bluuebunny/crossref_metadata_2025 huggingface dataset:
Transporter Associated with Antigen Processing Preselection of Peptides Binding to the MHC: A Bioinformatic Evaluation
Authors: Doytchinova Irini , Hemsley Shelley , Flower Darren R.
Published: April 2014

TAP is responsible for the transit of peptides from the cytosol to the lumen of the endoplasmic reticulum. In an immunological context, this event is followed by the binding of peptides to MHC molecules before export to the cell surface and recognition by T cells. Because TAP transport precedes MHC binding, TAP preferences may make a significant contribution to epitope selection. To assess the impact of this preselection, we have developed a scoring function for TAP affinity prediction using the additive method, have used it to analyze and extend the TAP binding motif, and have evaluated how well this model acts as a preselection step in predicting MHC binding peptides. To distinguish between MHC alleles that are exclusively dependent on TAP and those exhibiting only a partial dependence on TAP, two sets of MHC binding peptides were examined: HLA-A*0201 was selected as a representative of partially TAP-dependent HLA alleles, and HLA-A*0301 represented fully TAP-dependent HLA alleles. TAP preselection has a greater impact on TAP-dependent alleles than on TAP-independent alleles. The reduction in the number of nonbinders varied from 10% (TAP-independent) to 33% (TAP-dependent), suggesting that TAP preselection is an important component in the successful in silico prediction of T cell epitopes.
SHIP2 Is Recruited to the Cell Membrane upon Macrophage Colony-Stimulating Factor (M-CSF) Stimulation and Regulates M-CSF-Induced Signaling
Authors: Wang Yijie , Keogh Rosemary J. , Hunter Melissa G. , Mitchell Christina A. , Frey Randall S. , Javaid Kamran , Malik Asrar B. , Schurmans Stéphane , Tridandapani Susheela , Marsh Clay B.
Published: April 2014

The Src homology 2-containing inositol phosphatase SHIP1 functions in hemopoietic cells to limit activation events mediated by PI3K products, including Akt activation and cell survival. In contrast to the limited cellular expression of SHIP1, the related isoform SHIP2, is widely expressed in both parenchymal and hemopoietic cells. The goal of this study was to determine how SHIP2 functions to regulate M-CSF signaling. We report that 1) SHIP2 was tyrosine-phosphorylated in M-CSF-stimulated human alveolar macrophages, human THP-1 cells, murine macrophages, and the murine macrophage cell line RAW264; 2) SHIP2 associated with the M-CSF receptor after M-CSF stimulation; and 3) SHIP2 associated with the actin-binding protein filamin and localization to the cell membrane, requiring the proline-rich domain, but not on the Src homology 2 domain of SHIP2. Analyzing the function of SHIP2 in M-CSF-stimulated cells by expressing either wild-type SHIP2 or an Src homology 2 domain mutant of SHIP2 reduced Akt activation in response to M-CSF stimulation. In contrast, the expression of a catalytically deficient mutant of SHIP2 or the proline-rich domain of SHIP2 enhanced Akt activation. Similarly, the expression of wild-type SHIP2 inhibited NF-κB-mediated gene transcription. Finally, fetal liver-derived macrophages from SHIP2 gene knockout mice enhanced activation of Akt in response to M-CSF treatment. These data suggest a novel regulatory role for SHIP2 in M-CSF-stimulated myeloid cells.
The Glycosylation of Human Serum IgD and IgE and the Accessibility of Identified Oligomannose Structures for Interaction with Mannan-Binding Lectin
Authors: Arnold James N. , Radcliffe Catherine M. , Wormald Mark R. , Royle Louise , Harvey David J. , Crispin Max , Dwek Raymond A. , Sim Robert B. , Rudd Pauline M.
Published: April 2014

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn354 are inaccessible because the complex glycans at Asn445 block access to the site. On IgE, the additional CH2 hinge domain blocks access to the oligomannose glycans at Asn394 on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man5GlcNAc2 glycans, which are the trimmed products of the Glc3Man9GlcNAc2 oligomannose precursor. The presence of these structures suggests that the CH2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man5GlcNAc2 during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.
The Membrane-Proximal Immunoreceptor Tyrosine-Based Inhibitory Motif Is Critical for the Inhibitory Signaling Mediated by Siglecs-7 and -9, CD33-Related Siglecs Expressed on Human Monocytes and NK Cells
Authors: Avril Tony , Floyd Helen , Lopez Frederic , Vivier Eric , Crocker Paul R.
Published: April 2014

Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and -9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are able to inhibit the FcεRI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that “inside-out” signaling may occur to regulate ligand binding.
RON Receptor Tyrosine Kinase, a Negative Regulator of Inflammation, Inhibits HIV-1 Transcription in Monocytes/Macrophages and Is Decreased in Brain Tissue from Patients with AIDS
Authors: Lee Eileen S. , Kalantari Parisa , Tsutsui Shigeki , Klatt Alicia , Holden Janet , Correll Pamela H. , Power Christopher , Henderson Andrew J.
Published: April 2014

Activation of macrophages and microglia cells after HIV-1 infection and their production of inflammatory mediators contribute to HIV-associated CNS diseases. The mechanisms that initiate and maintain inflammation after HIV-1 infection in the brain have not been well studied. Furthermore, it is not understood why in HIV-associated CNS disease, macrophages and microglia are biased toward inflammation rather than production of mediators that control inflammation. We have focused on the receptor tyrosine kinase RON, a critical negative regulator of macrophage function and inflammation, to determine whether this receptor regulates HIV-1 expression. Overexpressing RON in monocytes/macrophages demonstrates that RON inhibits HIV-1 proviral transcription in part by decreasing the binding activity of NF-κB to the HIV-1 long terminal repeat. Because macrophages and microglia cells are a critical reservoir for HIV-1 in the CNS, we examined brain tissues for RON expression and detected RON in astrocytes, cortical neurons, and monocytoid cells. RON was detected in all control patients who were HIV seronegative (n = 7), whereas six of nine brain samples obtained from AIDS patients exhibited reduced RON protein. These data suggest that RON initiates signaling pathways that negatively regulate HIV-1 transcription in monocytes/macrophages and that HIV-1 suppresses RON function by decreasing protein levels in the brain to assure efficient replication. Furthermore, HIV-1 infection would compromise the ability of RON to protect against inflammation and consequent CNS damage.
Activation of Virus-Specific CD8+ T Cells by Lipopolysaccharide-Induced IL-12 and IL-18
Authors: Raué Hans-Peter , Brien James D. , Hammarlund Erika , Slifka Mark K.
Published: April 2014

Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-γ production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8+ T cells directly ex vivo. Following acute viral infection, 70–80% of virus-specific T cells will produce IFN-γ after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-γ production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.
TLR-Independent Induction of Dendritic Cell Maturation and Adaptive Immunity by Negative-Strand RNA Viruses
Authors: López Carolina B. , Moltedo Bruno , Alexopoulou Lena , Bonifaz Laura , Flavell Richard A. , Moran Thomas M.
Published: April 2014

TLR signaling leads to dendritic cell (DC) maturation and immunity to diverse pathogens. The stimulation of TLRs by conserved viral structures is the only described mechanism leading to DC maturation after a virus infection. In this report, we demonstrate that mouse myeloid DCs mature normally after in vivo and in vitro infection with Sendai virus (SeV) in the absence of TLR3, 7, 8, or 9 signaling. DC maturation by SeV requires virus replication not necessary for TLR-mediated triggering. Moreover, DCs deficient in TLR signaling efficiently prime for Th1 immunity after infection with influenza or SeV, generating IFN-γ-producing T cells, CTLs and antiviral Abs. We have previously demonstrated that SeV induces DC maturation independently of the presence of type I IFN, which has been reported to mature DCs in a TLR-independent manner. The data presented here provide evidence for the existence of a novel intracellular pathway independent of TLR-mediated signaling responsible for live virus triggering of DC maturation and demonstrate its critical role in the onset of antiviral immunity. The revelation of this pathway should stimulate invigorating research into the mechanism for virus-induced DC maturation and immunity.
Nitric Oxide Inhibits IgE-Dependent Cytokine Production and Fos and Jun Activation in Mast Cells
Authors: Davis Beverley J. , Flanagan Brian F. , Gilfillan Alasdair M. , Metcalfe Dean D. , Coleman John W.
Published: April 2014

