bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Identification of GL50, a Novel B7-Like Protein That Functionally Binds to ICOS Receptor
Authors: Ling Vincent , Wu Paul W. , Finnerty Heather F. , Bean Kevin M. , Spaulding Vicki , Fouser Lynette A. , Leonard John P. , Hunter Sharon E. , Zollner Richard , Thomas Jenifer L. , Miyashiro Joy S. , Jacobs Kenneth A. , Collins Mary
Published: April 2014

By the genetic selection of mouse cDNAs encoding secreted proteins, a B7-like cDNA clone termed mouse GL50 (mGL50) was isolated encoding a 322-aa polypeptide identical with B7h. Isolation of the human ortholog of this cDNA (hGL50) revealed a coding sequence of 309 aa residues with 42% sequence identity with mGL50. Northern analysis indicated GL50 to be present in many tissues including lymphoid, embryonic yolk sac, and fetal liver samples. Of the CD28, CTLA4, and ICOS fusion constructs tested, flow cytometric analysis demonstrated only mouse ICOS-IgG binding to mGL50 cell transfectants. Subsequent phenotyping demonstrated high levels of ICOS ligand staining on splenic CD19+ B cells and low levels on CD3+ T cells. These results indicate that GL50 is a specific ligand for the ICOS receptor and suggest that the GL50-ICOS interaction functions in lymphocyte costimulation.
Recognition of a Sequestered Self Peptide by Influenza Virus-Specific CD8+ Cytolytic T Lymphocytes
Authors: Fan Rong , Tykodi Scott S. , Braciale Thomas J.
Published: April 2014

The Ag receptors on CD8+ CTL recognize foreign antigenic peptides associated with cell surface MHC class I molecules. Peptides derived from self proteins are also normally presented by MHC class I molecules. Here we report that an H-2Kd-restricted murine CD8+ CTL clone directed to an influenza hemagglutinin epitope can recognize a peptide derived from the murine mitochondrial aconitase enzyme in association with H-2Kd molecules. Surprisingly, this self peptide is not normally displayed on the cell surface associated with the restricting MHC class I molecule. Several lines of evidence suggest that this self peptide, although requiring association with the Kd molecule for CTL recognition, is not associated with this or other MHC class I allele under physiologic conditions in intact cells. Rather, it is sequestered in the cytoplasm associated with a carrier protein and is released only upon cell disruption. These results suggest a means of restricting the entry of self peptide into the class I pathway. In addition, this finding raises the possibility that self peptides sequestered within the cell can, after release from damaged cells, interact with MHC class I molecules on bystander cells and trigger autoimmune injury by virus-specific CTLs during viral infection.
Transgenic Expression of Fas Ligand on Thyroid Follicular Cells Prevents Autoimmune Thyroiditis
Authors: Batteux Frédéric , Lores Patrick , Bucchini Danièle , Chiocchia Gilles
Published: April 2014

“Immune privilege” is defined as tissue resistance to aggression by specifically activated lymphocytes, and involves the interaction between Fas expressed on infiltrating cells and Fas ligand (FasL) constitutively expressed on the target tissue. To test whether ectopic expression of FasL on thyrocytes could prevent autoimmune aggression of the thyroid by activated lymphoid cells, three lines of transgenic mice expressing low, intermediate, and high levels of functional FasL on thyroid follicular cells were generated. Experimental autoimmune thyroiditis was induced by immunization with mouse thyroglobulin. In all of the experiments, the effects were dependent on the level of FasL expression. Low and intermediate expression had no or only weak preventive effects, respectively, whereas high FasL expression strongly inhibited lymphocytic infiltration of the thyroid. Anti-mouse thyroglobulin-proliferative and cytotoxic T cell responses, as well as autoantibody production, were diminished in transgenic mice expressing high levels of FasL relative to controls. Furthermore, in these latter mice Th1 responses to mouse thyroglobulin were profoundly down-regulated, uncovering a new potential role for FasL in peripheral tolerance to organ-specific Ags. In sum, the prevention of experimental autoimmune thyroiditis by FasL on thyrocytes is dependent on the level of FasL expression.
TNF-α Is the Critical Mediator of the Cyclic AMP-Induced Apoptosis of CD8+4+ Double-Positive Thymocytes
Authors: Guevara Patiño José A. , Ivanov Vladimir N. , Lacy Elizabeth , Elkon Keith B. , Marino Michael W. , Nikolić-Žugić Janko
Published: April 2014

