bluuebunny/crossref_metadata_2025 huggingface dataset:
ABrugia malayiHomolog of Macrophage Migration Inhibitory Factor Reveals an Important Link Between Macrophages and Eosinophil Recruitment During Nematode Infection
Authors: Falcone Franco H. , Loke P’ng , Zang Xingxing , MacDonald Andrew S. , Maizels Rick M. , Allen Judith E.
Published: April 2014

Infections with the helminth parasite Brugia malayi share many key features with Th2-mediated allergic diseases, including recruitment of eosinophils. We have investigated the dynamics of inflammatory cell recruitment under type 2 cytokine conditions in mice infected with B. malayi. Among the cells recruited to the site of infection is a novel population of “alternatively activated” macrophages that ablate cell proliferation and enhance Th2 differentiation. By profiling gene expression in this macrophage population, we found a dramatic up-regulation of a recently described eosinophil chemotactic factor, eosinophil chemotactic factor-L/Ym1, representing over 9% of clones randomly selected from a cDNA library. Because B. malayi is known to secrete homologs (Bm macrophage migration inhibitory factor (MIF)-1 and -2) of the human cytokine MIF, we chose to investigate the role this cytokine mimic may play in the development of the novel macrophage phenotype observed during infection. Strikingly, administration of soluble recombinant Bm-MIF-1 was able to reproduce the effects of live parasites, leading both to the up-regulation of Ym1 by macrophages and a marked recruitment of eosinophils in vivo. Because activity of Bm-MIF-1 is dependent upon an amino-terminal proline, this residue was mutated to glycine; the resultant recombinant (Bm-MIF-1G) was unable to induce Ym1 transcription in macrophages or to mediate the recruitment of eosinophils. These data suggest that macrophages may provide a crucial link between helminth parasites, their active cytokine mimics, and the recruitment of eosinophils in infection.
Loss of Pentameric Symmetry of C-Reactive Protein Is Associated with Promotion of Neutrophil-Endothelial Cell Adhesion
Authors: Zouki Christine , Haas Barbara , Chan John S. D. , Potempa Lawrence A. , Filep János G.
Published: April 2014

The classic acute-phase reactant C-reactive protein (CRP) is a cyclic pentameric protein that diminishes neutrophil accumulation in inflamed tissues. When the pentamer is dissociated, CRP subunits undergo conformational rearrangement that results in expression of a distinctive isomer with unique antigenic and physicochemical characteristics (termed modified CRP (mCRP)). Recently, mCRP was detected in the wall of normal human blood vessels. We studied the impact and mechanisms of action of mCRP on expression of adhesion molecules on human neutrophils and their adhesion to human coronary artery endothelial cells. Both CRP and mCRP (0.1–200 μg/ml) down-regulated neutrophil l-selectin expression in a concentration-dependent fashion. Furthermore, mCRP, but not CRP, up-regulated CD11b/CD18 expression and stimulated neutrophil extracellular signal-regulated kinase activity, which was accompanied by activation of p21ras oncoprotein, Raf-1, and mitogen-activated protein kinase kinase. These actions of mCRP were sensitive to the mitogen-activated protein kinase kinase inhibitor PD98059. mCRP markedly enhanced attachment of neutrophils to LPS-activated human coronary artery endothelial when added together with neutrophils. This effect of mCRP was attenuated by an anti-CD18 mAb. Thus, loss of pentameric symmetry in CRP is associated with appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at inflamed or injured vascular sites.
Role of Sulfation in CD44-Mediated Hyaluronan Binding Induced by Inflammatory Mediators in Human CD14+ Peripheral Blood Monocytes
Authors: Brown Kelly L. , Maiti Arpita , Johnson Pauline
Published: April 2014

Activation of T cells by Ag or stimulation of monocytes with inflammatory cytokines induces CD44 to bind to hyaluronan (HA), an adhesion event implicated in leukocyte-leukocyte, leukocyte-endothelial cell, and leukocyte-stromal cell interactions. We have previously shown that TNF-α induces CD44 sulfation in a leukemic cell line, which correlated with the induction of HA binding and CD44-mediated adhesion. In this study, we establish that TNF-α and IFN-γ induce HA binding and the sulfation of CD44 in CD14+ PBMC, whereas no induced HA binding or CD44 sulfation was observed in CD14− PBMC stimulated with TNF-α. Treatment of cells with NaClO3, an inhibitor of sulfation, prevented HA binding in a significant percentage of CD14+ PBMC induced by TNF-α, LPS, IL-1β, or IFN-γ. Furthermore, stimulation with TNF-α or IFN-γ in the presence of NaClO3 reduced the ability of isolated CD44H to bind HA, demonstrating a direct effect of CD44H sulfation on HA binding. In contrast, the transient induction of HA binding in T cells by PHA was not affected by NaClO3, suggesting that activated T cells do not use sulfation as a mechanism to regulate HA binding. Overall, these results demonstrate that inducible sulfation of CD44H is one mechanism used by CD14+ peripheral blood monocytes to induce HA binding in response to inflammatory agents such as TNF-α and IFN-γ.
Cross-Talk Between IL-1 and IL-6 Signaling Pathways in Rheumatoid Arthritis Synovial Fibroblasts
Authors: Deon David , Ahmed Simi , Tai Katy , Scaletta Nicholas , Herrero Carmen , Lee In-Hong , Krause Anja , Ivashkiv Lionel B.
Published: April 2014