NO is a cell-derived radical reported to inhibit mast cell degranulation and subsequent allergic inflammation, although whether its action is nonspecific or occurs via specific molecular mechanisms remains unknown. To examine this question, we set out to determine whether NO inhibits mast cell cytokine production, and, if so, whether it also alters FcεRI-dependent signal transduction. As hypothesized, the radical inhibited IgE/Ag-induced IL-4, IL-6, and TNF production. Although NO did not influence phosphorylated JNK, p38 MAPK, or p44/42 MAPK, it did inhibit phosphorylation of phospholipase Cγ1 and the AP-1 transcription factor protein c-Jun, but not NF-κB or CREB. NO further completely abrogated IgE/Ag-induced DNA-binding activity of the nuclear AP-1 proteins Fos and Jun. These results show that NO is capable of inhibiting FcεRI-dependent mast cell cytokine production at the level of gene regulation, and suggest too that NO may contribute to resolution of allergic inflammation.
Platelet-Activating Factor and Kinin-Dependent Vascular Leakage as a Novel Functional Activity of the Soluble Terminal Complement Complex
Authors: Bossi Fleur , Fischetti Fabio , Pellis Valentina , Bulla Roberta , Ferrero Elisabetta , Mollnes Tom Eirik , Regoli Domenico , Tedesco Francesco
Published: April 2014

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.
A Novel Mechanism for TNF-α Regulation by p38 MAPK: Involvement of NF-κB with Implications for Therapy in Rheumatoid Arthritis
Authors: Campbell Jamie , Ciesielski Cathleen J. , Hunt Abigail E. , Horwood Nicole J. , Beech Jonathan T. , Hayes Louise A. , Denys Agnes , Feldmann Marc , Brennan Fionula M. , Foxwell Brian M. J.
Published: April 2014

TNF-α is a key factor in a variety of inflammatory diseases. This study examines the role of p38 MAPK in the regulation of TNF-α in primary human cells relevant to inflammation, e.g., macrophages and rheumatoid synovial cells. Using a dominant negative variant (D168A) of p38 MAPK and a kinase inhibitor, SB203580, we confirm in primary human macrophages that p38 MAPK regulates TNF-α production using a posttranscriptional mechanism requiring the 3′ untranslated region of the gene. However, in LPS-activated primary human macrophages we also detect a second previously unidentified mechanism, the p38 MAPK modulation of TNF-α transcription. This is mediated through p38 MAPK regulation of NF-κB. Interestingly this mechanism was not observed in rheumatoid synovial cells. Importantly however, the dominant negative mutant of p38 MAPK, but not SB203580 was effective at inhibiting spontaneous TNF-α production in these ex vivo rheumatoid synovial cell cultures. These data indicate there are potential major differences in the role of p38 MAPK in inflammatory signaling that have a bearing on the use of this kinase as a target for therapy. These results indicate despite disappointing results with p38 MAPK inhibitors in the clinic, this kinase is a valid target in rheumatoid disease.
Interaction of Soluble Form of Recombinant Extracellular TLR4 Domain with MD-2 Enables Lipopolysaccharide Binding and Attenuates TLR4-Mediated Signaling
Authors: Hyakushima Naoki , Mitsuzawa Hiroaki , Nishitani Chiaki , Sano Hitomi , Kuronuma Koji , Konishi Masanori , Himi Tetsuo , Miyake Kensuke , Kuroki Yoshio
Published: April 2014

TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu24-Lys631. MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of KD = 6.29 × 10−8 M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-κB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu24-Lys631 enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.
RhoA/Rho-Associated Kinase Pathway Selectively Regulates Thrombin-Induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells via Activation of IκB Kinase β and Phosphorylation of RelA/p65
Authors: Anwar Khandaker N. , Fazal Fabeha , Malik Asrar B. , Rahman Arshad
Published: April 2014

We investigated the involvement of the RhoA/Rho-associated kinase (ROCK) pathway in regulating ICAM-1 expression in endothelial cells by the procoagulant, thrombin. Exposure of HUVECs to C3 exoenzyme, a selective inhibitor of Rho, markedly reduced thrombin-induced ICAM-1 expression. Inhibition of ROCK, the downstream effector of Rho, also prevented thrombin-induced ICAM-1 expression. Blockade of thrombin-induced ICAM-1 expression was secondary to inhibition of NF-κB activity, the key regulator of ICAM-1 expression in endothelial cells. In parallel studies we observed that inhibition of the RhoA/ROCK pathway by the same pharmacological and genetic approaches failed to inhibit TNF-α-induced NF-κB activation and ICAM-1 expression. The effect of RhoA/ROCK inhibition on thrombin-induced NF-κB activation was secondary to inhibition of IκB kinase activation and subsequent IκBα degradation and nuclear uptake and the DNA binding of NF-κB. Inhibition of the RhoA/ROCK pathway also prevented phosphorylation of Ser536 within the transactivation domain 1 of NF-κB p65/RelA, a critical event conferring transcriptional competency to the bound NF-κB. Thus, the RhoA/ROCK pathway signals thrombin-induced ICAM-1 expression through the activation of IκB kinase, which promotes NF-κB binding to ICAM-1 promoter and phosphorylation of RelA/p65, thus mediating the transcriptional activation of bound NF-κB.
Lipopolysaccharide Induces Matrix Metalloproteinase-9 Expression via a Mitochondrial Reactive Oxygen Species-p38 Kinase-Activator Protein-1 Pathway in Raw 264.7 Cells
Authors: Woo Chang-Hoon , Lim Jae-Hyang , Kim Jae-Hong
Published: April 2014

