cells generated in DR-BB rats infected with recombinant vaccinia virus expressing KRV-specific structural and nonstructural proteins could not induce diabetes, indicating that molecular mimicry is not the mechanism by which KRV induces autoimmune diabetes. Alternatively, we asked whether KRV infection of DR-BB rats could disrupt the finely tuned immune balance and activate autoreactive T cells that are cytotoxic to β cells, resulting in T cell-mediated autoimmune diabetes. We found that both Th1-like CD45RC+CD4+ and cytotoxic CD8+ T cells were up-regulated, whereas Th2-like CD45RC−CD4+ T cells were down-regulated, and that isolated and activated CD45RC+CD4+ and CD8+ T cells from KRV-infected DR-BB rats induced autoimmune diabetes in young diabetes-prone BioBreeding (DP-BB) rats. We conclude that KRV-induced autoimmune diabetes in DR-BB rats is not due to molecular mimicry, but is due to a breakdown of the finely tuned immune balance of Th1-like CD45RC+CD4+ and Th2-like CD45RC−CD4+ T cells, resulting in the selective activation of β cell-cytotoxic effector T cells.
IL-1-Independent Role of IL-17 in Synovial Inflammation and Joint Destruction During Collagen-Induced Arthritis
Authors: Lubberts Erik , Joosten Leo A. B. , Oppers Birgitte , van den Bersselaar Liduine , Coenen-de Roo Christina J. J. , Kolls Jay K. , Schwarzenberger Paul , van de Loo Fons A. J. , van den Berg Wim B.
Published: April 2014

T cell IL-17 displays proinflammatory properties and is expressed in the synovium of patients with rheumatoid arthritis. Its contribution to the arthritic process has not been identified. Here, we show that blocking of endogenous IL-17 in the autoimmune collagen-induced arthritis model results in suppression of arthritis. Also, joint damage was significantly reduced. In contrast, overexpression of IL-17 enhanced collagen arthritis. Moreover, adenoviral IL-17 injected in the knee joint of type II collagen-immunized mice accelerated the onset and aggravated the synovial inflammation at the site. Radiographic and histologic analysis showed markedly increased joint destruction. Elevated levels of IL-1β protein were found in synovial tissue. Intriguingly, blocking of IL-1αβ with neutralizing Abs had no effect on the IL-17-induced inflammation and joint damage in the knee joint, implying an IL-1 independent pathway. This direct potency of IL-17 was underscored in the unabated IL-17-induced exaggeration of bacterial cell wall-induced arthritis in IL-1β−/− mice. In conclusion, this data shows that IL-17 contributes to joint destruction and identifies an IL-1-independent role of IL-17. These findings suggest IL-17 to be a novel target for the treatment of destructive arthritis and may have implications for tissue destruction in other autoimmune diseases.
Functional Expression of IL-12 Receptor by Human Eosinophils: IL-12 Promotes Eosinophil Apoptosis
Authors: Nutku Esra , Zhuang Qianli , Soussi-Gounni Abdelillah , Aris Fadi , Mazer Bruce D. , Hamid Qutayba
Published: April 2014

In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rβ1 and -β2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rβ1 and -β2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis.
Tissue-Type Plasminogen Activator Deficiency Exacerbates Arthritis
Authors: Yang Yuan H. , Carmeliet Peter , Hamilton John A.
Published: April 2014

Fibrin deposition, cell migration, and tissue remodeling are key components in the lesions of inflammatory joint diseases, such as rheumatoid arthritis. The plasminogen activators (PAs), namely, tissue-type PA (t-PA) and urokinase PA, are implicated in these aspects of an inflammatory response, although their precise roles are yet to be defined. We therefore used gene-deficient mice to explore their role in a two-stage arthritis model involving intraarticular methylated BSA injection, followed by systemic IL-1 treatment. We report in this study that both t-PA and urokinase PA are protective for the mild arthritis induced by intraarticular methylated BSA injection alone, since absence of either of them exacerbates the response; following s.c. IL-1 injection, t-PA−/− mice had particularly severe disease. Fibrin deposition appeared to parallel disease severity under the various conditions, suggesting that PA-mediated fibrinolysis may be normally playing a protective role in inflammatory joint disease.
Adenoviral Delivery of Human and Viral IL-10 in Murine Sepsis
Authors: Minter Rebecca M. , Ferry Maria A. , Murday Michelle E. , Tannahill Cynthia L. , Bahjat Frances R. , Oberholzer Caroline , Oberholzer Andreas , LaFace Drake , Hutchins Beth , Wen Shufen , Shinoda Jeremy , Copeland Edward M. , Moldawer Lyle L.
Published: April 2014

Adenovirus (Ad) gene therapy has been proposed as a drug-delivery system for the targeted administration of protein-based therapies, including growth factors and biological response modifiers. However, inflammation associated with Ad transduction has raised concern about its safety and efficacy in acute inflammatory diseases. In the present report, intratracheal and i.v. administration of a first-generation adenoviral recombinant (E1,E3 deleted) either containing an empty cassette or expressing the anti-inflammatory cytokines viral or human IL-10 (IL-10) was administered to mice subjected to zymosan-induced multisystem organ failure or to acute necrotizing pancreatitis. Pretreatment of mice with the intratracheal instillation of Ad expressing human IL-10 or viral IL-10 reduced weight loss, attenuated the proinflammatory cytokine response, and reduced mortality in the zymosan-induced model, whereas pretreatment with a control adenoviral recombinant did not significantly exacerbate the response. Pretreatment of mice with pancreatitis using adenoviral vectors expressing IL-10 significantly reduced the degree of pancreatic and liver injury and liver inflammation when administered systemically, but not intratracheally. We conclude that adenoviral vectors can be administered prophylactically in acute inflammatory syndromes, and expression of the anti-inflammatory protein IL-10 can be used to suppress the underlying inflammatory process.