The balance between pro- and anti-inflammatory cytokines plays an important role in determining the severity of inflammation in rheumatoid arthritis (RA). Antagonism between opposing cytokines at the level of signal transduction plays an important role in many other systems. We have begun to explore the possible contribution of signal transduction cross-talk to cytokine balance in RA by examining the effects of IL-1, a proinflammatory cytokine, on the signaling and action of IL-6, a pleiotropic cytokine that has both pro- and anti-inflammatory actions, in RA synovial fibroblasts. Pretreatment with IL-1 suppressed Janus kinase-STAT signaling by IL-6, modified patterns of gene activation, and blocked IL-6 induction of tissue inhibitor of metalloproteases 1 expression. These results suggest that proinflammatory cytokines may contribute to pathogenesis by modulating or blocking signal transduction by pleiotropic or anti-inflammatory cytokines. The mechanism of inhibition did not require de novo gene activation and did not depend upon tyrosine phosphatase activity, but, instead, was dependent on the p38 stress kinase. These results identify a molecular basis for IL-1 and IL-6 cross-talk in RA synoviocytes and suggest that, in addition to levels of cytokine expression, modulation of signal transduction also plays a role in regulating cytokine balance in RA.
Dominant Autoimmune Epitopes Recognized by Pemphigus Antibodies Map to the N-Terminal Adhesive Region of Desmogleins
Authors: Sekiguchi Maiko , Futei Yuko , Fujii Yoshiko , Iwasaki Toshiro , Nishikawa Takeji , Amagai Masayuki
Published: April 2014

Desmoglein (Dsg) is a cadherin-type adhesion molecule found in desmosomes. Dsg1 and Dsg3 are the target Ags in the autoimmune blistering diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. To map conformational epitopes of Dsg1 and Dsg3 in PF and PV, we generated Dsg1- and Dsg3-domain-swapped molecules and point-mutated Dsg3 molecules with Dsg1-specific residues by baculovirus expression. The swapped domains were portions of the N-terminal extracellular domains of Dsg1 (1–496) and Dsg3 (1–566), which have similar structures but distinct epitopes. The binding of autoantibodies to the mutant molecules was assessed by competition ELISAs. Domain-swapped molecules containing the N-terminal 161 residues of Dsg1 and Dsg3 yielded >50% competition in 30/43 (69.8%) PF sera and 31/40 (77.5%) PV sera, respectively. Furthermore, removal of Abs against the 161 N-terminal residues of Dsg1 by immunoadsorption eliminated the ability of PF sera to induce cutaneous blisters in neonatal mice. Within these N-terminal regions, most of the epitopes were mapped to residues 26–87 of Dsg1 and 25–88 of Dsg3. Furthermore, a point-mutated Dsg3 molecule containing Dsg1-specific amino acid substitutions (His25, Cys28, Ala29) reacted with anti-Dsg1 IgG, thus defining one of the epitopes of Dsg1. Using the predicted three-dimensional structure of classic cadherins as a model, these findings suggest that the dominant autoimmune epitopes in both PF and PV are found in the N-terminal adhesive surfaces of Dsgs.
Autoantibodies to the Amino-Terminal Fragment of β-Fodrin Expressed in Glandular Epithelial Cells in Patients with Sjögren’s Syndrome
Authors: Kuwana Masataka , Okano Tetsuroh , Ogawa Yoko , Kaburaki Junichi , Kawakami Yutaka
Published: April 2014

Sjögrens’s syndrome (SS) is an autoimmune disease characterized by destruction of lacrimal and salivary glands, but the mechanisms underlying the disease process are unclear. By immunoscreening a HepG2 cDNA library with serum from an SS patient we isolated a cDNA encoding amino-terminal 616 aa of β-fodrin, a membrane skeleton protein associated with ion channels and pumps. Serum Ab to the amino-terminal fragment of β-fodrin was frequently detected in SS patients compared with rheumatic disease patients without SS or healthy controls (70 vs 12 or 4%; p < 0.00001). All the anti-β-fodrin-positive sera recognized the amino-terminal fragment with no homology to α-fodrin. Anti-β-fodrin Abs in patients’ sera as well as mouse polyclonal sera raised against the amino-terminal β-fodrin fragment did not react with intact β-fodrin, but recognized the 65-kDa amino-terminal fragment generated through cleavage by caspase-3 or granzyme B. When expression of intact and fragmented β-fodrin in lacrimal glands was assessed by immunohistochemistry, the antigenic amino-terminal fragment was distributed diffusely in acinar epithelial cell cytoplasm, whereas the carboxyl-terminal fragment and/or intact β-fodrin were localized in peripheral cytoplasm, especially at the basal membrane, in SS patients. In contrast, intact β-fodrin was detected primarily at the apical membrane of epithelia, and the amino-terminal fragment was scarcely detected in control patients with chronic graft-vs-host disease. These findings suggest that cleavage and altered distribution of β-fodrin in glandular epithelial cells may induce impaired secretory function and perpetuate an autoimmune response to β-fodrin, leading to autoantibody production and glandular destruction in SS.
Molecular and Immunological Characterization of Arginine Kinase from the Indianmeal Moth,Plodia interpunctella, a Novel Cross-Reactive Invertebrate Pan-Allergen
Authors: Binder Marina , Mahler Vera , Hayek Brigitte , Sperr Wolfgang R. , Schöller Matthias , Prozell Sabine , Wiedermann Gerhard , Valent Peter , Valenta Rudolf , Duchêne Michael
Published: April 2014

IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals. We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest. Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P. interpunctella larvae. cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated. Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography. Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions. It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients’ IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel. Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens. Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals.
Cutting Edge: IL-10-Producing CD4+ T Cells Mediate Tumor Rejection
Authors: Segal Benjamin M. , Glass Deborah D. , Shevach Ethan M.
Published: April 2014

IL-10 has potent immunosuppressive properties, and IL-10-producing CD4+ Tr1 cells have been characterized as regulators of Th1-mediated immunity. In this study, using a s.c. model of glioma cell growth in mice, we demonstrate that CD4+, but not CD8+, T cells play a critical role in tumor rejection following vaccination with irradiated glioma cells. Surprisingly, glioma-specific CD4+ T cells produce IL-10 but neither IL-4 nor IFN-γ, and glioma rejection is compromised in IL-10−/− hosts. Hence, our findings demonstrate that IL-10-producing CD4+ T cells can manifest antitumor functions and suggest that IL-10 may have proinflammatory effects in disease states.
Human Immature Dendritic Cells Efficiently Bind and Take up Secretory IgA Without the Induction of Maturation
Authors: Heystek Heleen C. , Moulon Corinne , Woltman Andrea M. , Garonne Pierre , van Kooten Cees
Published: April 2014

Immature dendritic cells (DC) reside in peripheral tissues, where they pick up and process incoming pathogens via scavenger receptors or FcR such as FcγR and FcεR. At mucosal surfaces, IgA is the main Ig to protect the body from incoming pathogens. In addition, DC are present in high numbers at these sites. We detected expression of FcαR (CD89) on the CD14+ population of CD34+ progenitor-derived DC and on monocyte-derived DC (MoDC). However, CD89 expression was strongly decreased upon differentiation from monocyte to DC. We found only minimal binding of serum IgA to MoDC but strong binding of secretory IgA (SIgA). The SIgA binding to MoDC could not be blocked by anti-CD89 blocking Abs. DC efficiently internalized SIgA, but not serum IgA, and uptake of SIgA could be blocked by specific sugars or partially by Ab reactive with mannose receptor. Importantly, binding and uptake of SIgA was not accompanied by signs of DC maturation, such as increased expression of CD86 and CD83 or induction of cytokine secretion. These data indicate that SIgA can interact with DC not via CD89, but via carbohydrate-recognizing receptors like mannose receptor and suggest that uptake of SIgA-containing immune complexes by immature DC may be a mechanism to modulate mucosal immune responses.
TCRβ Transmembrane Tyrosines Are Required for Pre-TCR Function
Authors: Spain Lisa M. , Liu Pinghu
Published: April 2014

The pre-TCR promotes thymocyte development in the αβ lineage. Productive rearrangement of the TCRβ locus triggers the assembly of the pre-TCR, which includes the pTα chain and CD3 εγδζ subunits. This complex receptor signals the up-regulation of CD4 and CD8 expression, thymocyte proliferation/survival, and the cessation of TCRβ rearrangements (allelic exclusion). In this study, we investigate the function of two conserved tyrosine residues located in the TCRβ chain transmembrane region of the pre-TCR. We show that replacement of both tyrosines with alanine and expression of the mutant receptor in RAG-1null thymocytes prevents surface expression and abolishes pre-TCR function relative to wild-type receptor. Replacement of both tyrosines with phenylalanines (YF double mutant) generates a complex phenotype in which thymocyte survival and proliferation are severely disrupted, differentiation is moderately disrupted, and allelic exclusion is unaffected. We further show that the YF double mutant receptor is expressed on the cell surface and associates with pTα and CD3ε at the same level as does wild-type TCRβ, while association of the YF double mutant with CD3ζ is slightly reduced relative to wild type. These data demonstrate that pre-TCR signaling pathways leading to proliferation and survival, differentiation, and allelic exclusion are differently sensitive to subtle mutation-induced alterations in pre-TCR structure.
Lactobacilli Differentially Modulate Expression of Cytokines and Maturation Surface Markers in Murine Dendritic Cells
Authors: Christensen Hanne R. , Frøkiær Hanne , Pestka James J.
Published: April 2014

Dendritic cells (DC) play a pivotal immunoregulatory role in the Th1, Th2, and Th3 cell balance and are present throughout the gastrointestinal tract. Thus, DC may be targets for modulation by gut microbes, including ingested probiotics. In the present study, we tested the hypothesis that species of Lactobacillus, important members of the gut flora, differentially activate DC. Bone marrow-derived murine DC were exposed to various lethally irradiated Lactobacillus spp. and resultant culture supernatants were analyzed for IL-6, IL-10, IL-12, and TNF-α. Substantial differences were found among strains in the capacity to induce IL-12 and TNF-α production in the DC. Similar but less pronounced differences were observed among lactobacilli in the induction of IL-6 and IL-10. Although all strains up-regulated surface MHC class II and B7-2 (CD86), which is indicative of DC maturation, those lactobacilli with greatest capacity to induce IL-12 were most effective. Remarkably, Lactobacillus reuteri DSM12246, a poor IL-12 inducer, inhibited IL-12, IL-6, and TNF-α induction by the otherwise strong cytokine inducer L. casei CHCC3139, while IL-10 production remained unaltered. In analogous fashion, L. reuteri reduced L. casei-induced up-regulation of B7-2. These results suggest that different species of Lactobacillus exert very different DC activation patterns and, furthermore, at least one species may be capable of inhibiting activities of other species in the genus. Thus, the potential exists for Th1/Th2/Th3-driving capacities of the gut DC to be modulated according to composition of gut microflora, including ingested probiotics.
Chronic Soluble Antigen Sensitization Primes a Unique Memory/Effector T Cell Repertoire Associated with Th2 Phenotype Acquisition In Vivo
Authors: Foucras Gilles , Gallard Alexandra , Coureau Christiane , Kanellopoulos Jean-M. , Guéry Jean-Charles
Published: April 2014