We have identified a novel signaling pathway that leads to expression of matrix metalloproteinase-9 (MMP-9) in murine macrophages in response to the bacterial endotoxin, LPS. We showed that p38 kinase was essential for this induction and observed that LPS-induced MMP-9 expression was sensitive to rottlerin, a putative protein kinase Cδ (PKCδ) inhibitor. However neither infection with a retrovirus expressing a dominant negative mutant of PKCδ nor down-regulation of PKCδ by prolonged PMA treatment affected MMP-9 expression, thus excluding involvement of PKCδ. Interestingly, LPS-induced MMP-9 expression and p38 kinase phosphorylation were shown to be suppressed by the antioxidant N-acetylcysteine and the flavoenzyme inhibitor diphenyleneiodonium chloride, but not by pyrrolidine dithiocarbamate, an NF-κB inhibitor. In addition, LPS was found to induce the production of mitochondrial reactive oxygen species (ROS) and this effect was rottlerin-sensitive, suggesting an inhibitory effect of rottlerin on mitochondrial ROS. LPS-induced MMP-9 expression and p38 kinase phosphorylation were also inhibited by rotenone, a specific inhibitor of mitochondrial complex I, supporting the role of mitochondrial ROS in LPS signaling to MMP-9. Finally, we showed that the ROS-p38 kinase cascade targets the transcription factor AP-1. Taken together, our findings identify a ROS-p38 kinase-AP-1 cascade as a novel pathway mediating LPS signaling to MMP-9 expression in macrophages.
Inhibition of Fractalkine Ameliorates Murine Collagen-Induced Arthritis
Authors: Nanki Toshihiro , Urasaki Yasuyo , Imai Toshio , Nishimura Miyuki , Muramoto Kenzo , Kubota Tetsuo , Miyasaka Nobuyuki
Published: April 2014

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-γ by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.
A Novel HLA-A*3303-Restricted Minor Histocompatibility Antigen Encoded by an Unconventional Open Reading Frame of Human TMSB4Y Gene
Authors: Torikai Hiroki , Akatsuka Yoshiki , Miyazaki Mikinori , Warren Edus H. , Oba Taku , Tsujimura Kunio , Motoyoshi Kazuo , Morishima Yasuo , Kodera Yoshihisa , Kuzushima Kiyotaka , Takahashi Toshitada
Published: April 2014

Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompatibility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5′-untranslated region of the TMSB4Y gene. An HLA-A*3303-restricted CD8+ CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide, EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon. Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope. Tetramer analysis continued to detect TMSB4Y/A33-specific CD8+ T cells at least up to 700 days post-HSCT. This finding underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame products.
Anti-Phospholipid Antibodies Restore Mesenteric Ischemia/Reperfusion-Induced Injury in Complement Receptor 2/Complement Receptor 1-Deficient Mice
Authors: Fleming Sherry D. , Egan Ryan P. , Chai Chunyan , Girardi Guillermina , Holers V. Michael , Salmon Jane , Monestier Marc , Tsokos George C.
Published: April 2014

Complement receptor 2-deficient (Cr2−/−) mice are resistant to mesenteric ischemia/reperfusion (I/R) injury because they lack a component of the natural Ab repertoire. Neither the nature of the Abs that are involved in I/R injury nor the composition of the target Ag, to which recognition is lacking in Cr2−/− mice, is known. Because anti-phospholipid Abs have been shown to mediate fetal growth retardation and loss when injected into pregnant mice, we performed experiments to determine whether anti-phospholipid Abs can also reconstitute I/R injury and, therefore, represent members of the injury-inducing repertoire that is missing in Cr2−/− mice. We demonstrate that both murine and human monoclonal and polyclonal Abs against negatively charged phospholipids can reconstitute mesenteric I/R-induced intestinal and lung tissue damage in Cr2−/− mice. In addition, Abs against β2 glycoprotein I restore local and remote tissue damage in the Cr2−/− mice. Unlike Cr2−/− mice, reconstitution of I/R tissue damage in the injury-resistant Rag-1−/− mouse required the infusion of both anti-β2-glycoprotein I and anti-phospholipid Ab. We conclude that anti-phospholipid Abs can bind to tissues subjected to I/R insult and mediate tissue damage.
Self-Reactive T Cell Receptor-Reactive CD8+ T Cells Inhibit T Cell Lymphoma Growth In Vivo
Authors: Gonthier Marie , Llobera Régine , Arnaud Jacques , Rubin Bent
Published: April 2014