Apoptosis is one of the key regulatory mechanisms in tissue modeling and development. In the thymus, 95–98% of all thymocytes die by apoptosis because they failed to express a TCR with an optimal affinity for the selecting intrathymic peptide-MHC complexes. We studied the possible role of two prominent nerve growth factor (NGF-TNF) family member systems, Fas ligand (FasL)-Fas receptor (FasR) and TNF-α-TNFR, in apoptosis of murine CD8+4+ double-positive (DP) thymocytes induced via TCR-CD3- and cAMP-mediated signaling. TCR-CD3ε-mediated apoptosis of DP thymocytes was found not to be dependent on either of the two systems. The FasL-FasR system was also found to be dispensable for the cAMP-mediated apoptosis. By contrast, cAMP agonists (dibutyryl-cAMP and forskolin) induced apoptosis via TNF-α, as evidenced by 1) the ability of anti-TNF-α mAbs to abrogate cAMP analogue-induced DP apoptosis in a dose-dependent manner; and 2) increased resistance of DP thymocytes from TNF-α−/− and TNFR I−/−II−/− animals to cAMP agonist-mediated apoptosis. cAMP agonists induced DP thymocyte death by a combination of two mechanisms: first, they induced selective up-regulation of TNF-α production, and, second, they sensitized DP thymocytes to TNF-α. The latter effect may be due to the down-regulation of TNFR-associated factor 2 protein. These results identify TNF-α as the critical mediator of cAMP-induced apoptosis in thymocytes and provide a molecular explanation for how the cAMP stimulators, including the sex steroids, may modulate T cell production output, as observed under physiological and pharmacological conditions.
IL-4 Suppression of In Vivo T Cell Activation and Antibody Production
Authors: Morris Suzanne C. , Gause William C. , Finkelman Fred D.
Published: April 2014

Injection of mice with a foreign anti-IgD Ab stimulates B and T cell activation that results in large cytokine and Ab responses. Because most anti-IgD-activated B cells die before they can be stimulated by activated T cells, and because IL-4 prolongs the survival of B cells cultured with anti-Ig, we hypothesized that treatment with IL-4 at the time of anti-IgD Ab injection would decrease B cell death and enhance anti-IgD-induced Ab responses. Instead, IL-4 treatment before or along with anti-IgD Ab suppressed IgE and IgG1 responses, whereas IL-4 injected after anti-IgD enhanced IgE responses. The suppressive effect of early IL-4 treatment on the Ab response to anti-IgD was associated with a rapid, short-lived increase in IFN-γ gene expression but decreased CD4+ T cell activation and decreased or delayed T cell production of other cytokines. We examined the possibilities that IL-4 stimulation of IFN-γ production, suppression of IL-1 or IL-2 production, or induction of TNF-α or Fas-mediated apoptosis could account for IL-4’s suppressive effect. The suppressive effect of IL-4 was not reversed by IL-1, IL-2, or anti-TNF-α or anti-IFN-γ mAb treatment, or mimicked by treatment with anti-IL-2Rα (CD25) and anti-IL-2Rβ (CD122) mAbs. Early IL-4 treatment failed to inhibit anti-IgD-induced Ab production in Fas-defective lpr mice; however, the poor responsiveness of lpr mice to anti-IgD made this result difficult to interpret. These observations indicate that exposure to IL-4, while T cells are first being activated by Ag presentation, can inhibit T cells activation or promote deletion of responding CD4+ T cells.
Inhibition of Th1 Immune Response by Glucocorticoids: Dexamethasone Selectively Inhibits IL-12-Induced Stat4 Phosphorylation in T Lymphocytes
Authors: Franchimont Denis , Galon Jérôme , Gadina Massimo , Visconti Roberta , Zhou Y.-J. , Aringer Martin , Frucht David M. , Chrousos George P. , O’Shea John J.
Published: April 2014

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-γ secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.
Activation of Macrophage CD8: Pharmacological Studies of TNF and IL-1β Production
Authors: Lin Tong-Jun , Hirji Nadir , Stenton Grant R. , Gilchrist Mark , Grill Brock J. , Schreiber Alan D. , Befus A. Dean
Published: April 2014

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8α (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1β and promoted TNF and IL-1β secretion. Similarly, OX8 Ab (CD8α) stimulated NR8383 cells to secrete TNF, IL-1β, and NO. Activation of CD8β (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1β and stimulated secretion of TNF, but not IL-1β. Interestingly, anti-CD8 Abs did not stimulate IFN-γ or PGE2 production, or phagocytosis by macrophages. OX8 (CD8α)-induced TNF and IL-1β production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1β, stimulation by CD8α is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1β production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8α (OX8)-induced TNF and IL-1β production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1β expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.
IFN-γ Activates the C3G/Rap1 Signaling Pathway
Authors: Alsayed Yazan , Uddin Shahab , Ahmad Sarfraz , Majchrzak Beata , Druker Brian J. , Fish Eleanor N. , Platanias Leonidas C.
Published: April 2014