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vβ repertoire, different from the TCR Vβ profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IAd-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.
Elevated NF-κB Activation in Nonobese Diabetic Mouse Dendritic Cells Results in Enhanced APC Function
Authors: Poligone Brian , Weaver Donald J. , Sen Pradip , Baldwin Albert S. , Tisch Roland
Published: April 2014

We have recently demonstrated that dendritic cells (DC) prepared from nonobese diabetic (NOD) mice, a spontaneous model for insulin-dependent diabetes mellitus, exhibit elevated levels of NF-κB activation upon stimulation. In the current study, we investigated the influence of dysregulation of NF-κB activation on the APC function of bone marrow-derived DC prepared from NOD vs BALB/c and nonobese diabetes-resistant mice. NOD DC pulsed with either peptide or virus were found to be more efficient than BALB/c DC at stimulating in vitro naive Ag-specific CD8+ T cells. The T cell stimulatory capacity of NOD DC was suppressed by gene transfer of a modified form of IκBα, indicating a direct role for NF-κB in this process. Furthermore, neutralization of IL-12(p70) to block autocrine-mediated activation of DC also significantly reduced the capacity of NOD DC to stimulate T cells. Despite a reduction in low molecular mass polypeptide-2 expression relative to BALB/c DC, no effect on proteasome-dependent events associated with the NF-κB signaling pathway or Ag processing was detected in NOD DC. Finally, DC from nonobese diabetes-resistant mice, a strain genotypically similar to NOD yet disease resistant, resembled BALB/c and not NOD DC in terms of the level of NF-κB activation, secretion of IL-12(p70) and TNF-α, and the capacity to stimulate T cells. Therefore, elevated NF-κB activation and enhanced APC function are specific for the NOD genotype and correlate with the progression of insulin-dependent diabetes mellitus. These results also provide further evidence indicating a key role for NF-κB in regulating the APC function of DC.
Contributions from Self-Renewal and Trafficking to the Uterine NK Cell Population of Early Pregnancy
Authors: Chantakru Sirirak , Miller Craig , Roach Lindsay E. , Kuziel William A. , Maeda Nobuyo , Wang Wan-Chao , Evans Sharon S. , Croy B. Anne
Published: April 2014

Uterine NK (uNK) cells are abundant in human and murine uteri during decidualization. It is unclear whether precursors of uNK (pre-uNK) cells self-renew or are recruited from other sites. To assess self-renewal of pre-uNK cells, uterine segments from NK cell-competent mice were grafted orthotopically into NK/uNK cell-deficient or wild-type mice. Only in wild-type recipients did decidualized grafts contain uNK cells, indicating that pre-uNK cells do not self-renew in uterus. To identify pre-uNK cell sources, thymus, bone marrow, lymph node, or spleen cells were grafted from virgin or pregnant NK cell-competent donors into mated NK/uNK cell-deficient recipients. Cells from secondary lymphoid tissues of pregnant donors gave high level uNK cell reconstitution, which was independent of chemokine receptors CCR2 or CCR5. Pregnancy-induced changes to lymphocyte-endothelial cell interactions were documented using adhesion of human lymphocytes to frozen mouse tissue sections under shear. A dynamic increase was observed in L-selectin- and α4 integrin-dependent adhesion of CD56bright NK cells to decidualizing uterus and in human PBL adhesion to lymph node endothelium. These data support a model that attributes the dramatic increases in human and murine uNK cells during decidualization to precursor cell recruitment.
Influence of the Isotype of the Light Chain on the Properties of IgG
Authors: Montaño Ramon F. , Morrison Sherie L.
Published: April 2014

It is widely appreciated that the isotype of the H chain of the Ab molecule influences its functional properties. We have now investigated the contribution of the isotype of the L chain to the structural and functional properties of the Ab molecule. In these studies, the L chain variable region of a murine anti-dansyl Ab was joined to either human κ or λ constant region domains and expressed with mouse-human chimeric H chains of the four human IgG isotypes. The resulting Abs were secreted as fully assembled molecules although, as has been previously observed, IgG4 with either κ or λ L chains was also secreted as HL half-molecules. However, the isotype of the L chain can influence the kinetics of intracellular assembly with IgG1λ, IgG2λ, and IgG4λ assembling more slowly than their κ counterparts. The isotype of the L chain also influenced the susceptibility of the interchain disulfide bonds to attack by reducing agents with variable effects, depending on the isotype of the H chains. For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain influenced the rate of clearance in mice, with IgG2λ having a shorter in vivo half-life than IgG2κ. Only slight differences were also observed between λ and κ molecules in their kinetics of binding to and dissociation from the hapten dansyl. These studies demonstrate that the isotype of the L chain has only a slight impact on the structural and functional properties of variable region identical Abs.
HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) Use the Same Anchor Residues for Binding an Immunodominant Peptide Derived from Human Type II Collagen
Authors: Rosloniec Edward F. , Whittington Karen B. , Zaller Dennis M. , Kang Andrew H.
Published: April 2014