Syngenic C57BL/6 mice (H-2b) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8+ T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-γ and TNF-α, but little or no IL-4. By means of TCRβ-negative variant L12R4 cells, P3.3, and TCR-Vβ2 cDNA-transfected and TCR-Vβ2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the Vβ12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-Kb, and anti-Db mAb. Furthermore, vaccination with Vβ12 peptide p67–78, which binds to both Kb and Db MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-Vβ-specific, Kb-, or Db-restricted CD8+ T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.
TLR4 Contributes to Disease-Inducing Mechanisms Resulting in Central Nervous System Autoimmune Disease
Authors: Kerfoot Steven M. , Long Elizabeth M. , Hickey Michael J. , Andonegui Graciela , Lapointe Benoit M. , Zanardo Renata C. O. , Bonder Claudine , James Will G. , Robbins Stephen M. , Kubes Paul
Published: April 2014

Environmental factors strongly influence the development of autoimmune diseases, including multiple sclerosis. Despite this clear association, the mechanisms through which environment mediates its effects on disease are poorly understood. Pertussis toxin (PTX) functions as a surrogate for environmental factors to induce animal models of autoimmunity, such as experimental autoimmune encephalomyelitis. Although very little is known about the molecular mechanisms behind its function in disease development, PTX has been hypothesized to facilitate immune cell entry to the CNS by increasing permeability across the blood-brain barrier. Using intravital microscopy of the murine cerebromicrovasculature, we demonstrate that PTX alone induces the recruitment of leukocytes and of active T cells to the CNS. P-selectin expression was induced by PTX, and leukocyte/endothelial interactions could be blocked with a P-selectin-blocking Ab. P-selectin blockade also prevented PTX-induced increase in permeability across the blood-brain barrier. Therefore, permeability is a secondary result of recruitment, rather than the primary mechanism by which PTX induces disease. Most importantly, we show that PTX induces intracellular signals through TLR4, a receptor intimately associated with innate immune mechanisms. We demonstrate that PTX-induced leukocyte recruitment is dependent on TLR4 and give evidence that the disease-inducing mechanisms initiated by PTX are also at least partly dependent on TLR4. We propose that this innate immune pathway is a novel mechanism through which environment can initiate autoimmune disease of the CNS.
Intranasal HIV-1-gp160-DNA/gp41 Peptide Prime-Boost Immunization Regimen in Mice Results in Long-Term HIV-1 Neutralizing Humoral Mucosal and Systemic Immunity
Authors: Devito Claudia , Zuber Bartek , Schröder Ulf , Benthin Reinhold , Okuda Kenji , Broliden Kristina , Wahren Britta , Hinkula Jorma
Published: April 2014

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1–9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse’s life span.
Regulatory Cells and Infectious Agents: Détentes Cordiale and Contraire
Authors: Rouse Barry T. , Suvas Susmit
Published: April 2014

This brief review describes the types of interactions that occur between CD4+CD25+ regulatory T cells (Treg) and microbial pathogens. These interactions range from one of mutual benefit (détente cordiale) such as occurs in Leishmania major infection of resistant mouse strains, to instances where the Treg response appears to mainly favor the pathogen and be detrimental to the host (détente contraire). Examples of the latter include chronic persistent infections with retroviruses, perhaps including HIV, and hepatitis C virus. The Treg response also hampers the effectiveness of immunity against some acute virus infections such as HSV. Evidence is also discussed showing that Treg can play a benevolent role to limit the severity of bystander tissue damage in circumstances where the immune response to pathogens is immunopathological. Finally, emerging approaches are discussed that either blunt or activate Treg and that could be used practically to manage host-pathogen interaction.
Cutting Edge: LFA-1 Integrin-Dependent T Cell Adhesion Is Regulated by Both Ag Specificity and Sensitivity
Authors: Mueller Kristen L. , Daniels Mark A. , Felthauser Alicia , Kao Charlly , Jameson Stephen C. , Shimizu Yoji
Published: April 2014