IFN-γ transduces signals by activating the IFN-γ receptor-associated Jak-1 and Jak-2 kinases and by inducing tyrosine phosphorylation and activation of the Stat-1 transcriptional activator. We report that IFN-γ activates a distinct signaling cascade involving the c-cbl protooncogene product, CrkL adapter, and small G protein Rap1. During treatment of NB-4 human cells with IFN-γ, c-cbl protooncogene product is rapidly phosphorylated on tyrosine and provides a docking site for the src homology 2 domain of CrkL, which also undergoes IFN-γ-dependent tyrosine phosphorylation. CrkL then regulates activation of the guanine exchange factor C3G, with which it interacts constitutively via its N terminus src homology 3 domain. This results in the IFN-γ-dependent activation of Rap1, a protein known to exhibit tumor suppressor activity and mediate growth inhibitory responses. In a similar manner, Rap1 is also activated in response to treatment of cells with type I IFNs (IFN-α, IFN-β), which also engage CrkL in their signaling pathways. On the other hand, IFN-γ does not induce formation of nuclear CrkL-Stat5 DNA-binding complexes, which are induced by IFN-α and IFN-β, indicating that pathways downstream of CrkL are differentially regulated by different IFN subtypes. Taken altogether, our data demonstrate that, in addition to activating the Stat pathway, IFN-γ activates a distinct signaling cascade that may play an important role in the generation of its growth inhibitory effects on target cells.
Cloning and Characterization of IL-10-Related T Cell-Derived Inducible Factor (IL-TIF), a Novel Cytokine Structurally Related to IL-10 and Inducible by IL-9
Authors: Dumoutier Laure , Louahed Jamila , Renauld Jean-Christophe
Published: April 2014

IL-9 is a Th2 cytokine active on various cell types such as T and B lymphocytes, mast cells, and eosinophils, and potentially involved in allergy and asthma. To understand better the molecular mechanisms underlying the activity of this cytokine, we used a cDNA subtraction method to identify genes specifically induced by IL-9 in mouse T cells. One of the IL-9-regulated genes isolated by this approach turned out to encode a 180-amino acid long protein, including a potential signal peptide, and showing 22% amino acid identity with IL-10. This protein, designated IL-10-related T cell-derived inducible factor (IL-TIF), is induced by IL-9 in thymic lymphomas, T cells, and mast cells, and by lectins in freshly isolated splenocytes. Experiments concerning the mechanism regulating IL-TIF expression in T cells indicate that IL-9 induction is rapid (within 1 h), does not require protein synthesis, and depends on the activation of the Janus kinase (JAK)-STAT pathway. In vivo, constitutive expression of IL-TIF was detected by RT-PCR in thymus and brain, suggesting that the role of this new factor is not restricted to the immune system. Transfection of HEK293 cells with the IL-TIF cDNA resulted in the production of a glycosylated protein of about 25 kDa that was found to induce STAT activation in mesangial and neuronal cell lines. Further studies will have to address the possibility that some of the IL-9 activities may be mediated by IL-TIF.
Regulator of G Protein Signaling 1 (RGS1) Markedly Impairs Giα Signaling Responses of B Lymphocytes
Authors: Moratz Chantal , Kang Veronica H. , Druey Kirk M. , Shi Chong-Shan , Scheschonka Astrid , Murphy Philip M. , Kozasa Tohru , Kehrl John H.
Published: April 2014

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-α. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various Gα subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Giα and Gqα GTPase-activating protein and a potential G12α effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12α. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.
Membrane Cofactor Protein (MCP; CD46): Isoform-Specific Tyrosine Phosphorylation
Authors: Wang Guixian , Liszewski M. Kathryn , Chan Andrew C. , Atkinson John P.
Published: April 2014

Membrane cofactor protein (MCP; CD46) is a widely expressed type 1 transmembrane glycoprotein that inhibits complement activation on host cells. It also is a receptor for several pathogens including measles virus, Streptococcus pyogenes, Neisseria gonorrhea, and Neisseria meningitidis. That MCP may have signaling capability was suggested by its microbial interactions. That is, binding of MCP on human monocytes by measles virus hemagglutinin or cross-linking by an anti-MCP Ab resulted in IL-12 down-regulation, while binding to MCP by Neisseria on epithelial cells produced a calcium flux. Through alternative splicing, MCP is expressed on most cells with two distinct cytoplasmic tails of 16 (CYT-1) or 23 (CYT-2) amino acids. These play pivotal roles in intracellular precursor processing and basolateral localization. We investigated the putative signal transduction pathway mediated by MCP and demonstrate that CYT-2, but not CYT-1, is phosphorylated on tyrosine. We examined MCP tail peptides and performed Ab cross-linking experiments on several human cell lines and MCP isoform transfectants. We found an MCP peptide of CYT-2 was phosphorylated by a src kinase system. Western blots of the cells lines demonstrated that cells bearing CYT-2 were also phosphorylated on tyrosine. Additionally, we provide genetic and biochemical evidence that the src family of kinases is responsible for the latter phosphorylation events. In particular, the src kinase, Lck, is required for phosphorylation of MCP in the Jurkat T cell line. Taken together, these studies suggest a src family-dependent pathway for signaling through MCP.
Single-Cytokine-Producing CD4 Memory Cells Predominate in Type 1 and Type 2 Immunity
Authors: Karulin Alexey Y. , Hesse Maike D. , Tary-Lehmann Magdalena , Lehmann Paul V.
Published: April 2014