Rheumatoid arthritis is an autoimmune disease in which susceptibility is strongly associated with the expression of specific HLA-DR haplotypes, including DR1 (DRB1*0101) and DR4 (DRB1*0401). As transgenes, both of these class II molecules mediate susceptibility to an autoimmune arthritis induced by immunization with human type II collagen (hCII). The dominant T cell response of both the DR1 and DR4 transgenic mice to hCII is focused on the same determinant core, CII(263–270). Peptide binding studies revealed that the affinity of DR1 and DR4 for CII(263–270) was at least 10 times less than that of the model Ag HA(307–319), and that the affinity of DR4 for the CII peptide is 3-fold less than that of DR1. As predicted based on the crystal structures, the majority of the CII-peptide binding affinity for DR1 and DR4 is controlled by the Phe263; however, unexpectedly the adjacent Lys264 also contributed significantly to the binding affinity of the peptide. Only these two CII amino acids were found to provide binding anchors. Amino acid substitutions at the remaining positions had either no effect or significantly increased the affinity of the hCII peptide. Affinity-enhancing substitutions frequently involved replacement of a negative charge, or Gly or Pro, hallmark amino acids of CII structure. These data indicate that DR1 and DR4 bind this CII peptide in a nearly identical manner and that the primary structure of CII may dictate a different binding motif for DR1 and DR4 than has been described for other peptides that bind to these alleles.
How H13 Histocompatibility Peptides Differing by a Single Methyl Group and Lacking Conventional MHC Binding Anchor Motifs Determine Self-Nonself Discrimination
Authors: Ostrov David A. , Roden Matthew M. , Shi Wuxian , Palmieri Edith , Christianson Gregory J. , Mendoza Lisa , Villaflor Gilbert , Tilley Darcie , Shastri Nilabh , Grey Howard , Almo Steven C. , Roopenian Derry , Nathenson Stanley G.
Published: April 2014

The mouse H13 minor histocompatibility (H) Ag, originally detected as a barrier to allograft transplants, is remarkable in that rejection is a consequence of an extremely subtle interchange, P4Val/Ile, in a nonamer H2-Db-bound peptide. Moreover, H13 peptides lack the canonical P5Asn central anchor residue normally considered important for forming a peptide/MHC complex. To understand how these noncanonical peptide pMHC complexes form physiologically active TCR ligands, crystal structures of allelic H13 pDb complexes and a P5Asn anchored pDb analog were solved to high resolution. The structures show that the basis of TCRs to distinguish self from nonself H13 peptides is their ability to distinguish a single solvent-exposed methyl group. In addition, the structures demonstrate that there is no need for H13 peptides to derive any stabilization from interactions within the central C pocket to generate fully functional pMHC complexes. These results provide a structural explanation for a classical non-MHC-encoded H Ag, and they call into question the requirement for contact between anchor residues and the major MHC binding pockets in vaccine design.
Ceramide-Rich Membrane Rafts Mediate CD40 Clustering
Authors: Grassmé Heike , Jendrossek Verena , Bock Jürgen , Riehle Andrea , Gulbins Erich
Published: April 2014

Many receptor systems use receptor clustering for transmembrane signaling. In this study, we show that acid sphingomyelinase (ASM) is essential for the clustering of CD40. Stimulation of lymphocytes via CD40 ligation results in ASM translocation from intracellular stores, most likely vesicles, into distinct membrane domains on the extracellular surface of the plasma membrane. Surface ASM initiates a release of extracellularly oriented ceramide, which in turn mediates CD40 clustering in sphingolipid-rich membrane domains. ASM, ceramide, and CD40 colocalize in the cap-like structure of stimulated cells. Deficiency of ASM, destruction of sphingolipid-rich rafts, or neutralization of surface ceramide prevents CD40 clustering and CD40-initiated cell signaling. These findings indicate that the ASM-mediated release of ceramide and/or metabolites of ceramide regulate clustering of CD40, which seems to be a prerequisite for cellular activation via CD40.
Regulation of Human β-Defensin-2 in Gingival Epithelial Cells: The Involvement of Mitogen-Activated Protein Kinase Pathways, But Not the NF-κB Transcription Factor Family
Authors: Krisanaprakornkit Suttichai , Kimball Janet R. , Dale Beverly A.
Published: April 2014

Stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by neutrophils and epithelial factors, including antimicrobial peptides of the β-defensin family. Previous work has shown that multiple signaling pathways are involved in human β-defensin (hBD)-2 mRNA regulation in human gingival epithelial cells stimulated with a periodontal bacterium, Fusobacterium nucleatum, and other stimulants. The goal of this study was to further characterize these pathways. The role of NF-κB in hBD-2 regulation was investigated initially due to its importance in inflammation and infection. Nuclear translocation of p65 and NF-κB activation was seen in human gingival epithelial cells stimulated with F. nucleatum cell wall extract, indicating possible involvement of NF-κB in hBD-2 regulation. However, hBD-2 induction by F. nucleatum was not blocked by pretreatment with two NF-κB inhibitors, pyrrolidine dithiocarbamate and the proteasome inhibitor, MG132. To investigate alternative modes of hBD-2 regulation, we explored involvement of mitogen-activated protein kinase pathways. F. nucleatum activated p38 and c-Jun NH2-terminal kinase (JNK) pathways, whereas it had little effect on p44/42. Furthermore, inhibition of p38 and JNK partially blocked hBD-2 mRNA induction by F. nucleatum, and the combination of two inhibitors completely blocked expression. Our results suggest that NF-κB is neither essential nor sufficient for hBD-2 induction, and that hBD-2 regulation by F. nucleatum is via p38 and JNK, while phorbol ester induces hBD-2 via the p44/42 extracellular signal-regulated kinase pathway. Studies of hBD-2 regulation provide insight into how its expression may be enhanced to control infection locally within the mucosa and thereby reduce microbial invasion into the underlying tissue.
IFN-α Secretion by Type 2 Predendritic Cells Up-Regulates MHC Class I in the HIV-1-Infected Thymus
Authors: Keir Mary E. , Stoddart Cheryl A. , Linquist-Stepps Valerie , Moreno Mary E. , McCune Joseph M.
Published: April 2014

The ability of HIV-1 to evade the host immune response leads to the establishment of chronic infection. HIV-1 has been reported to up-regulate MHC I molecules on the surface of thymocytes from HIV-1-infected thymus. We demonstrate in this study that HIV-1 up-regulates MHC I on both HIV-1-infected and uninfected thymocytes in a manner that is independent of Nef, proportional to viral replication, and entirely mediated by IFN-α. IL-3Rα+ type 2 predendritic cells (preDC2) resident in the thymic medulla secrete IFN-α, which acts on IFN-αβR-expressing immature thymocytes to induce MHC I expression. Furthermore, thymic preDC2 are permissive for HIV-1 infection and positive for intracellular p24. These data demonstrate the ability of IFN-α secreted by preDC2 to induce MHC I up-regulation in the HIV-1-infected human thymus.
Intensity of the Vaccine-Elicited Immune Response Determines Tumor Clearance
Authors: Perez-Diez Ainhoa , Spiess Paul J. , Restifo Nicholas P. , Matzinger Polly , Marincola Francesco M.
Published: April 2014

Tumor Ag-specific vaccines used for cancer immunotherapy can generate specific CD8 responses detectable in PBMCs and in tumor-infiltrating lymphocytes. However, human studies have shown that detection of a systemic vaccine-induced response does not necessarily correlate with the occasional instances of tumor rejection. Because this discrepancy might partially be attributable to the genetic heterogeneity of human cancers, as well as to the immunosuppressive effects of previous treatments, we turned to a mouse model in which these variables could be controlled to determine whether a relationship exists between the strength of vaccine-induced immune responses and tumor rejection. We challenged mice with the β-galactosidase (β-gal)-expressing tumor cells, C25.F6, vaccinated them with β-gal-carrying viral vectors, and used quantitative RT-PCR to measure the vaccine-induced immune response of splenocytes directly ex vivo. We found that the strength of the response increased with increasing doses of β-gal-carrying vector and/or upon boosting with a heterologous β-gal-carrying virus. Most importantly, we found that the strength of the detected immune response against this foreign Ag strongly correlated with reduction in the number of lung metastases. The results from this mouse model have major implications for the implementation of tumor vaccines in humans.
The Protective Role of Nitric Oxide in a Neurotoxicant- Induced Demyelinating Model
Authors: Arnett Heather A. , Hellendall Ron P. , Matsushima Glenn K. , Suzuki Kinuko , Laubach Victor E. , Sherman Paula , Ting Jenny P.-Y.
Published: April 2014

Demyelination is often associated with acute inflammatory events involving the recruitment-activation of microglia/macrophage, astrocytes, and leukocytes. The ultimate role of inflammatory products in demyelinating disease and in the survival of oligodendrocytes, the myelin forming cells, is unresolved. The current study examines the role of inducible NO synthase (iNOS)-derived NO in a neurotoxicant-induced model of demyelination. NO levels were greatly elevated in the midline corpus callosum during demyelination in genetically intact C57BL/6 mice, and this NO was due solely to the induction of iNOS, as the correlates of NO were not found in mice lacking iNOS. C57BL/6 mice lacking iNOS exhibited more demyelination, but did not display an increased overall cellularity in the corpus callosum, attributable to an unimpeded microglia/macrophage presence. An enhanced course of pathology was noted in mice lacking iNOS. This was associated with a greater depletion of mature oligodendrocytes, most likely due to apoptosis of oligodendrocytes. Microglia and astrocytes did not undergo apoptosis during treatment. Our results suggest a moderately protective role for NO during acute inflammation-association demyelination.
Multilevel Dysregulation of STAT3 Activation in Anaplastic Lymphoma Kinase-Positive T/Null-Cell Lymphoma
Authors: Zhang Qian , Raghunath Puthryaveett N. , Xue Liquan , Majewski Miroslaw , Carpentieri David F. , Odum Niels , Morris Stephan , Skorski Tomasz , Wasik Mariusz A.
Published: April 2014

Accumulating evidence indicates that expression of anaplastic lymphoma kinase (ALK), typically due to t(2;5) translocation, defines a distinct type of T/null-cell lymphoma (TCL). The resulting nucleophosmin (NPM) /ALK chimeric kinase is constitutively active and oncogenic. Downstream effector molecules triggered by NPM/ALK remain, however, largely unidentified. Here we report that NPM/ALK induces continuous activation of STAT3. STAT3 displayed tyrosine phosphorylation and DNA binding in all (four of four) ALK+ TCL cell lines tested. The activation of STAT3 was selective because none of the other known STATs was consistently tyrosine phosphorylated in these cell lines. In addition, malignant cells in tissue sections from all (10 of 10) ALK+ TCL patients expressed tyrosine-phosphorylated STAT3. Transfection of BaF3 cells with NPM/ALK resulted in tyrosine phosphorylation of STAT3. Furthermore, STAT3 was constitutively associated with NPM/ALK in the ALK+ TCL cell lines. Additional studies into the mechanisms of STAT3 activation revealed that the ALK+ TCL cells expressed a positive regulator of STAT3 activation, protein phosphatase 2A (PP2A), which was constitutively associated with STAT3. Treatment with the PP2A inhibitor calyculin A abrogated tyrosine phosphorylation of STAT3. Finally, ALK+ T cells failed to express a negative regulator of activated STAT3, protein inhibitor of activated STAT3. These data indicate that NPM/ALK activates STAT3 and that PP2A and lack of protein inhibitor of activated STAT3 may be important in maintaining STAT3 in the activated state in the ALK+ TCL cells. These results also suggest that activated STAT3, which is known to display oncogenic properties, as well as its regulatory molecules may represent attractive targets for novel therapies in ALK+ TCL.
Cutting Edge: CpG DNA Inhibits Dendritic Cell Apoptosis by Up-Regulating Cellular Inhibitor of Apoptosis Proteins Through the Phosphatidylinositide-3′-OH Kinase Pathway
Authors: Park Yunji , Lee Seung Woo , Sung Young Chul
Published: April 2014

CpG DNA has been recognized as a powerful stimulant of dendritic cells (DCs). In this study, we demonstrate that CpG DNA inhibits spontaneous apoptosis of DCs. CpG DNA up-regulated cellular inhibitor of apoptosis proteins (cIAPs) as well as Bcl-2 and Bcl-xL, but down-regulated active caspase-3. Although CpG DNA activated p38 mitogen-activated protein kinase, extracellular signal-related kinase, and phosphatidylinositide-3′-OH kinase (PI3K), only the blocking of PI3K inhibited the CpG DNA-induced DC survival. Moreover, while the expression of Bcl-2 and Bcl-xL depends on both PI3K and p38 mitogen-activated protein kinase, the up-regulation of cIAPs and the down-regulation of active caspase-3 by CpG DNA require PI3K activation, suggesting PI3K-dependent up-regulation of cIAPs in the antiapoptotic activity of CpG DNA in DCs. This study indicates that CpG DNA provides a survival signal to DCs, which might be one of mechanisms by which bacterial DNA stimulates and maintains the innate immune responses.
Decreased IL-15 May Contribute to Elevated IgE and Acute Inflammation in Atopic Dermatitis
Authors: Ong Peck Y. , Hamid Qutayba A. , Travers Jeffrey B. , Strickland Ian , Kerithy Muhamed Al , Boguniewicz Mark , Leung Donald Y. M.
Published: April 2014

PBMC and acute skin lesions of patients with atopic dermatitis (AD) are characterized by increased IL-4 and IL-13, but decreased IFN-γ production. This bias toward an increased Th2 cytokine profile may contribute to the elevated IgE levels and acute skin inflammation seen in AD. In this study, we examined the levels of IL-15, a Th1-like cytokine, in the PBMC and the skin lesions of AD patients. IL-15 secretion by Staphylococcal enterotoxin B-treated PBMC of AD patients was significantly lower than that of normals and psoriasis patients (p < 0.001). Membrane-bound IL-15 expression as measured by mean fluorescence intensity and percentage of IL-15-positive cells in Staphylococcal enterotoxin B-treated monocytes of AD patients (644 ± 49% and 12.7 ± 0.6%, respectively) were significantly lower than that of normals (869 ± 56% and 15.8 ± 1.2%, respectively) and psoriasis patients (1488 ± 217% and 22.7 ± 0.8%, respectively; p < 0.0007 and p < 0.0001, respectively). The membrane-bound IL-15 expression was also significantly lower in the control monocytes of AD patients compared with that in normals and psoriasis patients. There was no significant difference in the absolute number or percentage of monocytes between the study subjects. However, psoriasis skin lesions were found to have significantly more IL-15 mRNA-expressing cells (22.4 ± 1.7) compared with that in acute AD (7.5 ± 1.7) and chronic AD (13.7 ± 1.7) skin lesions (p < 0.05). IL-15 enhanced IFN-γ production by the PBMC of AD patients (p < 0.01), but not by that of normal individuals or psoriasis patients. In addition, IL-15 was found to suppress IgE synthesis (p < 0.01) by the PBMC of AD patients. These data support the concept that reduced IL-15 expression may contribute to the pathogenesis of AD.
Identification of Multiple Isolated Lymphoid Follicles on the Antimesenteric Wall of the Mouse Small Intestine
Authors: Hamada Hiromasa , Hiroi Takachika , Nishiyama Yasuhiro , Takahashi Hidemi , Masunaga Yohei , Hachimura Satoshi , Kaminogawa Shuichi , Takahashi-Iwanaga Hiromi , Iwanaga Toshihiko , Kiyono Hiroshi , Yamamoto Hiroshi , Ishikawa Hiromichi
Published: April 2014

We have revealed that 100–200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+CD19+CD23+IgMlowIgDhighCD5−Mac-1− phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2−/−, TCR-β−/−, and Ig μ-chain mutant (μm−/−) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+B220− in RAG-2−/−, TCR-β−/−, and μm−/− mice. ILF develop normally in the progeny of transplacentally manipulated Peyer’s patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Rα−/− PPnull mice. Neither ILF nor PP are detectable in lymphotoxin α−/− and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor γ-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.
Cutting Edge: Ectopic Expression of CD40 Ligand on B Cells Induces Lupus-Like Autoimmune Disease
Authors: Higuchi Tetsuya , Aiba Yuichi , Nomura Takashi , Matsuda Junichiro , Mochida Keiji , Suzuki Misao , Kikutani Hitoshi , Honjo Tasuku , Nishioka Kiyoshi , Tsubata Takeshi
Published: April 2014

CD40 ligand (CD40L) is ectopically expressed on B cells in patients with systemic lupus erythematosus (SLE) and lupus-prone BXSB mice. To assess the role of the ectopic CD40L expression in development of SLE, we have established transgenic mice expressing CD40L on B cells. Some of the 12- to 14-mo-old CD40L-transgenic mice spontaneously produced autoantibodies such as antinuclear Abs, anti-DNA Abs, and antihistone Abs. Moreover, approximately half of the transgenic mice developed glomerulonephritis with immune-complex deposition, whereas the kidneys of the normal littermates showed either no pathological findings or only mild histological changes. These results indicate that CD40L on B cells causes lupus-like disease in the presence of yet unknown environmental factors that by themselves do not induce the disease. Thus, ectopic CD40L expression on B cells may play a crucial role in development of SLE.
Activation-Induced Expression of Carcinoembryonic Antigen-Cell Adhesion Molecule 1 Regulates Mouse T Lymphocyte Function
Authors: Nakajima Atsushi , Iijima Hideki , Neurath Markus F. , Nagaishi Takashi , Nieuwenhuis Edward E. S. , Raychowdhury Raktima , Glickman Jonathan , Blau Dianna M. , Russell Sara , Holmes Kathryn V. , Blumberg Richard S.
Published: April 2014

Carcinoembryonic Ag cell adhesion molecule 1 (CEACAM1) consists of highly related homologs in humans and rodents that are characterized by significant alternate splicing generating isoforms capable of negative intracellular signaling by virtue of two immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic (cyt) tail. Although human T cells have been recently observed to express CEACAM1, the expression and function of CEACAM1 in mouse T cells have not been defined. Although resting mouse spleen T cells exhibited no evidence of CEACAM1 on the cell surface, CEACAM1 was rapidly up-regulated on CD4+ and CD8+ T cells after activation with either Con A or anti-CD3 without a requirement for either de novo transcription or translation due to the fact that CEACAM1 was present intracellularly before activation. Using a GST-CEACAM1-cytoplasmic tail fusion protein, it was shown that the cytoplasmic tail of CEACAM1 bound the src homology domain-containing phosphatase 1 and adaptor protein 1 complex in its phosphorylated and nonphosphorylated states, respectively. CEACAM1 ligation with an anti-CEACAM1 mAb resulted in inhibition of an allogeneic MLR and anti-CD3 plus anti-CD28 Ab-induced proliferation of spleen T cells in vitro and inhibition of a delayed-type hypersensitivity response to oxazolone in vivo. Inhibition of the delayed-type hypersensitivity response required that the anti-CEACAM1-specific mAb be present at the time of T cell sensitization. These studies support a role for CEACAM1 as a novel class of immunoreceptor tyrosine-based inhibition motif-bearing regulatory molecules on T cells that are active during early phases of the immune response in mice.
Does CD40 Ligation Induce B Cell Negative Selection?
Authors: Martínez-Barnetche Jesús , Madrid-Marina Vicente , Flavell Richard A. , Moreno José
Published: April 2014

Binding of CD154 to its receptor, CD40, provides costimulation for mature B cell activation and differentiation in response to Ag receptor signals. In mice, early B cell precursors express CD40, but its function at this stage is unknown. We examined the effects of CD40 ligation during B cell ontogeny in transgenic mice constitutively expressing CD154 on B cells (κEP-CD154). Precursors beyond pro-B cells were absent in adult bone marrow but were increased in the fetal liver. Newborn κEP-CD154 mice had largely increased numbers of peripheral B cells, which were CD154+, and that 36 h after birth expressed high surface levels of CD23 and MHC class II, resembling activated mature B cells. Nevertheless, κEP-CD154 mice were hypogammaglobulinemic, indicating that the expanded population of apparently activated B cells was nonfunctional. Further analysis revealed that soon after birth, κEP-CD154 mice-derived B cells became CD5+/Fas+, after which progressively decreased in the periphery in a CD154-CD40-dependent manner. These results indicate that CD40 ligation during B cell ontogeny induces negative selection characterized by either hyporesponsiveness or an arrest in maturation depending on the time of analysis and the anatomic site studied.