Ab stimulation of the TCR rapidly enhances the functional activity of the LFA-1 integrin. Although TCR-mediated changes in LFA-1 activity are thought to promote T cell-APC interactions, the Ag specificity and sensitivity of TCR-mediated triggering of LFA-1 is not clear. We demonstrate that peptide/MHC (pMHC) tetramers rapidly enhance LFA-1-dependent adhesion of OT-I TCR transgenic CD8+ T cells to purified ICAM-1. Inhibition of src family tyrosine kinase or PI3K activity blocked pMHC tetramer- and anti-CD3-stimulated adhesion. These effects are highly specific because partial agonist and antagonist pMHC tetramers are unable to stimulate OT-I T cell adhesion to ICAM-1. The Ag thresholds required for T cell adhesion to ICAM-1 resemble those of early T cell activation events, because optimal LFA-1 activation occurs at tetramer concentrations that fail to induce maximal T cell proliferation. Thus, TCR signaling to LFA-1 is highly Ag specific and sensitive to low concentrations of Ag.
Cutting Edge: Differential Roles for Phosphoinositide 3-Kinases, p110γ and p110δ, in Lymphocyte Chemotaxis and Homing
Authors: Reif Karin , Okkenhaug Klaus , Sasaki Takehiko , Penninger Joseph M. , Vanhaesebroeck Bart , Cyster Jason G.
Published: April 2014

Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110γ-deficient T cells, but not B cells, show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110δ, a major PI3K isoform in lymphocytes. B cells deficient in p110δ showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110δ-deficient B cells revealed diminished homing to Peyer’s patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer’s patches. These observations establish that p110γ and p110δ function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.
Cutting Edge: Prolonged Antigen Presentation after Herpes Simplex Virus-1 Skin Infection
Authors: Stock Angus T. , Mueller Scott N. , van Lint Allison L. , Heath William R. , Carbone Francis R.
Published: April 2014

It has been reported that MHC class I-restricted Ag presentation persists for only a short period following infection with certain pathogens, declining in parallel with the emergence of specific CTL activity. We have examined this issue in the case of murine infection with HSV-1. We found that the period of Ag presentation capable of priming naive CD8+ T cells is comparatively prolonged, persisting for at least 7 days after infection, and continuing despite the appearance of localized CTL activity. Ag presentation was abbreviated to 3 or 4 days postinfection by surgical excision of the inoculation site early after infection. This intervention attenuated the size of the primary CTL response, implying that prolonged presentation is necessary to drive maximal CTL expansion. Combined, these data show that, in some types of infection, CTL priming can extend well beyond the first 24–48 h after primary inoculation.
TNF Family Member B Cell-Activating Factor (BAFF) Receptor-Dependent and -Independent Roles for BAFF in B Cell Physiology
Authors: Sasaki Yoshiteru , Casola Stefano , Kutok Jeffery L. , Rajewsky Klaus , Schmidt-Supprian Marc
Published: April 2014

The cytokine TNF family member B cell-activating factor (BAFF; also termed BLyS) is essential for B cell generation and maintenance. Three receptors have been identified that bind to BAFF: transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Recently, it was shown that A/WySnJ mice, which contain a dramatically reduced peripheral B cell compartment due to decreased B cell life span, express a mutant BAFF-R. This finding, together with normal or enhanced B cell generation in mice deficient for BCMA or TACI, respectively, suggested that the interaction of BAFF with BAFF-R triggers signals essential for the generation and maintenance of mature B cells. However, B cells in mice deficient for BAFF differ phenotypically and functionally from A/WySnJ B cells. Residual signaling through the mutant BAFF-R could account for these differences. Alternatively, dominant-negative interference by the mutant receptor could lead to an overestimation of the importance of BAFF-R. To resolve this issue, we generated BAFF-R-null mice. Baff-r−/− mice display strongly reduced late transitional and follicular B cell numbers and are essentially devoid of marginal zone B cells. Overexpression of Bcl-2 rescues mature B cell development in Baff-r−/− mice, suggesting that BAFF-R mediates a survival signal. CD21 and CD23 surface expression are reduced on mature Baff-r−/− B cells, but not to the same extent as on mature B cells in BAFF-deficient mice. In addition, we found that Baff-r−/− mice mount significant, but reduced, Ag-specific Ab responses and are able to form spontaneous germinal centers in mesenteric lymph nodes. The reduction in Ab titers correlates with the reduced B cell numbers in the mutant mice.
B7-CD28 Interaction Promotes Proliferation and Survival but Suppresses Differentiation of CD4−CD8− T Cells in the Thymus
Authors: Zheng Xincheng , Gao Jian-Xin , Chang Xing , Wang Yin , Liu Yan , Wen Jing , Zhang Huiming , Zhang Jian , Liu Yang , Zheng Pan
Published: April 2014

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25+CD4+ regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4−CD8− (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25+CD44− (DN3) to CD25−CD44− (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.
A Stroma-Derived Defect in NF-κB2−/− Mice Causes Impaired Lymph Node Development and Lymphocyte Recruitment
Authors: Carragher Damian , Johal Ramneek , Button Adele , White Andrea , Eliopoulos Aristides , Jenkinson Eric , Anderson Graham , Caamaño Jorge
Published: April 2014

The NF-κB family of transcription factors is vital to all aspects of immune function and regulation in both the hemopoietic and stromal compartments of immune environments. Recent studies of mouse models deficient for specific members of the NF-κB family have revealed critical roles for these proteins in the process of secondary lymphoid tissue organogenesis. In this study, we investigate the role of NF-κB family member NF-κB2 in lymph node development and lymphocyte recruitment. Inguinal lymph nodes in nfκb2−/− mice are reduced in size and cellularity, most notably in the B cell compartment. Using in vitro and in vivo lymph node grafting assays, we show that the defect resides in the stromal compartment. Further examination of the nfκb2−/− inguinal lymph nodes revealed that expression of peripheral node addressin components CD34 and glycosylation-dependent cell adhesion molecule-1 along with the high endothelial venule-restricted sulfotransferase HEC-GlcNAc6ST was markedly reduced. Furthermore, expression of the lymphocyte homing chemokines CCL19, CCL21, and CXCL13 was down-regulated. These data highlight the role of NF-κB2 in inguinal lymph node organogenesis and recruitment of lymphocytes to these organs due to its role in up-regulation of essential cell adhesion molecules and chemokines, while suggesting a potential role for NF-κB2 in organization of lymph node endothelium.
STAT5 Is Required for Thymopoiesis in a Development Stage-Specific Manner
Authors: Kang Joonsoo , DiBenedetto Brian , Narayan Kavitha , Zhao Hang , Der Sandy D. , Chambers Cynthia A.
Published: April 2014

Diverse cytokines necessary for normal lymphopoiesis and lymphocyte homeostasis activate STAT5 in responder cells. Although STAT5 has been suggested to be a central molecular effecter of IL-7 function, its essential role during IL-7-dependent T cell development in vivo remained unclear. Using Stat5−/− mice we now show that STAT5 is essential for various functions ascribed to IL-7 in vivo. STAT5 is required for embryonic thymocyte production, TCRγ gene transcription, and Peyer’s patch development. In sharp contrast, normal STAT5 is dispensable for adult thymopoiesis. In peripheral lymphocytes, STAT5 is primarily required for the generation and/or maintenance of γδ T cells and TCRγδ+ intraepithelial lymphocytes. Collectively, these results demonstrate that STAT5 is critical for many, but not all, aspects of steady state lymphoid lineage development and maintenance and suggest the existence of previously undocumented cytokine signaling traits and/or cytokine milieu during adult thymopoiesis.
Regulated Expression of the Receptor-Like Tyrosine Phosphatase CD148 on Hemopoietic Cells
Authors: Lin Joseph , Zhu Jing W. , Baker Jeanne E. , Weiss Arthur
Published: April 2014

CD148 is a receptor-like protein tyrosine phosphatase expressed on a wide variety of cell types. Through the use flow cytometry and immunofluorescence microscopy on tissue sections, we examined the expression of CD148 on multiple murine hemopoietic cell lineages. We found that CD148 is moderately expressed during all stages of B cell development in the bone marrow, as well as peripheral mature B cells. In contrast, CD148 expression on thymocytes and mature T cells is substantially lower. However, stimulation of peripheral T cells through the TCR leads to an increase of CD148 expression. This up-regulation on T cells can be partially inhibited by reagents that block the activity of src family kinases, calcineurin, MEK, or PI3K. Interestingly, CD148 levels are elevated on freshly isolated T cells from MRL lpr/lpr and CTLA-4-deficient mice, two murine models of autoimmunity. Together, these expression data along with previous biochemical data suggest that CD148 may play an important regulatory role to control an immune response.
B Cell-Activating Factor Belonging to the TNF Family Acts through Separate Receptors to Support B Cell Survival and T Cell-Independent Antibody Formation
Authors: Shulga-Morskaya Svetlana , Dobles Max , Walsh Meghan E. , Ng Lai Guan , MacKay Fabienne , Rao Sambasiva P. , Kalled Susan L. , Scott Martin L.
Published: April 2014