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN-γ to be rare (0–10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Th1 or Th2 cells but by single-cytokine-producing memory cells.
Transgenic Expression of Cyclin D1 in Thymic Epithelial Precursors Promotes Epithelial and T Cell Development
Authors: Klug David B. , Crouch Elizabeth , Carter Carla , Coghlan Lezlee , Conti Claudio J. , Richie Ellen R.
Published: April 2014

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5− TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3ε transgenic and hCD3ε/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.
Heavy Chain Diversity Region Segments of the Channel Catfish: Structure, Organization, Expression and Phylogenetic Implications
Authors: Hayman J. Russell , Lobb Craig J.
Published: April 2014

Circular DNA, derived from lymphocytes of juvenile channel catfish, was used to construct λ libraries that were screened to identify the products of immunoglobulin DH-JH excision events. Clones were characterized that contained DH to JH recombination signal joints. The signal joints represented 23-bp recombination signal sequences (RSS) identical to germline JH segments that were adjacent to DH 12-bp RSS elements. DH flanking regions within the clones were used to probe a genomic library. Three germline DH gene segments containing 11–19 bp coding regions flanked by 12-bp RSS elements with conserved heptamers and nonamers were identified. The DH locus is closely linked to the JH locus, and Southern blots indicate that the DH segments represent different single member gene families. Analysis of H chain cDNA shows that each germline DH segment was expressed in functional VDJ recombination events involving different JH segments and members of different VH families. Several aspects of CDR3 junctional diversity were evident, including deletion of coding region nucleotides, N- and P-region nucleotide additions, alternate DH reading frame utilization, and point mutations. Coding region motifs of catfish DH segments are phylogenetically conserved in some DH segments of higher vertebrates. These studies indicate that the structure, genomic organization, and recombination patterns of DH segments typically associated with higher vertebrates evolved early in vertebrate phylogeny at the level of the bony fish.
The Nucleotide-Replacement Spectrum Under Somatic Hypermutation Exhibits Microsequence Dependence That Is Strand-Symmetric and Distinct from That Under Germline Mutation
Authors: Cowell Lindsay G. , Kepler Thomas B.
Published: April 2014

Somatic mutation is a fundamental component of acquired immunity. Although its molecular basis remains undetermined, the sequence specificity with which mutations are introduced has provided clues to the mechanism. We have analyzed data representing over 1700 unselected mutations in V gene introns and nonproductively rearranged V genes to identify the sequence specificity of the mutation spectrum—the distribution of resultant nucleotides. In other words, we sought to determine what effects the neighboring bases have on what a given base mutates “to.” We find that both neighboring bases have a significant effect on the mutation spectrum. Their influences are complicated, but much of the effect can be characterized as enhancing homogeneity of the mutated DNA sequence. In contrast to what has been reported for the sequence specificity of the “targeting” mechanism, that of the spectrum is notably symmetric under complementation, indicating little if any strand bias. We compared the spectrum to that found previously for germline mutations as revealed by analyzing pseudogene sequences. We find that the influences of nearest neighbors are quite different in the two datasets. Altogether, our findings suggest that the mechanism of somatic hypermutation is complex, involving two or more stages: introduction of mis-pairs and their subsequent resolution, each with distinct sequence specificity and strand bias.
Intestinal Intraepithelial Lymphocytes Exert Potent Protective Cytotoxic Activity During an Acute Virus Infection
Authors: Müller Stefan , Bühler-Jungo Myriam , Mueller Christoph
Published: April 2014

After systemic infection of mice with 104 PFU of lymphocytic choriomeningitis virus (LCMV), infected cells are detected simultaneously in various organs, including spleen and intestinal mucosa. Most notably, virus-infected cells are also present among CD11c+ dendritic cells in the subepithelial area of the small intestinal mucosa. Some of these virus-infected cells are in close spatial association with intestinal intraepithelial lymphocytes (IEL). Therefore, we compared virus-specific cytotoxic activity of CD8 splenocytes with that of IEL subsets. While ex vivo isolated TCRαβ+CD8αα+ IEL exert only minimal virus-specific cytotoxicity, maximum specific killing mediated by TCRαβ+CD8αβ+ IEL on day 8 postinfection exceeds maximum cytotoxic activity observed with CD8 splenocytes when assessed in vitro. Maximum cytotoxic activity of IEL is preceded by peak perforin and granzyme B mRNA expression in IEL around day 6 postinfection, suggesting a recent activation in situ. The antivirus cytotoxicity of in vivo primed IEL is further demonstrated by the protection from virus production in the spleen of mice infected with LCMV 10 h before adoptive cell transfer. These data indicate a potent priming of LCMV-specific IEL in situ after systemic LCMV infection and suggest that cytotoxic IEL markedly contribute to the elimination of virus-infected cells in the intestinal mucosa.
Selection of Babesia bovis-Infected Erythrocytes for Adhesion to Endothelial Cells Coselects for Altered Variant Erythrocyte Surface Antigen Isoforms
Authors: O’Connor Roberta M. , Allred David R.
Published: April 2014

