bluuebunny/crossref_metadata_2025 huggingface dataset:
B7-H1 Is Expressed by Human Endothelial Cells and Suppresses T Cell Cytokine Synthesis
Authors: Mazanet Melissa M. , Hughes Christopher C. W.
Published: April 2014

Human endothelial cells (ECs) provide costimulatory signals sufficient to activate resting memory T cells to produce IL-2 and IFN-γ, at least in part through CD58-CD2 interactions. Recently, the B7-like molecule, B7-H1 (PD-L1), was described and shown to regulate T cell activation; however, there are conflicting reports on whether it stimulates or inhibits T cell cytokine synthesis. B7-H1 is not expressed constitutively by ECs; however, it is rapidly induced by IFN-γ, and synergistically by IFN-γ and TNF. In inflamed skin, B7-H1 is expressed by a subset of microvessels, and by keratinocytes, but is barely detectable in normal skin. Blocking the interaction of EC-expressed B7-H1 with its T cell ligand, programmed death-1 (PD-1), using a PD-1-Fc fusion protein, or by blocking B7-H1 expression with morpholino antisense oligonucleotides, augments expression of IL-2 and IFN-γ, implicating B7-H1 as a negative regulator of cytokine synthesis. However, signaling through PD-1 does not affect induction of the activation markers CD25 or CD69 on T cells, suggesting that its effects are specific to cytokine synthesis. The suppressive effects of B7-H1 on cytokine expression are proportional to the strength of the primary stimulus, allowing for B7-H1 to determine the level of T cell activation in response to ECs. Our results demonstrate that B7-H1 negatively regulates cytokine synthesis in T cells activated by ECs.
Morphine Regulates Gene Expression of α- and β-Chemokines and Their Receptors on Astroglial Cells Via the Opioid μ Receptor
Authors: Mahajan Supriya D. , Schwartz Stanley A. , Shanahan Thomas C. , Chawda Ram P. , Nair Madhavan P. N.
Published: April 2014

The brain is a target organ for recreational drugs and HIV-1. Epidemiological data demonstrate that opioid abuse is a risk factor for HIV-1 infection and progression to AIDS. Chemokines and their receptors have been implicated in the neuropathogenesis of HIV-1 infections. However, little is known about the effects of opioids on the expression of chemokines and their receptors (the latter also are HIV-1 coreceptors) by cells of the CNS. Herein we describe the effects of morphine on gene expression of the α- and β-chemokines and their receptors by the astrocytoma cell line U87 and by primary normal human astrocyte (NHA) cultures. U87 cells treated with morphine showed significant down-regulation of IL-8 gene expression, whereas expression of the IL-8 receptor CXCR2 was reciprocally up-regulated as detected by RT-PCR. Treatment of NHAs with morphine suppressed IL-8 and macrophage-inflammatory protein-1β gene expression, whereas expression of their receptor genes, CCR3 and CCR5, was simultaneously enhanced. These morphine-induced effects on U87 and NHA cells were reversed by the opioid μ receptor antagonist β-funaltrexamine. Morphine also enhanced the constitutive expression of the opioid μ receptor on astroglial cells. Our results support the hypothesis that opioids play a significant role in the susceptibility of the CNS to HIV-1 infection and subsequent encephalopathy by inhibiting local production of HIV-1-protective chemokines (IL-8 and macrophage-inflammatory protein-1β) and enhancing expression of HIV-1 entry coreceptor genes (CCR3, CCR5, and CXCR2) within the CNS. These effects of opioids appear to be mediated through the opioid μ receptor that we demonstrated on astroglial cells.
MHC Alloantigens Elicit Secondary, But Not Primary, Indirect In Vitro Proliferative Responses
Authors: Haller Gary W. , Lima Brian , Kunisaki Shaun M. , Germana Sharon , Leguern Christian , Huang Christene A. , Sachs David H.
Published: April 2014

The relative contributions of direct and indirect pathways of allorecognition to graft rejection remain controversial. Recent reports suggest that the indirect pathway may play a prominent role in both acute and chronic allograft rejection. Such studies suggest that MHC-derived allopeptides are more immunogenic than those derived from minor histocompatibility or other nominal Ags. The aim of this study was to characterize the immunogenicity of MHC alloantigens in MHC-defined miniature swine via primary and secondary MLR culture assays. APCs were selectively depleted from either responder or stimulator cell populations to specifically analyze direct and indirect proliferative responses, respectively. Radio-resistant cytokine secretion and subsequent backstimulation of responder cells was eliminated by using stimulators that were either lysed or unresponsive to the responder MHC haplotypes. When the effect of backstimulation was eliminated from MLR culture assays, indirect proliferative responses were not observed among naive responders. Only after in vivo priming of responder animals could indirect proliferation be detected. These data do not refute the potential importance of indirect allorecognition in graft rejection. However, they suggest that MHC-derived alloantigens behave similarly in vitro to minor histocompatibility Ags, with comparable immunogenicity. These data also suggest that the MLR culture assay does not accurately reflect the importance of indirect mechanisms that have previously been reported in experimental models of graft rejection. A greater understanding of the indirect pathway and the associated immunogenicity of MHC allopeptides has the potential benefit of enabling the development of therapeutic interventions to prevent or halt allograft rejection.
μ-Opioid Receptor Mediates Chronic Restraint Stress-Induced Lymphocyte Apoptosis
Authors: Wang Jinghua , Charboneau Richard , Barke Roderick A. , Loh Horace H. , Roy Sabita
Published: April 2014