The TNF-related ligand, B cell-activating factor belonging to the TNF family (BAFF), is necessary for normal B cell development and survival, and specifically binds the receptors transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), B cell maturation Ag (BCMA), and BAFF-R. Similarities between mice completely lacking BAFF and A/WySnJ strain mice that express a naturally occurring mutant form of BAFF-R suggest that BAFF acts primarily through BAFF-R. However, the nearly full-length BAFF-R protein expressed by A/WySnJ mice makes unambiguous interpretation of receptor function in these animals impossible. Using homologous recombination we created mice completely lacking BAFF-R and compared them directly to A/WySnJ mice and to mice lacking BAFF. BAFF-R-null mice exhibit loss of mature B cells similar to that observed in BAFF−/− and A/WySnJ mice. Also, mice lacking both TACI and BCMA simultaneously exhibit no B cell loss, thus confirming that BAFF-R is the primary receptor for transmitting the BAFF-dependent B cell survival signal. However, while BAFF-R-null mice cannot carry out T cell-dependent Ab formation, they differ from BAFF-deficient mice in generating normal levels of Ab to at least some T cell-independent Ags. These studies clearly demonstrate that BAFF regulates Ab responses in vivo through receptors in addition to BAFF-R.
IL-10-Producing B220+CD11c− APC in Mouse Spleen
Authors: Burke Fiona , Stagg Andrew J. , Bedford Penelope A. , English Nicholas , Knight Stella C.
Published: April 2014

APC acting at the early stages of an immune response can shape the nature of that response. Such APC will include dendritic cells (DCs) but may also include populations of B cells such as marginal zone B cells in the spleen. In this study, we analyze APC populations in mouse spleen and compare the phenotype and function of B220+CD11c− populations with those of CD11c+ spleen DC subsets. Low-density B220+ cells had morphology similar to DCs and, like DCs, they could stimulate naive T cells, and expressed high levels of MHC and costimulatory molecules. However, the majority of the B220+ cells appeared to be of B cell lineage as demonstrated by coexpression of CD19 and surface Ig, and by their absence from RAG-2−/− mice. The phenotype of these DC-like B cells was consistent with that of B cells in the marginal zone of the spleen. On bacterial stimulation, they preferentially produced IL-10 in contrast to the DCs, which produced IL-12. Conventional B cells did not produce IL-10. The DC-like B cells could be induced to express low levels of the DC marker CD11c with maturational stimuli. A minority of the B220+CD11c− low-density cells did not express CD19 and surface Ig and may be a DC subset; this population also produced IL-10 on bacterial stimulation. B220+ APC in mouse spleen that stimulate naive T cells and preferentially produce IL-10 may be involved in activating regulatory immune responses.
Activation through Cannabinoid Receptors 1 and 2 on Dendritic Cells Triggers NF-κB-Dependent Apoptosis: Novel Role for Endogenous and Exogenous Cannabinoids in Immunoregulation
Authors: Do Yoonkyung , McKallip Robert J. , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: April 2014

The precise role of cannabinoid receptors (CB)1 and CB2, as well as endogenous ligands for these receptors, on immune cells remains unclear. In the current study, we examined the effect of endogenous and exogenous cannabinoids on murine bone marrow-derived dendritic cells (DCs). Addition of Δ9-tetrahydrocannabinol (THC), a major psychoactive component found in marijuana or anandamide, an endogenous cannabinoid, to DC cultures induced apoptosis in DCs. DCs expressed CB1 and CB2 receptors and the engagement of both receptors was necessary to trigger apoptosis. Treatment with THC induced caspase-2, -8, and -9 activation, cleavage of Bid, decreased mitochondrial membrane potential, and cytochrome c release, suggesting involvement of death-receptor and mitochondrial pathways. DCs from Bid-knockout mice were sensitive to THC-induced apoptosis thereby suggesting that Bid was dispensable. There was no induction of p44/p42 MAPK, p38 MAPK, or stress-activated protein/JNK pathway in THC-treated DCs. However, THC treatment induced phosphorylation of IκB-α, and enhanced the transcription of several apoptotic genes regulated by NF-κB. Moreover, inhibition of NF-κB was able to block THC-induced apoptosis in DCs. Lastly, in vivo treatment of mice with THC caused depletion of splenic DCs. Together, our study demonstrates for the first time that endogenous and exogenous cannabinoids may suppress the immune response through their ability to induce apoptosis in DCs.