Sequestration of Babesia bovis-infected erythrocytes (IRBCs) in the host microvasculature is thought to constitute an important mechanism of immune evasion. Since Ig is considered to be important for protection from disease, an in vitro assay of B. bovis sequestration was used to explore the ability of anti-B. bovis Ig to interfere with IRBC cytoadhesion, and to identify IRBC surface Ags acting as endothelial cell receptors. Bovine infection sera reactive with the IRBC surface inhibited and even reversed the binding of IRBCs to bovine brain capillary endothelial cells (BBECs). This activity is at least partially attributable to serum IgG. IgG isolated from inhibitory serum captured the variant erythrocyte surface ag 1 (VESA1) in surface-specific immunoprecipitations of B. bovis-IRBCs. Selection for the cytoadhesive phenotype concurrently selected for antigenic and structural changes in the VESA1 Ag. In addition, the anti-VESA1 mAb, 4D9.1G1, proved capable of effectively inhibiting and reversing binding of adhesive, mAb-reactive parasites to BBECs, and by immunoelectron microscopy localized VESA1 to the external tips of the IRBC membrane knobs. These data are consistent with a link between antigenic variation and cytoadherence in B. bovis and suggest that the VESA1 Ag acts as an endothelial cell ligand on the B. bovis-IRBC.
Ras, Protein Kinase Cζ, and IκB Kinases 1 and 2 Are Downstream Effectors of CD44 During the Activation of NF-κB by Hyaluronic Acid Fragments in T-24 Carcinoma Cells
Authors: Fitzgerald Katherine A. , Bowie Andrew G. , Skeffington Barbara Sheehy , O’Neill Luke A. J.
Published: April 2014

We have investigated the ability of hyaluronic acid (HA) fragments to activate the transcription factor NF-κB. HA fragments activated NF-κB in the cell lines T-24, HeLa, MCF7, and J774. Further studies in T-24 cells demonstrated that HA fragments also induced IκBα phosphorylation and degradation, κB-linked reporter gene expression, and ICAM-1 promoter activity in an NF-κB-dependent manner. The effect of HA was size dependent as neither disaccharide nor native HA were active. CD44, the principal cellular receptor for HA, was critical for the response because the anti-CD44 Ab IM7.8.1 blocked the effect on NF-κB. HA fragments activated the IκB kinase complex, and the effect on a κB-linked reporter gene was blocked in T-24 cells expressing dominant negative IκB kinases 1 or 2. Activation of protein kinase C (PKC) was required because calphostin C inhibited NF-κB activation and IκBα phosphorylation. In particular, PKCζ was required because transfection of cells with dominant negative PKCζ blocked the effect of HA fragments on κB-linked gene expression and HA fragments increased PKCζ activity. Furthermore, damnacanthal and manumycin A, two mechanistically distinct inhibitors of Ras, blocked NF-κB activation. Transfection of T-24 cells with dominant negative Ras (RasN17) blocked HA fragment-induced κB-linked reporter gene expression, and HA fragments activated Ras activity within 5 min. Taken together, these studies establish a novel signal transduction cascade eminating from CD44 to Ras, PKCζ, and IκB kinase 1 and 2.
Human Toll-Like Receptor 2 Mediates Monocyte Activation byListeria monocytogenes, But Not by Group B Streptococci or Lipopolysaccharide
Authors: Flo Trude H. , Halaas Øyvind , Lien Egil , Ryan Liv , Teti Giuseppe , Golenbock Douglas T. , Sundan Anders , Espevik Terje
Published: April 2014