Psychological stress is associated with immunosuppression in both humans and animals. Although it was well established that psychological stressors stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, resulting in the release of various hormones and neurotransmitters, the mechanisms underlying these phenomena are poorly understood. In this study, μ-opioid receptor knockout (MORKO) mice were used to investigate whether the μ-opioid receptor mediates the immunosuppression induced by restraint stress. Our results showed that wild-type (WT) mice subjected to chronic 12-h daily restraint stress for 2 days exhibited a significant decrease in splenocyte number with a substantial increase in apoptosis and CD95 (Fas/APO-1) expression of splenocytes. The effects are essentially abolished in MORKO mice. Furthermore, inhibition of splenic lymphocyte proliferation, IL-2, and IFN-γ production induced by restraint stress in WT mice was also significantly abolished in MORKO mice. Interestingly, both stressed WT and MORKO mice showed a significant elevation in plasma corticosterone and pituitary proopiomelanocortin mRNA expression, although the increase was significantly lower in MORKO mice. Adrenalectomy did not reverse restraint stress-induced immunosuppression in WT mice. These data clearly established that the μ-opioid receptor is involved in restraint stress-induced immune alterations via a mechanism of apoptotic cell death, and that the effect is not mediated exclusively through the glucocorticoid pathway.
B6 Strain Ly49I Inhibitory Gene Expression on T Cells in FVB.Ly49IB6 Transgenic Mice Fails to Prevent Normal T Cell Functions
Authors: Morris Margaret A. , Liu Jingxuan , Arora Veera , George Thaddeus C. , Klem Jennifer , Schatzle John D. , Kumar Vinay , Bennett Michael
Published: April 2014

Inhibitory Ly49 receptors expressed on NK cells provide a mechanism for tolerance to normal self tissues. The immunoregulatory tyrosine-based inhibitory motifs present in some Ly49s are able to transmit an inhibitory signal upon ligation by MHC class I ligands. In our system, as well as others, mice transgenic for inhibitory Ly49 receptors express these receptors on both NK and T cells. FVB (H2q) mice transgenic for the B6 strain Ly49I (Ly49IB6) express the inhibitory Ly49 receptor on the surface of both T and NK cells. Although Ly49I functions to prevent NK-mediated rejection of H2b donor bone marrow cells in this transgenic mouse strain, the T cells do not appear to be affected by the expression of the Ly49I transgene. FVB.Ly49I T cells have normal proliferative capabilities both in vitro and in vivo in response to the Ly49I ligand, H2b. In vivo functional T cell assays were also done, showing that transgenic T cells were not functionally affected. T cells in these mice also appear to undergo normal T cell development and activation. Only upon stimulation with suboptimal doses of anti-CD3 in the presence of anti-Ly49I is T cell proliferation inhibited. These data are in contrast with findings in Ly49A, and Ly49G2 receptor transgenic models. Perhaps Ly49I-H2b interactions are weaker or of lower avidity than Ly49A-H-2Dd interactions, especially in T cells.
Inducible Costimulator Costimulates Cytotoxic Activity and IFN-γ Production in Activated Murine NK Cells
Authors: Ogasawara Kouetsu , Yoshinaga Steven K. , Lanier Lewis L.
Published: April 2014

The functions of NK cells are regulated by the balance of activating and inhibitory signals. The inhibitory NK cell receptors are well understood; however, less is known about the activating signaling pathways. To explore whether a costimulatory receptor, inducible costimulator (ICOS), is involved in NK cell function, we assessed the role of ICOS in NK cell-mediated cytotoxicity and cytokine production. In addition, to determine whether ICOS contributes to the elimination of tumors in vivo, we examined the tumor growth survival of mice injected with a tumor expressing the ICOS ligand, B7RP-1. We found that ICOS was up-regulated by cytokine stimulation in murine NK cells. Consistent with ICOS expression on activated NK cells, ICOS-dependent cytotoxicity and IFN-γ production were observed, and appeared to require signaling through the phosphoinositide 3-kinase pathway. Interestingly, ICOS-mediated stimulation allowed activated NK cells to kill more efficiently tumor cells expressing MHC class I. Furthermore, fewer metastases appeared in the liver and spleen of mice injected with the ICOS ligand-expressing tumor compared with mice bearing the parental tumor. These results indicate that NK cell functions are regulated by ICOS.
Altered IL-4 mRNA Stability Correlates with Th1 and Th2 Bias and Susceptibility to Hypersensitivity Pneumonitis in Two Inbred Strains of Mice
Authors: Butler Noah S. , Monick Martha M. , Yarovinsky Timur O. , Powers Linda S. , Hunninghake Gary W.
Published: April 2014