Human Toll like receptor (TLR) 2 has been implicated as a signaling receptor for LPS from Gram-negative bacteria and cell wall components from Gram-positive organisms. In this study, we investigated whether TLR2 can signal cell activation by the heat-killed group B streptococci type III (GBS) and Listeria monocytogenes (HKLM). HKLM, but not GBS, showed a time- and dose-dependent activation of Chinese hamster ovary cells transfected with human TLR2, as measured by translocation of NF-κB and induction of IL-6 production. A mAb recognizing a TLR2-associated epitope (TL2.1) was generated that inhibited IL-6 production from Chinese hamster ovary-TLR2 cells stimulated with HKLM or LPS. The TL2.1 mAb reduced HKLM-induced TNF production from human monocytes by 60%, whereas a CD14 mAb (3C10) reduced the TNF production by 30%. However, coadministrating TL2.1 and 3C10 inhibited the TNF response by 80%. In contrast to this, anti-CD14 blocked LPS-induced TNF production from monocytes, whereas anti-TLR2 showed no inhibition. Neither TL2.1 nor 3C10 affected GBS-induced TNF production. These results show that TLR2 can function as a signaling receptor for HKLM, possibly together with CD14, but that TLR2 is unlikely to be involved in cell activation by GBS. Furthermore, although LPS can activate transfected cell lines through TLR2, this receptor does not seem to be the main transducer of LPS activation of human monocytes. Thus, our data demonstrate the ability of TLR2 to distinguish between different pathogens.
Administration of IgG Fc Fragments Prevents Glomerular Injury in Experimental Immune Complex Nephritis
Authors: Gómez-Guerrero Carmen , Duque Natalia , Casado María Teresa , Pastor Carlos , Blanco Julia , Mampaso Francisco , Vivanco Fernando , Egido Jesús
Published: April 2014

Most human nephritis is due to glomerular deposition and/or formation of immune complexes (IC). In cultured mesangial cells, Fc receptor stimulation induces proliferation, matrix synthesis, and release of several mediators implicated in the initiation and progression of glomerular injury. Since Ig Fc fragments in vitro modified these phenomena, we studied the effects of systemic administration of IgG Fc fragments on the evolution of experimental IC nephritis. Fc fragment injection (1 mg/day i.p.) to rats with ongoing nephritis (proteinuria 20–50 mg/24 h vs 9 ± 0.2 mg/24 h in controls) markedly ameliorates proteinuria, renal function, and morphological renal lesions. This was accompanied by a reduction in the renal synthesis of chemokines (monocyte chemoattractant protein-1, IFN-inducible protein-10, and cytokine-induced neutrophil chemoattractant-1), matrix proteins, and growth factors (platelet-derived growth factor, and TGF-β), and in the activity of transcription factors. The treatment did not affect the glomerular deposition of IgG IC and complement C1q. In contrast, a decrease in the renal expression and production of C3 was observed without changes in serum complement levels. In vitro, very low complement consumption and no C3b covalent interaction were observed with Fc fragments, confirming that they did not modify systemic complement activity. These results indicate that the administration of Fc fragments prevents the development of glomerular damage in an aggressive model of proliferative glomerulonephritis through mechanisms involving a reduced local generation of complement, chemokines and growth factors. Modulation of IC-mesangial cell interaction by Fc fragment administration could represent a new approach to the treatment of severe immune nephritis.
Requirement of B7 Costimulation for Th1-Mediated Inflammatory Bone Resorption in Experimental Periodontal Disease
Authors: Kawai Toshihisa , Eisen-Lev Ronit , Seki Makoto , Eastcott Jean W. , Wilson Mark E. , Taubman Martin A.
Published: April 2014

The CD28 costimulation at TCR signaling plays a pivotal role in the regulation of the T cell response. To elucidate the role of T cells in periodontal disease, a system of cell transfer with TCR/CD28-dependent Th1 or Th2 clones was developed in rats. Gingival injection of specific Ag, Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein, and LPS could induce local bone resorption 10 days after the transfer of Ag-specific Th1 clone cells, but not after transfer of Th2 clone cells. Interestingly, the presence of LPS was required not only for the induction of bone resorption but also for Ag-specific IgG2a production. LPS injection elicited the induction of expression of both B7-1 and B7-2 expression on gingival macrophages, which otherwise expressed only MHC class II when animals were injected with Ag alone. The expression of B7 molecules was observed for up to 3 days, which corresponded to the duration of retention of T clone cells in gingival tissues. Either local or systemic administration of CTLA4Ig, a functional antagonist of CD28 binding to B7, could abrogate the bone resorption induced by Th1 clone cells combined with gingival challenge with both Ag and LPS. These results suggest that local Ag-specific activation of Th1-type T cells by B7 costimulation appeared to trigger inflammatory bone resorption, whereas inhibition of B7 expression by CTLA4Ig might be a therapeutic approach for intervention with inflammatory bone resorption.
Activated Macrophages Direct Apoptosis and Suppress Mitosis of Mesangial Cells
Authors: Duffield Jeremy S. , Erwig Lars-Peter , Wei Xiao-quing , Liew Foo Y. , Rees Andrew J. , Savill John S.
Published: April 2014

During inflammation in the glomerulus, the complement of resident myofibroblast-like mesangial cells is regulated by mitosis and apoptosis, but the cellular mechanisms controlling the size of mesangial cell populations have remained obscure. Prompted by studies of development, we sought evidence that macrophages regulate mesangial cell number. Rat bone marrow-derived macrophages primed with IFN-γ then further activated in coculture with LPS or TNF-α elicited a 10-fold induction of rat mesangial cell apoptosis and complete suppression of mitosis, effects inhibitable by the NO synthase inhibitors l-monomethyl arginine and l-N6-(1-iminoethyl) lysine dihydrochloride. Complete dependence upon macrophage-derived NO was observed in comparable experiments employing activated bone marrow macrophages from wild-type and NO synthase 2−/− mice. Nevertheless, when mesangial cells were primed with IFN-γ plus TNF-α, increased induction by activated macrophages of mesangial apoptosis exhibited a NO-independent element. The use of gld/gld macrophages excluded a role for Fas ligand in this residual kill, despite increased expression of Fas and increased susceptibility to soluble Fas ligand exhibited by cytokine-primed mesangial cells. Finally, activated macrophages isolated from the glomeruli of rats with nephrotoxic nephritis also induced apoptosis and suppressed mitosis in mesangial cells by an l-monomethyl arginine-inhibitable mechanism. These data demonstrate that activated macrophages, via the release of NO and other mediators, regulate mesangial cell populations in vitro and may therefore control the mesangial cell complement at inflamed sites.
The Effect of IL-5 and Eotaxin Expression in the Lung on Eosinophil Trafficking and Degranulation and the Induction of Bronchial Hyperreactivity
Authors: Mould Arne W. , Ramsay Alistair J. , Matthaei Klaus I. , Young Ian G. , Rothenberg Marc E. , Foster Paul S.
Published: April 2014

The mechanisms regulating the selective migration and degranulation of eosinophils in the asthmatic lung and the subsequent development of airways hyperreactivity (AHR) have not been fully delineated. In this investigation, we have employed a novel transgene model to facilitate the dissection of the contributions of IL-5 and/or eotaxin to eosinophil function in the absence of complex tissue signals derived from the allergic lung. Gene transfer of IL-5 and/or eotaxin to the lungs of naive mice induced a pronounced and selective airways eosinophilia, but did not result in eosinophil degranulation or AHR. Airways eosinophilia occurred independently of the induction of a blood eosinophilia, but was markedly augmented by the coexpression of both cytokines and/or by the transient mobilization of eosinophils from the bone marrow by the administration of i.v. IL-5. However, for eosinophil degranulation and AHR to occur, the inhalation of Ag was required in association with IL-5 and eotaxin expression. Investigations in IL-5-deficient mice linked eosinophilia, and not solely IL-5 and eotaxin, with the induction of AHR. Furthermore, eosinophil degranulation and AHR were dependent on CD4+ T cells. Importantly, this investigation shows that IL-5 regulates eosinophilia within the lung as well as in the circulation and also amplifies eotaxin-induced chemotaxis in the airway compartment. Moreover, the interplay between these cytokines, CD4+ T cells, and factors generated by Ag inhalation provides fundamental signals for eosinophil degranulation and the induction of AHR.
Role of p38 Mitogen-Activated Protein Kinase in a Murine Model of Pulmonary Inflammation
Authors: Nick Jerry A. , Young Scott K. , Brown Kevin K. , Avdi Natalie J. , Arndt Patrick G. , Suratt Benjamin T. , Janes Michael S. , Henson Peter M. , Worthen G. Scott
Published: April 2014

Early inflammatory events include cytokine release, activation, and rapid accumulation of neutrophils, with subsequent recruitment of mononuclear cells. The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway plays a central role in regulating a wide range of inflammatory responses in many different cells. A murine model of mild LPS-induced lung inflammation was developed to investigate the role of the p38 MAPK pathway in the initiation of pulmonary inflammation. A novel p38 MAPK inhibitor, M39, was used to determine the functional consequences of p38 MAPK activation. In vitro exposure to M39 inhibited p38 MAPK activity in LPS-stimulated murine and human neutrophils and macrophages, blocked TNF-α and macrophage inflammatory protein-2 (MIP-2) release, and eliminated migration of murine neutrophils toward the chemokines MIP-2 and KC. In contrast, alveolar macrophages required a 1000-fold greater concentration of M39 to block release of TNF-α and MIP-2. Systemic inhibition of p38 MAPK resulted in significant decreases in the release of TNF-α and neutrophil accumulation in the airspaces following intratracheal administration of LPS. Recovery of MIP-2 and KC from the airspaces was not affected by inhibition of p38 MAPK, and accumulation of mononuclear cells was not significantly reduced. When KC was instilled as a proinflammatory stimulus, neutrophil accumulation was significantly decreased by p38 MAPK inhibition independent of TNF-α or LPS. Together, these results demonstrate a much greater dependence on the p38 MAPK cascade in the neutrophil when compared with other leukocytes, and suggest a means of selectively studying and potentially modulating early inflammation in the lung.
Engagement of the OX-40 Receptor In Vivo Enhances Antitumor Immunity
Authors: Weinberg Andrew D. , Rivera Martin-Muy , Prell Rodney , Morris Arden , Ramstad Trygg , Vetto John T. , Urba Walter J. , Alvord Gregory , Bunce Campbell , Shields John
Published: April 2014