Previously, we have shown in a model of hypersensitivity pneumonitis that Th1-biased C57BL/6 mice are susceptible and Th2-biased DBA/2 mice are resistant to disease. We also showed that this was explained in part by differential regulation of IL-12 by IL-4. For these reasons, we postulated that C57BL/6 and DBA/2 mice differentially express IL-4. In this study, we show that C57BL/6 immune cells express Th2 but not Th1 cytokines at lower levels than DBA/2 cells. We also found that C57BL/6 splenocytes exhibit decreased mRNA stability of Th2 cytokines, relative to DBA/2 splenocytes. Stability of IL-2 and IFN-γ were similar in the two strains of mice. Differences in Th2 cytokine mRNA stability between C57BL/6 and DBA/2 cells were not due to sequence polymorphism at specific regions of the IL-4/IL-13 locus. Furthermore, expression of Th1- and Th2-specific transcription factors T-bet and GATA-3, as well as the nuclear factor of activated T cells transcription factor, NFATc, was not significantly different between the two mice. Our data suggest that decreased mRNA stability of Th2 cytokines in C57BL/6 splenocytes may underlie the differential susceptibility to hypersensitivity pneumonitis between C57BL/6 and DBA/2 mice. Moreover, our results indicate that regulation of mRNA stability may serve as an important mechanism underlying Th1/Th2 immune polarization.
A Novel Costimulation Pathway Via the 4C8 Antigen for the Induction of CD4+ Regulatory T Cells
Authors: Masuyama Jun-ichi , Kaga Shuji , Kano Shogo , Minota Seiji
Published: April 2014

CD4+CD25+ regulatory T (Treg) cells naturally occur in mice and humans, and similar Treg cells can be induced in vivo and in vitro. However, the molecular mechanisms that mediate the generation of these Treg cell populations remain unknown. We previously described anti-4C8 mAbs that inhibit the postadhesive transendothelial migration of T cells through human endothelial cell monolayers. We demonstrate in this work that Treg cells are induced by costimulation of CD4+ T cells with anti-CD3 plus anti-4C8. The costimulation induced full activation of CD4+ T cells with high levels of IL-2 production and cellular expansion that were comparable to those obtained on costimulation by CD28. However, upon restimulation, 4C8-costimulated cells produced high levels of IL-10 but no IL-2 or IL-4, and maintained high expression levels of CD25 and intracellular CD152, as compared to CD28-costimulated cells. The former cells showed hyporesponsiveness to anti-CD3 stimulation and suppressed the activation of bystander T cells depending on cell contact but not IL-10 or TGF-β. The suppressor cells developed from CD4+CD25−CD45RO+ cells. The results suggest that 4C8 costimulation induces the generation of Treg cells that share phenotypic and functional features with CD4+CD25+ T cells, and that CD25− memory T cells may differentiate into certain Treg cell subsets in the periphery.
T Cell Receptor-γ Allele-Specific Selection of Vγ1/Vδ4 Cells in the Intestinal Epithelium
Authors: Grigoriadou Kalliopi , Boucontet Laurent , Pereira Pablo
Published: April 2014

Previous genetic analyses have shown that the relative representation of subsets of γδ intestinal intraepithelial lymphocytes (i-IELs) is influenced by genes linked to the TCRγ, TCRδ, and MHC loci. Here, we have analyzed V-gene use in γδ i-IELs from C57BL/6 (B6) and C57BL/10 (B10) mice and from their F1 and F2 progenies with a larger panel of Vγ- and Vδ-specific mAbs and have shown that the influence of TCRγ-linked genes operates at two levels: one influencing the representation of Vγ1 (or Vγ7) i-IELs and other acting specifically on the Vγ1/Vδ4 i-IEL subset, which represents 3% and 15% of the γδ i-IELs in B6 and B10 mice, respectively. Analysis of mice transgenic for a rearranged Vγ1Jγ4Cγ4 chain of B6 origin demonstrated that the TCRγ-linked genes influencing the representation of the Vγ1/Vδ4 i-IEL subset are the structural genes of TCRγ chains. This influence is allele specific and cell autonomous, as evidenced by the different behavior of Vγ1/Vδ4 cells bearing either parental allele in F1 mice. The representation of Vγ1/Vδ4 cells among γδ thymocytes is similar in B6 and B10 mice, demonstrating that the Vδ4 chain can pair well with both alleles of the Vγ1Jγ4Cγ4 chain and strongly suggesting that a cellular selection mechanism is responsible for the observed differences. The Vγ1-Jγ4 junctional amino acid sequences of B6 Vγ1/Vδ4 i-IELs are diverse but display less variation in length than those found in similar cells from B10 mice, indicating that B6 Vγ1/Vδ4 cells are the target of this cellular selection event.
c-Cbl Is Involved in Met Signaling in B Cells and Mediates Hepatocyte Growth Factor-Induced Receptor Ubiquitination
Authors: Taher Taher E. I. , Tjin Esther P. M. , Beuling Esther A. , Borst Jannie , Spaargaren Marcel , Pals Steven T.
Published: April 2014

Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13–16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis.
Bcl-2 Rescues the Germinal Center Response But Does Not Alter the V Gene Somatic Hypermutation Spectrum in MSH2-Deficient Mice
Authors: Alabyev Boris , Manser Tim
Published: April 2014

Ab V genes in mice deficient for the postreplication mismatch repair factor MutS homolog (MSH2) have been reported to display an abnormal bias for hypermutations at G and C nucleotides and hotspots. We previously showed that the germinal center (GC) response is severely attenuated in MSH2-deficient mice. This suggested that premature death of GC B cells might preclude multiple rounds of hypermutation necessary to generate a normal spectrum of base changes. To test this hypothesis, we created MSH2-deficient mice in which Bcl-2 expression was driven in B cells from a transgene. In such mice, the elevated levels of intra-GC apoptosis and untimely GC dissolution characteristic of MSH2-deficient mice are suppressed. However, the spectrum of hypermutation is unchanged. These data indicate that the effects of MSH2 deficiency on GC B cell viability and the hypermutation process are distinct.
Mac-1 (CD11b/CD18) as Accessory Molecule for FcαR (CD89) Binding of IgA
Authors: van Spriel Annemiek B. , Leusen Jeanette H. W. , Vilé Henriëtte , van de Winkel Jan G. J.
Published: April 2014

IgA, the principal ligand for FcαRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcαRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the β2 integrin Mac-1 (Mac-1−/−) were unable to bind SIgA, despite expression of FcαRI. Consistent with this, serum IgA stimulated Mac-1−/− PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1−/− PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcαRI interactions.
DNA-Encoded Fetal Liver Tyrosine Kinase 3 Ligand and Granulocyte Macrophage-Colony-Stimulating Factor Increase Dendritic Cell Recruitment to the Inoculation Site and Enhance Antigen-Specific CD4+ T Cell Responses Induced by DNA Vaccination of Outbred Animals
Authors: Mwangi Waithaka , Brown Wendy C. , Lewin Harris A. , Howard Chris J. , Hope Jayne C. , Baszler Timothy V. , Caplazi Patrick , Abbott Jeffrey , Palmer Guy H.
Published: April 2014

DNA-based immunization is a contemporary strategy for developing vaccines to prevent infectious diseases in animals and humans. Translating the efficacy of DNA immunization demonstrated in murine models to the animal species that represent the actual populations to be protected remains a significant challenge. We tested two hypotheses directed at enhancing DNA vaccine efficacy in outbred animals. The first hypothesis, that DNA-encoding fetal liver tyrosine kinase 3 ligand (Flt3L) and GM-CSF increases dendritic cell (DC) recruitment to the immunization site, was tested by intradermal inoculation of calves with plasmid DNA encoding Flt3L and GM-CSF followed by quantitation of CD1+ DC. Peak DC recruitment was detected at 10–15 days postinoculation and was significantly greater (p < 0.05) in calves in the treatment group as compared with control calves inoculated identically, but without Flt3L and GM-CSF. The second hypothesis, that DNA encoding Flt3L and GM-CSF enhances immunity to a DNA vector-expressed Ag, was tested by analyzing the CD4+ T lymphocyte response to Anaplasma marginale major surface protein 1a (MSP1a). Calves immunized with DNA-expressing MSP1a developed strong CD4+ T cell responses against A. marginale, MSP1a, and specific MHC class II DR-restricted MSP1a epitopes. Administration of DNA-encoding Flt3L and GM-CSF before MSP1a DNA vaccination significantly increased the population of Ag-specific effector/memory cells in PBMC and significantly enhanced MSP1a-specific CD4+ T cell proliferation and IFN-γ secretion as compared with MHC class II DR-matched calves vaccinated identically but without Flt3L and GM-CSF. These results support use of these growth factors in DNA vaccination and specifically indicate their applicability for vaccine testing in outbred animals.
The β-Glucan Receptor, Dectin-1, Is Predominantly Expressed on the Surface of Cells of the Monocyte/Macrophage and Neutrophil Lineages
Authors: Taylor Philip R. , Brown Gordon D. , Reid Delyth M. , Willment Janet A. , Martinez-Pomares Luisa , Gordon Siamon , Wong Simon Y. C.
Published: April 2014

We recently identified dectin-1 (βGR) as a major β-glucan receptor on leukocytes and demonstrated that it played a significant role in the non-opsonic recognition of soluble and particulate β-glucans. Using a novel mAb (2A11) raised against βGR, we show here that the receptor is not dendritic cell-restricted as first reported, but is broadly expressed, with highest surface expression on populations of myeloid cells (monocyte/macrophage (Mφ) and neutrophil lineages). Dendritic cells and a subpopulation of T cells also expressed the βGR, but at lower levels. Alveolar Mφ, like inflammatory Mφ, exhibited the highest surface expression of βGR, indicative of a role for this receptor in immune surveillance. In contrast, resident peritoneal Mφ expressed much lower levels of βGR on the cell surface. Characterization of the nonopsonic recognition of zymosan by resident peritoneal Mφ suggested the existence of an additional β-glucan-independent mechanism of zymosan binding that was not observed on elicited or bone marrow-derived Mφ. Although this recognition could be inhibited by mannan, we were able to exclude involvement of the Mφ mannose receptor and complement receptor 3 in this process. These observations imply the existence of an additional mannan-dependent receptor involved in the recognition of zymosan by resident peritoneal Mφ.
IL-1 Receptor-Associated Kinase and Low Molecular Weight GTPase RhoA Signal Molecules Are Required for Bacterial Lipopolysaccharide-Induced Cytokine Gene Transcription
Authors: Chen Ling-Yu , Zuraw Bruce L. , Liu Fu-Tong , Huang Shuang , Pan Zhixing K.
Published: April 2014

Proinflammatory cytokines such as IL-1, TNF, IL-6, and IL-8 are produced by leukocytes in response to bacteria or bacterial components. A great deal has been learned during the past few years about the synthesis and release of proinflammatory cytokines by leukocytes; however, relatively little is known about the intracellular events that lead to leukocyte proinflammatory cytokine gene transcription. This study examined the signal transduction pathway of IL-8 induction by bacterial LPS. Stimulation of monocytes with LPS rapidly activated RhoA, and pretreatment of monocytes with a RhoA inhibitor, C3 transferase exoenzyme, effectively blocked LPS-induced IL-8 gene expression. Overexpression of dominant negative RhoA (T19N) or IL-1R-associated kinase completely inhibited LPS-stimulated reporter gene expression. Induction of IL-8 was also inhibited by dominant negative IκB kinase and myeloid differentiation protein (MyD88). These results indicate that RhoA and IL-1R-associated kinase are novel signal transducers for LPS-induced Toll-like receptor 4-mediated proinflammatory cytokine synthesis in human monocytes.
NF-κB Regulation in Human Neutrophils by Nuclear IκBα: Correlation to Apoptosis
Authors: Castro-Alcaraz Susana , Miskolci Veronika , Kalasapudi Bharati , Davidson Dennis , Vancurova Ivana
Published: April 2014

Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-κB plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-κB inhibitor, IκBα, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of IκBα in human neutrophils and to determine whether increased accumulation of IκBα in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of IκBα that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of IκBα that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of IκBα in stimulated neutrophils by blocking the IκBα nuclear export, and this is associated with inhibition of NF-κB activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-κB regulation in human neutrophils by nuclear IκBα. Our results demonstrate that the NF-κB activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of IκBα in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.
Cellular Activation, Phagocytosis, and Bactericidal Activity Against Group B Streptococcus Involve Parallel Myeloid Differentiation Factor 88-Dependent and Independent Signaling Pathways
Authors: Henneke Philipp , Takeuchi Osamu , Malley Richard , Lien Egil , Ingalls Robin R. , Freeman Mason W. , Mayadas Tanya , Nizet Victor , Akira Shizuo , Kasper Dennis L. , Golenbock Douglas T.
Published: April 2014

Group B streptococci (GBS) vigorously activate inflammatory responses. We reported previously that a secreted GBS “factor” activates phagocytes via Toll-like receptor (TLR)2 and TLR6, but that GBS cell walls activate cells independently of these receptors. We hypothesized that the phagocytic immune functions in response to GBS, such as inflammation, uptake, and elimination of bacteria, occur through a coordinated engagement of TLRs, along with the coreceptors CD14 and CD11b/CD18. Using various knockout mice we show that GBS-induced activation of p38 and NF-κB depends upon the expression of the cytoplasmic TLR adapter protein, myeloid differentiation factor 88 (MyD88), but not TLR2 and/or TLR4. Macrophages with deletions of CD14 and complement receptor 3 had a normal cytokine response to whole bacteria, although the response to GBS factor was abrogated in CD14-null cells. The intracellular formation of bactericidal oxygen species proved to be MyD88 dependent; however, uptake of GBS, a prerequisite for intracellular killing by O2 radicals, occurred independently of MyD88. While deletion of complement receptor 3 greatly diminished the uptake of opsonized GBS, it did not affect the formation of bactericidal O2 radicals or inflammatory signaling intermediates. We conclude that the inflammatory, bactericidal, and phagocytic responses to GBS occur via parallel but independent processes.
Accelerated Onset and Increased Severity of Acute Graft-Versus-Host Disease Following Adoptive Transfer of DR6-Deficient T Cells
Authors: Liu Jinqi , Heuer Josef G. , Na Songqing , Galbreath Elizabeth , Zhang Tonghai , Yang Derek D. , Glasebrook Andrew , Song Ho Yeong
Published: April 2014

DR6 is a recently identified member of the TNFR family. In a previous study, we have shown that DR6 KO mice have enhanced CD4+ T cell proliferation and Th2 cytokine production. Acute graft-vs-host disease (GVHD) results from the activation and expansion of alloreactive donor T cells following bone marrow transplantation. In this article, we demonstrate that the transfer of donor T cells from DR6 KO mice into allogeneic recipient mice in a parent into an F1 model of acute GVHD results in a more rapid onset of GVHD with increased severity. Recipients of DR6 KO T cells exhibit earlier systemic symptoms of GVHD, more rapid weight loss, earlier histopathological organ damage in the thymus, spleen, and intestines, and earlier mortality. The rapid onset of GVHD in these mice may be attributable to the enhanced activation and expansion of DR6 KO CD4+ and CD8+ T cells. Our findings support the hypothesis that DR6 serves as an important regulatory molecule in T cell immune responses. The identification and use of DR6 ligands and/or agonistic Abs to DR6 may represent useful therapeutics in the treatment of T cell-mediated diseases such as GVHD.
Dual Roles for IFN-γ, But Not for IL-4, in Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice
Authors: Yu Shiguang , Sharp Gordon C. , Braley-Mullen Helen
Published: April 2014

Spontaneous autoimmune thyroiditis (SAT) is an organ-specific autoimmune disease characterized by chronic inflammation of the thyroid by T and B lymphocytes. To investigate the roles of Th1 and Th2 cytokines in the pathogenesis of SAT, IFN-γ−/− and IL-4−/− NOD.H-2h4 mice were generated. IL-4−/− mice developed lymphocytic SAT (L-SAT) comparable to that of wild-type (WT) mice. They produced little anti-mouse thyroglobulin (MTg) IgG1, but had levels of anti-MTg IgG2b comparable to WT mice. Compared with WT mice, IFN-γ−/− mice produced significantly less anti-MTg IgG1 and IgG2b. Absence of IFN-γ resulted in abnormal proliferation of thyroid epithelial cells with minimal lymphocyte infiltration. Thyroids of IFN-γ−/− mice had markedly reduced B lymphocyte chemoattractant expression, B cell and plasma cell infiltration, and decreased MHC class II expression on thyrocytes compared with WT mice. Adoptive transfer of WT splenocytes to IFN-γ−/− mice restored the capacity to develop typical L-SAT, enhanced anti-MTg IgG1 and IgG2b production, up-regulated MHC class II expression on thyrocytes and decreased thyrocyte proliferation. These results suggest that IFN-γ plays a dual role in the development of SAT. IFN-γ is required for development of L-SAT, and it also functions to inhibit thyroid epithelial cell proliferation.
Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Evidence for Functional Expression ofSle3/5by Non-T Cells
Authors: Sobel Eric S. , Morel Laurence , Baert Raquel , Mohan Chandra , Schiffenbauer Joel , Wakeland Edward K.
Published: April 2014

On the non-autoimmune C57BL/6 (B6) background, the chromosome 7-derived lupus susceptibility loci Sle3 and Sle5 have been shown to mediate an elevated CD4:CD8 ratio with an increase in activated CD4+ T cells, decreased susceptibility to apoptosis, and a break in humoral tolerance. Development of subcongenic strains has subsequently shown that the elevated CD4:CD8 ratio is due to Sle3 but that both loci contribute to the development of autoantibodies. To elucidate the functional expression patterns of these loci, adoptive transfer experiments were conducted. All possible combinations of bone marrow reconstitution, including syngenic, were conducted between the congenic B6 and B6.Sle3/5 strains. It was found that the Sle3/5 locus was functionally expressed by bone marrow-derived cells, but not by host cells, and that the elevated CD4:CD8 phenotype could be reconstituted in radiation chimeras. Using Ly5-marked congenic strains and B6 host mice, additional experiments surprisingly demonstrated that the elevated CD4:CD8 ratio was neither an intrinsic property of the T cells nor of single positive thymocytes. Allotype-marked chimeras indicated that autoantibody production by B cells was also an extrinsic property, as shown by the fact that B cells without the Sle3/5 interval contributed to autoantibody production. These experiments strongly suggest that a gene within the B6.Sle3/5 interval was expressed by a bone marrow-derived, nonlymphocyte population in the thymus and periphery and was affecting T cell selection and/or survival.
The Antisense Approach in Amyloid Light Chain Amyloidosis: Identification of Monoclonal Ig and Inhibition of Its Production by Antisense Oligonucleotides in In Vitro and In Vivo Models
Authors: Ohno Satoko , Yoshimoto Mitsuru , Honda Saho , Miyachi Sae , Ishida Tadao , Itoh Fumio , Endo Takao , Chiba Susumu , Imai Kohzoh
Published: April 2014

Primary amyloid L chain (AL) amyloidosis is a plasma cell disorder in which depositions of AL cause progressive organ failure. The lack of effective therapies for this fatal disease prompts exploration of newer treatment avenues. We have investigated the application of antisense oligonucleotides (AS) for the inhibition of monoclonal Ig production. The monoclonal L chain was identified by using primers designed for amplifying the human λ Ig V (Vλ) region. We demonstrated that AS against L chain complementarity-determining regions inhibited the production of L chain in vitro. RPMI 8226 myeloma cells injected in SCID mice developed s.c. tumors. RT-PCR analysis showed Vλ mRNA expression in the tumors. In addition, the presence of human Ig in the sera of mice given injection of RPMI 8226 cells was confirmed by ELISA. Administration of AS inhibited the expression of Vλ mRNA in the s.c. tumors and decreased the concentration of L chain in serum. Therefore, we have shown that it is possible to determine the sequence of Vλ mRNA and design specific complementary oligonucleotides, suggesting that treatment with Vλ antisense could represent a rational novel approach to improve treatment outcome in AL amyloidosis.
Cutting Edge: Murine UL16-Binding Protein-Like Transcript 1: A Newly Described Transcript Encoding a High-Affinity Ligand for Murine NKG2D
Authors: Carayannopoulos Leonidas N. , Naidenko Olga V. , Fremont Daved H. , Yokoyama Wayne M.
Published: April 2014

Murine NKG2D is known to recognize H60 and five RAE1 variants. The human homologue recognizes both inducible MHC class I chain-related gene and constitutive (UL16-binding protein (ULBP)) ligands. Widely expressed, the latter are thought to mark transformed or infected cells for destruction by NK cells in the context of down-regulated cell surface class I (i.e., the “missing self”-response). Unlike MIC and ULBP however, mRNA for the murine ligands appears only in very limited contexts in the mature animal. In this study, we describe a NKG2D ligand termed “murine ULBP-like transcript 1 (MULT1) whose mRNA appears to be widely expressed in adult parenchyma. This molecule possesses MHC class I-like α1 and α2 domains as well as a large cytoplasmic domain. Recombinant MULT1 binds NKG2D with relatively high affinity (KD ≈ 6 nM) and low koff (∼0.006s−1). Expression of MULT1 by normally resistant RMA cells results in their susceptibility to lysis by C57BL/6 splenocytes.
Cutting Edge: Suppression of T Cell Chemotaxis by Sphingosine 1-Phosphate
Authors: Graeler Markus , Shankar Geetha , Goetzl Edward J.
Published: April 2014

Murine CD4 and CD8 T cells express predominantly types 1 and 4 sphingosine 1-phosphate (S1P) G protein-coupled receptors (designated S1P1 and S1P4 or previously endothelial differentiation gene-encoded 1 and 6) for S1P, which has a normal plasma concentration of 0.1–1 μM. S1P now is shown to enhance chemotaxis of CD4 T cells to CCL-21 and CCL-5 by up to 2.5-fold at 10 nM to 0.1 μM, whereas 0.3–3 μM S1P inhibits this chemotaxis by up to 70%. Chemotaxis of S1P1, but not S1P4, transfectants to CXCL1 and CXCL4 was similarly affected by S1P. Activation of CD4 T cells, which decreases S1P receptor expression, suppressed effects of S1P on chemotaxis. Pretreatment of labeled CD4 T cells with S1P before reintroduction into mice inhibited by a maximum of 75% their migration into chemokine-challenged s.c. air pouches. The S1P-S1P1 receptor axis thus controls recruitment of naive T cells by maintaining their response threshold to diverse lymphotactic factors.
Subunits of IgM Reconstitute Defective Contact Sensitivity in B-1 Cell-DeficientxidMice: κ Light Chains Recruit T Cells Independent of Complement
Authors: Paliwal Vipin , Tsuji Ryohei F. , Szczepanik Marian , Kawikova Ivana , Campos Regis A. , Kneilling Manfred , Röcken Martin , Schuurman Janine , Redegeld Frank A. , Nijkamp Frans P. , Askenase Philip W.
Published: April 2014

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP κ L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while κ L chains act via another C-independent but mast cell-dependent pathway.
The Influence of Lysophosphatidic Acid on the Functions of Human Dendritic Cells
Authors: Panther Elisabeth , Idzko Marco , Corinti Silvia , Ferrari Davide , Herouy Yared , Mockenhaupt Maja , Dichmann Stefan , Gebicke-Haerter Peter , Di Virgilio Francesco , Girolomoni Giampiero , Norgauer Johannes
Published: April 2014

Lysophosphatidic acid (LPA) is a bioactive lipid mediator which is generated by secretory phospholipase A2. In this study, we studied the biological activity of LPA on human dendritic cells (DCs), which are specialized APCs characterized by their ability to migrate into target sites and secondary lymphoid organs to process Ags and activate naive T cells. We show that immature and mature DCs express the mRNA for different LPA receptors such as endothelial differentiation gene (EDG)-2, EDG-4, and EDG-7. In immature DCs, LPA stimulated pertussis toxin-sensitive Ca2+ increase, actin polymerization, and chemotaxis. During the maturation process, DCs lost their ability to respond toward LPA with Ca2+ transients, actin polymerization, and chemotaxis. However, LPA inhibited in a pertussis toxin-insensitive manner the secretion of IL-12 and TNFα as well as enhanced secretion of IL-10 from mature DCs. Moreover, LPA did not affect the endocytic or phagocytic capacities and the surface phenotype of DCs, although it increased the allostimulatory function of mature DC and inhibited their capacity to induce Th1 differentiation. In summary, our study implicates that LPA might regulate the trafficking, cytokine production, and T cell-activating functions of DCs.
Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor β-Chain-Deficient Mice
Authors: Minagawa Masahiro , Watanabe Hisami , Miyaji Chikako , Tomiyama Katsuhiro , Shimura Hideki , Ito Akiko , Ito Masaaki , Domen Jos , Weissman Irving L. , Kawai Kazuhiro
Published: April 2014

IL-2/IL-15Rβ-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rβ-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rβ-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rβ-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.
The Final N-Terminal Trimming of a Subaminoterminal Proline-Containing HLA Class I-Restricted Antigenic Peptide in the Cytosol Is Mediated by Two Peptidases
Authors: Lévy Frédéric , Burri Lena , Morel Sandra , Peitrequin Anne-Lise , Lévy Nicole , Bachi Angela , Hellman Ulf , Van den Eynde Benoît J. , Servis Catherine
Published: April 2014

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.
Endogenously Expressed nef Uncouples Cytokine and Chemokine Production from Membrane Phenotypic Maturation in Dendritic Cells
Authors: Messmer Davorka , Jacqué Jean-Marc , Santisteban Christine , Bristow Cynthia , Han Seol-Young , Villamide-Herrera Lorley , Mehlhop Erin , Marx Preston A. , Steinman Ralph M. , Gettie Agegnehu , Pope Melissa
Published: April 2014

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4+ T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-α, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4+ T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.
The Role of Bone Marrow-Derived Stromal Cells in the Maintenance of Plasma Cell Longevity
Authors: Wols Heather A. Minges , Underhill Gregory H. , Kansas Geoffrey S. , Witte Pamela L.
Published: April 2014

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6−/− mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.
T Lymphocytes Potentiate Murine Dendritic Cells to Produce IL-12
Authors: Rizzitelli Alexandra , Berthier Rolande , Collin Véronique , Candéias Serge M. , Marche Patrice N.
Published: April 2014

IL-12 is mainly produced by CD8α+ dendritic cells (DCs) and induces Th1 polarization of the immune response. We investigated the influence of lymphocytes on splenic DC (SDC) and thymic DC (TDC) development and on their IL-12 production capacity. First, CD3ε−/− mice, lacking T cells, and RAG-2−/− mice, lacking T and B cells, possess numbers of SDCs, TDCs, and CD8α+ SDCs similar to wild-type (WT) mice. Second, SDCs and TDCs from CD3ε−/− mice do not secrete IL-12 in vitro after different stimulations, whereas DCs from pTα−/− mice, possessing reduced T cell number, and RAG-2−/− mice, produce an IL-12 level similar to that of WT DCs. We show that T lymphocytes restore the capacity of DCs to produce IL-12 after stimulation in vivo by reconstitution of CD3ε−/− mice with WT T cells and in vitro by coculture of CD3ε−/− DCs with WT T cells. The regulation of IL-12 production occurred at the transcriptional level, with an increase of IL-12p35 transcripts and a decrease of IL-12p40 transcripts. Although IL-4 restores IL-12 production by CD3ε−/− SDCs, anti-IL-4 Abs inhibited only partially the IL-12 production in coculture of CD3ε−/− DCs and WT T cells. Taken together, these data show that T lymphocytes potentiate IL-12 production by DCs and that IL-4 is not solely involved in this regulation. In conclusion, B and T cells exert balanced actions on DCs by respectively inhibiting or promoting IL-12 production.