The OX-40 receptor (OX-40R), a member of the TNFR family, is primarily expressed on activated CD4+ T lymphocytes. Engagement of the OX-40R, with either OX-40 ligand (OX-40L) or an Ab agonist, delivers a strong costimulatory signal to effector T cells. OX-40R+ T cells isolated from inflammatory lesions in the CNS of animals with experimental autoimmune encephalomyelitis are the cells that respond to autoantigen (myelin basic protein) in vivo. We identified OX-40R+ T cells within primary tumors and tumor-invaded lymph nodes of patients with cancer and hypothesized that they are the tumor-Ag-specific T cells. Therefore, we investigated whether engagement of the OX-40R in vivo during tumor priming would enhance a tumor-specific T cell response. Injection of OX-40L:Ig or anti-OX-40R in vivo during tumor priming resulted in a significant improvement in the percentage of tumor-free survivors (20–55%) in four different murine tumors derived from four separate tissues. This anti-OX-40R effect was dose dependent and accentuated tumor-specific T cell memory. The data suggest that engagement of the OX-40R in vivo augments tumor-specific priming by stimulating/expanding the natural repertoire of the host’s tumor-specific CD4+ T cells. The identification of OX-40R+ T cells clustered around human tumor cells in vivo suggests that engagement of the OX-40R may be a practical approach for expanding tumor-reactive T cells and thereby a method to improve tumor immunotherapy in patients with cancer.
Leukemia Inhibitory Factor, Oncostatin M, IL-6, and Stem Cell Factor mRNA Expression in Human Thymus Increases with Age and Is Associated with Thymic Atrophy
Authors: Sempowski Gregory D. , Hale Laura P. , Sundy John S. , Massey Janice M. , Koup Richard A. , Douek Daniel C. , Patel Dhavalkumar D. , Haynes Barton F.
Published: April 2014

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR δ excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.
Variable Expression of Pathogenesis-Related Protein Allergen in Mountain Cedar (Juniperus ashei) Pollen
Authors: Midoro-Horiuti Terumi , Goldblum Randall M. , Kurosky Alexander , Wood Thomas G. , Brooks Edward G.
Published: April 2014

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.
CD47 Engagement Inhibits Cytokine Production and Maturation of Human Dendritic Cells
Authors: Demeure C. E. , Tanaka H. , Mateo V. , Rubio M. , Delespesse G. , Sarfati M.
Published: April 2014

Upon encounter with bacterial products, immature dendritic cells (iDCs) release proinflammatory cytokines and develop into highly stimulatory mature DCs. In the present study, we show that human monocyte-derived DCs functionally express the CD47 Ag, a thrombospondin receptor. Intact or F(ab′)2 of CD47 mAb suppress bacteria-induced production of IL-12, TNF-α, GM-CSF, and IL-6 by iDCs. 4N1K, a peptide derived from the CD47-binding site of thrombospondin, also inhibits cytokine release. The inhibition of IL-12 and TNF-α is IL-10-independent inasmuch as IL-10 production is down-modulated by CD47 mAb and blocking IL-10 mAb fails to restore cytokine levels. CD47 ligation counteracts the phenotypic and functional maturation of iDCs in that it prevents the up-regulation of costimulatory molecules, the loss of endocytic activity, and the acquisition of an increased capacity to stimulate T cell proliferation and IFN-γ production. Interestingly, regardless of CD47 mAb treatment during DC maturation, mature DC restimulated by soluble CD40 ligand and IFN-γ, to mimic DC/T interaction, produce less IL-12 and more IL-18 than iDCs. Finally, CD47 ligation on iDCs does not impair their capacity to phagocytose apoptotic cells. We conclude that following exposure to microorganisms, CD47 ligation may limit the intensity and duration of the inflammatory response by preventing inflammatory cytokine production by iDCs and favoring their maintenance in an immature state.
Modulation of NF-κB Activity and Apoptosis in Chronic Lymphocytic Leukemia B Cells
Authors: Furman Richard R. , Asgary Zahra , Mascarenhas John O. , Liou Hsiou-Chi , Schattner Elaine J.
Published: April 2014

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5+ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-κB proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-κB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-κB-binding activity in unstimulated CLL B cells relative to that detected in nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-κB activity and prolonged CLL cell survival in vitro. The principle NF-κB proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-κB activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound influence on NF-κB activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL.