bluuebunny/crossref_metadata_2025 huggingface dataset:
Toxoplasma gondiiTachyzoites Inhibit Proinflammatory Cytokine Induction in Infected Macrophages by Preventing Nuclear Translocation of the Transcription Factor NF-κB
Authors: Butcher Barbara A. , Kim Leesun , Johnson Peter F. , Denkers Eric Y.
Published: April 2014

Control of microbial infection requires regulated induction of NF-κB-dependent proinflammatory cytokines such as IL-12 and TNF-α. Activation of this important transcription factor is driven by phosphorylation-dependent degradation of the inhibitory IκB molecule, an event which enables NF-κB translocation from the cytoplasm to the nucleus. In this study, we show that intracellular infection of macrophages with the protozoan parasite Toxoplasma gondii induces rapid IκB phosphorylation and degradation. Nevertheless, NF-κB failed to translocate to the nucleus, enabling the parasite to invade cells without triggering proinflammatory cytokine induction. Infected cells subsequently subjected to LPS triggering were severely crippled in IL-12 and TNF-α production, a result of tachyzoite-induced blockade of NF-κB nuclear translocation. Our results are the first to demonstrate the ability of an intracellular protozoan to actively interfere with the NF-κB activation pathway in macrophages, an activity that may enable parasite survival within the host.
Protective Roles of Mast Cells Against Enterobacterial Infection Are Mediated by Toll-Like Receptor 4
Authors: Supajatura Volaluck , Ushio Hiroko , Nakao Atsuhito , Okumura Ko , Ra Chisei , Ogawa Hideoki
Published: April 2014

Toll-like receptors (TLRs) are mammalian homologues of the Drosophila Toll receptors and are thought to have roles in innate recognition of bacteria. We demonstrated that TLR 2, 4, 6, and 8 but not TLR5 were expressed on mouse bone marrow-derived mast cells (BMMCs). Using BMMCs from the genetically TLR4-mutated strain C3H/HeJ, we demonstrated that functional TLR4 was required for a full responsiveness of BMMCs to produce inflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-13) by LPS stimulation. TLR4-mediated stimulation of mast cells by LPS was followed by activation of NF-κB but not by stress-activated protein kinase/c-Jun NH2-terminal kinase signaling. In addition, in the cecal ligation and puncture-induced acute septic peritonitis model, we demonstrated that genetically mast cell-deficient W/Wv mice that were reconstituted with TLR4-mutated BMMCs had significantly higher mortality than W/Wv mice reconstituted with TLR4-intact BMMCs. Higher mortality of TLR4-mutated BMMC-reconstituted W/Wv mice was well correlated with defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Taken together, these observations provide definitive evidence that mast cells play important roles in exerting the innate immunity by releasing inflammatory cytokines and recruitment of neutrophils after recognition of enterobacteria through TLR4 on mast cells.
Taurine Chloramine Inhibits Inducible Nitric Oxide Synthase and TNF-α Gene Expression in Activated Alveolar Macrophages: Decreased NF-κB Activation and IκB Kinase Activity
Authors: Barua Madhabi , Liu Yong , Quinn Michael R.
Published: April 2014

Taurine prevents tissue damage in a variety of models that involve inflammation, including oxidant-induced lung damage. The mechanism of protection is uncertain, but is postulated to involve the actions of taurine chloramine (Tau-Cl) derived via halide-dependent myeloperoxidase associated with neutrophils. Understanding the influence of Tau-Cl on the production of inflammatory mediators by alveolar macrophages provides an opportunity for determining the mechanism of Tau-Cl action. The effects of Tau-Cl were evaluated on the production of NO and TNF-α in NR8383, a cloned cell line derived from rat alveolar macrophages (RAM), and in primary cultures of RAM. Production of NO and TNF-α, and expression of inducible NO synthase was inhibited by Tau-Cl in activated NR8383 cells as well as in RAM. Temporal (2, 4, 8, 24 h) expression of inducible NO synthase and TNF-α mRNAs was reduced by Tau-Cl in NR8383 cells. Tau-Cl depressed NF-κB migration into the nucleus of activated NR8383 cells and caused a more sustained presence of IκB in the cytoplasm. Stabilization of cytoplasmic IκB-α in Tau-Cl-treated cells resulted from decreased phosphorylation of IκB-α serine-32 and a lower activity of IκB kinase (IKK). Additional experiments demonstrated that Tau-Cl does not directly inhibit IKK activity. These results suggest that Tau-Cl exerts its effects at some level upstream of IKK in the signaling pathway and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-κB activation.
A Critical Role for Mouse CXC Chemokine(s) in Pulmonary Neutrophilia During Th Type 1-Dependent Airway Inflammation
Authors: Takaoka Akiko , Tanaka Yoshitaka , Tsuji Takemasa , Jinushi Takafumi , Hoshino Akihiko , Asakura Yumiko , Mita Yasuo , Watanabe Kazuhito , Nakaike Shiro , Togashi Yuji , Koda Toshiaki , Matsushima Kouji , Nishimura Takashi
Published: April 2014

Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.
Increased Frequency of Pre-germinal Center B Cells and Plasma Cell Precursors in the Blood of Children with Systemic Lupus Erythematosus
Authors: Arce Edsel , Jackson Deborah G. , Gill Michelle A. , Bennett Lynda B. , Banchereau Jacques , Pascual Virginia
Published: April 2014

We have analyzed the blood B cell subpopulations of children with systemic lupus erythematosus (SLE) and healthy controls. We found that the normal recirculating mature B cell pool is composed of four subsets: conventional naive and memory B cells, a novel B cell subset with pregerminal center phenotype (IgD+CD38+centerin+), and a plasma cell precursor subset (CD20−CD19+/lowCD27+/++ CD38++). In SLE patients, naive and memory B cells (CD20+CD38−) are ∼90% reduced, whereas oligoclonal plasma cell precursors are 3-fold expanded, independently of disease activity and modality of therapy. Pregerminal center cells in SLE are decreased to a lesser extent than conventional B cells, and therefore represent the predominant blood B cell subset in a number of patients. Thus, SLE is associated with major blood B cell subset alterations.
Cutting Edge: Identification of a Hybrid Cytokine Consisting of IL-7 and the β-Chain of the Hepatocyte Growth Factor/Scatter Factor
Authors: Lai Laijun , Goldschneider Irving
Published: April 2014

Pre-pro-B cell growth-stimulating factor (PPBSF) is a heterodimer of IL-7 and a 30-kDa cofactor. Unlike monomeric IL-7, PPBSF selectively induces proliferation and differentiation of pre-pro-B cells and up-regulates IL-7Rα-chain expression. Here we clone the PPBSF cofactor from bone marrow stromal cells and identify it as a variant β-chain of hepatocyte growth factor (HGF), a pleiotropic cytokine homologous to plasminogen that regulates cell growth, motility, and morphogenesis. We further demonstrate that, in the presence of low m.w. heparin sulfate-derived oligosaccharides, rHGFβ combines with rIL-7 to form a biologically active heterodimer having the properties of PPBSF. The results indicate that PPBSF is a novel form of cytokine (hybrid cytokine) consisting of the bioactive components of two unrelated cytokines. Based on its heparin-binding and mitogenic properties, we postulate that the HGFβ-chain in PPBSF enables IL-7 to participate in cognate interactions at the stromal cell surface and to transduce signals effectively at low levels of IL-7R.
Cutting Edge: Role of Toll-Like Receptor 9 in CpG DNA-Induced Activation of Human Cells
Authors: Takeshita Fumihiko , Leifer Cynthia A. , Gursel Ihsan , Ishii Ken J. , Takeshita Saoko , Gursel Mayda , Klinman Dennis M.
Published: April 2014

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.
Tumoricidal Activity of Monocyte-Derived Dendritic Cells: Evidence for a Caspase-8-Dependent, Fas-Associated Death Domain-Independent Mechanism
Authors: Vanderheyde Nathalie , Aksoy Ezra , Amraoui Zoulikha , Vandenabeele Peter , Goldman Michel , Willems Fabienne
Published: April 2014

Monocyte-derived dendritic cells (DC) were found to be cytotoxic for several tumor cell lines including Jurkat cells, which were killed through a calcium-independent pathway. K562 cells were resistant, excluding a NK cell-like activity. DC-mediated apoptosis did not involve classical death receptors because it was not reversed by blocking TNF/TNFR, CD95/CD95 ligand, or TNF-related apoptosis-inducing ligand/TNF-related apoptosis-inducing ligand receptor interactions. Fas-associated death domain-deficient, but not caspase-8 deficient, Jurkat cells were killed by DC. Indeed, caspase-8 cleavage was demonstrated in Jurkat cells cocultured with DC, and the use of specific caspase inhibitors confirmed that apoptosis triggered by DC was caspase-8 dependent. Furthermore, the involvement of Bcl-2 family members in the control of DC-mediated apoptosis was demonstrated by Bid cleavage in Jurkat cells cocultured with DC and resistance of Jurkat cells overexpressing Bcl-2 to DC-mediated cytotoxicity. Overall, these data indicate that monocyte-derived DC exert a caspase-8-dependent, Fas associated death domain-independent tumoricidal activity, a finding that could be relevant to their therapeutic use in cancer.
An Atypical Population of NK Cells That Spontaneously Secrete IFN-γ and IL-4 Is Present in the Intraepithelial Lymphoid Compartment of the Rat
Authors: Todd Derrick J. , Greiner Dale L. , Rossini Aldo A. , Mordes John P. , Bortell Rita
Published: April 2014

The intestinal lymphoid compartment of the rat is large and diverse, but the phenotype and functions of its constituent cell populations are not fully characterized. Using new methodology for the isolation and purification of rat intestinal intraepithelial lymphocytes (IELs), we previously identified a population of αβ- and γδ-TCR− NKR-P1A+ NK cells. These cells were almost completely restricted to the CD4−CD8− IEL population, and unlike peripheral NK cells in the rat, they were CD2−. We now report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, however, intraepithelial and splenic NK cells differ markedly in two respects. First, IENK cells express high levels of ADP-ribosyltransferase 2 (a marker of regulatory T cells in the rat) and CD25, whereas peripheral NK cells do not. Second, unlike splenic NK cells, a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. We conclude that the rat IEL compartment harbors a large population of NKR-P1A+CD3− cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive IENK cells may participate in the regulation of mucosal immunity.
Bm1–Bm5 Classification of Peripheral Blood B Cells Reveals Circulating Germinal Center Founder Cells in Healthy Individuals and Disturbance in the B Cell Subpopulations in Patients with Primary Sjögren’s Syndrome
Authors: Bohnhorst Janne Ø. , Bjørgan Marie B. , Thoen Jørn E. , Natvig Jacob B. , Thompson Keith M.
Published: April 2014

Analyses of B cells in the bone marrow and secondary lymphoid tissues have revealed a broad range of cell surface markers defining B cell subpopulations, but only a few of these have been used to analyze B cell subpopulations in peripheral blood (PB). We report here the delineation of circulating PB B cell subpopulations by staining for CD19, CD38, and IgD in combination with CD10, CD44, CD77, CD95, CD23, IgM, and the B cell memory marker CD27. The utility of this approach is shown by the demonstration of disturbances of circulating B cell subpopulations in patients with autoimmune disease. Five mature B cell (Bm) subpopulations were identified in normal PB that were comparable with the tonsillar Bm1, Bm2, early Bm5, Bm5 subpopulations and, surprisingly, to the germinal center (GC) founder cell subpopulation (Bm2′ and Bm3δ–4δ), suggesting that some GC founder cells are circulating. No PB B cells resembled the Bm3 and Bm4 GC cells. Remarkably, some cells with the CD38−IgD+ phenotype, previously known as naive Bm1 cells, expressed CD27. The CD38−IgD+ subpopulation therefore includes both naive Bm1 cells and IgD+ memory B cells. This new classification of B cell developmental stages reveals disturbances in the proportions of B cell subpopulations in primary Sjögren’s syndrome (pSS) patients compared with healthy donors and rheumatoid arthritis patients. Patients with pSS contained a significantly higher percentage of B cells in two activated stages, which might reflect a disturbance in B cell trafficking and/or alteration in B cell differentiation. These findings could be of diagnostic significance for pSS.
IL-10 Inhibits Granulocyte-Macrophage Colony-Stimulating Factor-Dependent Human Monocyte Survival at the Early Stage of the Culture and Inhibits the Generation of Macrophages
Authors: Hashimoto Shin-ichi , Komuro Iwao , Yamada Muneo , Akagawa Kiyoko S.
Published: April 2014

We previously demonstrated that IL-10 alone does not stimulate growth and differentiation of human monocytes, but enhances those of monocytes stimulated with M-CSF. We studied here the effect of IL-10 on human monocytes stimulated with GM-CSF. Monocytes stimulated with GM-CSF alone survived and developed into macrophages. Monocytes cultured with GM-CSF plus IL-10, however, died through apoptosis. IL-10 decreased expression of bcl-2, bcl-xL, and mcl-1- but not bax mRNA in monocytes stimulated with GM-CSF. IL-10 did not change the expression of mRNA of both GM-CSFR α-chain and β-chain, but inhibited tyrosine phosphorylation of STAT5 and extracellular signal-regulated kinases 1 and 2 in the monocytes. The inhibitory effect of IL-10 was restricted to treatment 48 h after stimulation with GM-CSF. Addition of IL-10 after that time induced neither apoptosis nor a decrease in expression of bcl-2, bcl-xL, and mcl-1 mRNA. IL-10, however, inhibited LPS-induced TNF-α production even in these cells, indicating that the cells still possessed responsiveness to IL-10. Monocytes pretreated for >48 h with GM-CSF became resistant to GM-CSF withdrawal, and the cells could survive without GM-CSF. These results indicate that IL-10 selectively inhibits GM-CSF-dependent monocyte survival by inhibiting the signaling events induced by GM-CSF, but the timing of addition of IL-10 is critical, and IL-10 had to be added within 48 h after stimulation with GM-CSF to achieve the inhibitory effect. These results taken together with our previous results indicate that IL-10 plays a pivotal role in monocyte survival and development into macrophages in concert with M-CSF and GM-CSF.
Mechanisms of Nuclear Import and Export That Control the Subcellular Localization of Class II Transactivator
Authors: Cressman Drew E. , O’Connor William J. , Greer Susanna F. , Zhu Xin-Sheng , Ting Jenny P.-Y.
Published: April 2014

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405–414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.
Delineation of Intrathymic T, NK, and Dendritic Cell (DC) Progenitors in Fetal and Adult Rats: Demonstration of a Bipotent T/DC Intermediate Precursor
Authors: Alonso-C. Luis M. , Muñoz Juan J. , Zapata Agustín G.
Published: April 2014

We previously published study results stating that the early rat fetal liver contains a high frequency of T/dendritic cells (DCs), but rarely T/NK bipotent common progenitors. Now, by using xenogenic rat/SCID mouse fetal thymic organ cultures, we extend these observations to the thymus, in which conflicting data have been published in human and mouse. On the one hand, enriched adult intrathymic CD45+CD2− triple negative for CD8, CD4, and CD3 Ag cell progenitors, which contained both rearranged TCRβ chain and pre-Tα chain transcripts, completely lacked NKR-P1A expressing cells, and upon limiting dilution conditions, generated T- and T/DC-containing lobes, but no T/NK or NK ones were found. On the other hand, the CD45+CD2− triple negative for CD8, CD4, and CD3 Ags cell population obtained from 15- and 16-day-old fetal rat thymus can be divided into NKR-P1A− and NKR-P1Alow cell subpopulations that differ in several aspects. Both cell subsets expressed pre-TCRα chain transcripts, but only the former contained fully rearranged TCRβ chain transcripts. Upon limiting dilution, T cell-committed progenitors were only found in the NKR-P1A− cell population, whereas NK-committed progenitors were present in the NKR-P1Alow population. More importantly, bipotential T/NK progenitors were very rare and were found only in the NKR-P1Alow cell population, whereas bipotential T/DC progenitors, only previously suggested in the adult mouse thymus, were observed frequently in the NKR-P1A−CD2− cell subpopulation. Our results demonstrate, therefore, that a common intrathymic T/DC intermediate represents the main T cell developmental pathway in rat thymus.
STAT5 Induces Macrophage Differentiation of M1 Leukemia Cells Through Activation of IL-6 Production Mediated by NF-κB p65
Authors: Kawashima Toshiyuki , Murata Ken , Akira Shizuo , Tonozuka Yukio , Minoshima Yukinori , Feng Sizhou , Kumagai Hidetoshi , Tsuruga Hiromichi , Ikeda Yasuo , Asano Shigetaka , Nosaka Tetsuya , Kitamura Toshio
Published: April 2014

We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21WAF/Cip1, and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-κB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-κB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines
Th1 Cytokine-Conditioned Bone Marrow-Derived Dendritic Cells Can Bypass the Requirement for Th Functions During the Generation of CD8+ CTL
Authors: Sato Marimo , Chamoto Kenji , Tsuji Takemasa , Iwakura Yoichiro , Togashi Yuji , Koda Toshiaki , Nishimura Takashi
Published: April 2014

Bone marrow-derived dendritic cell (BMDC) subsets have distinct immunoregulatory functions. Th1 cytokine-induced BMDC (BMDC1), compared with Th2 cytokine-induced BMDC2, have superior activities for the differentiation and expansion of CTL. To evaluate the cellular interactions between dendritic cells and CD8+ T cells for the induction of CTL, BALB/c-derived BMDC subsets were cocultured with purified CD8+ T cells from C57BL/6 mice. Our results demonstrate that BMDC1 support the generation of allogeneic CD8+ CTL in the absence of CD4+ Th cells. In contrast, BMDC0 (GM-CSF- plus IL-3-induced BMDC) and BMDC2 failed to promote the differentiation of CD8+ CTL. Using Ab-blocking experiments and studies with gene knockout mice, IL-2 and LFA-1 are demonstrated to be critical for BMDC1-induced CTL differentiation. Unexpectedly, BMDC1 were able to induce CTL from CD8+ T cells isolated from IFN-γ−/− and IFN-γ receptor−/− mice. However, BMDC1 produced higher levels of IFN-β than other BMDC subsets, and anti-IFN-β mAb blocked BMDC1-dependent CTL generation. These results indicated an indispensable role of IFN-β, but not IFN-γ, during BMDC1-induced CTL differentiation. We conclude that Th1-cytokine-conditioned BMDC1 can bypass Th cell function for the differentiation of naive CD8+ T cells into CTL.
Both CD34+38+ and CD34+38− Cells Home Specifically to the Bone Marrow of NOD/LtSZ scid/scid Mice but Show Different Kinetics in Expansion
Authors: Kerre Tessa C. C. , De Smet Greet , De Smedt Magda , Offner Fritz , De Bosscher José , Plum Jean , Vandekerckhove Bart
Published: April 2014

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38− umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38− cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38− cells.
The Rearranged VH Domain of a Physiologically Selected Anti-Single-Stranded DNA Antibody as a Precursor for Formation of IgM and IgG Antibodies to Diverse Antigens
Authors: Li Jing , Fernandez Luis , O’Connor Kevin C. , Imanishi-Kari Thereza , Stollar B. David
Published: April 2014

It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged VH domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by VH and VL gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1VH transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene VH DHJH was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive VH domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.
Examination of HY Response: T Cell Expansion, Immunodominance, and Cross-Priming Revealed by HY Tetramer Analysis
Authors: Millrain Maggie , Chandler Phillip , Dazzi Francesco , Scott Diane , Simpson Elizabeth , Dyson P. Julian
Published: April 2014

We have applied MHC class I tetramers representing the two H2b MHC class I-restricted epitopes of the mouse male-specific minor transplantation Ag, HY, to directly determine the extent of expansion and immunodominance within the CD8+ T cell compartment following exposure to male tissue. Immunization with male bone marrow (BM), spleen, dendritic cells (DCs) and by skin graft led to rapid expansion of both specificities occupying up to >20% of the CD8+ T cell pool. At a high dose, whole BM or spleen were found to be more effective at stimulating the response than BM-derived DCs. In vivo, immunodominance within the responding cell population was only observed following chronic Ag stimulation, whereas epitope immunodominance was established rapidly following in vitro restimulation. Peptide affinity for the restricting MHC molecule was greater for the immunodominant epitope, suggesting that this might be a factor in the emergence of immunodominance. Using tetramers, we were able to directly visualize the cross-primed CD8+ HY response, but we did not find it to be the principal route for MHC class I presentation. Immunization with female spleen or DCs coated with the full complement of defined HY peptides, including the Ab-restricted CD4+ Th cell determinant, failed to induce tetramer-reactive cells.
Ceramide Mediates Tumor-Induced Dendritic Cell Apoptosis
Authors: Kanto Tatsuya , Kalinski Pawel , Hunter Oriana C. , Lotze Michael T. , Amoscato Andrew A.
Published: April 2014

Induction of apoptosis in dendritic cells (DC) is one of the escape mechanisms of tumor cells from the immune surveillance system. This study aimed to clarify the underlying mechanisms of tumor-induced DC apoptosis. The supernatants (SN) of murine tumor cell lines B16 (melanoma), MCA207, and MCA102 (fibrosarcoma) increased C16 and C24 ceramide as determined by electrospray mass spectrometry and induced apoptosis in bone marrow-derived DC. N-oleoylethanolamine or d-l-threo 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits acid ceramidase or glucosylceramide synthase and then increases endogenous ceramide, enhanced DC apoptosis and ceramide levels in the presence of tumor SN. Pretreatment with l-cycloserine, an inhibitor of de novo ceramide synthesis, or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate reduced endogenous ceramide levels and protected DC from tumor-induced apoptosis. However, other DC survival factors, including LPS and TNF-α, failed to do so. The protective activity of 12-O-tetradecanoylphorbol-13-acetate is abrogated by pretreatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. Therefore, down-regulation of PI3K is the major facet of tumor-induced DC apoptosis. Tumor SN, N-oleoylethanolamine, or PDMP suppressed Akt, NF-κB, and bcl-xL in DC, suggesting that the accumulation of ceramide impedes PI3K-mediated survival signals. Taken together, ceramide mediates tumor-induced DC apoptosis by down-regulation of the PI3K pathway.
Oxidized Low-Density Lipoprotein Promotes Mature Dendritic Cell Transition from Differentiating Monocyte
Authors: Perrin-Cocon Laure , Coutant Fredéric , Agaugué Sophie , Deforges Séverine , André Patrice , Lotteau Vincent
Published: April 2014

Proinflammatory oxidized phospholipids are generated during oxidative modification of low-density lipoproteins (LDL). The production of these proinflammatory oxidized phospholipids is controlled by secreted enzymes that circulate as proteins complexed with LDL and high-density lipoprotein. During the acute phase response to tissue injury, profound changes occur in lipoprotein enzymatic composition that alter their anti-inflammatory function. Monocytes may encounter oxidized phospholipids in vivo during their differentiation to macrophages or dendritic cells (DC). In this study we show that the presence of oxidized LDL (oxLDL) at the first day of monocyte differentiation to DC in vitro yielded phenotypically atypical cells with some functional characteristics of mature DC. Addition of oxLDL during the late stage of monocyte differentiation gave rise directly to phenotypically mature DC with reduced uptake capacity, secreting IL-12 but not IL-10, and supporting both syngeneic and allogeneic T cell stimulation. In contrast to known mediators of DC activation, oxLDL did not trigger maturation of immature DC. An intriguing possibility is that a burst of oxidized phospholipids is an endogenous activation signal for the immune system, which is tightly controlled by lipoproteins during the acute phase response.
Developmentally Regulated Changes in Glucosidase II Association with, and Carbohydrate Content of, the Protein Tyrosine Phosphatase CD45
Authors: Baldwin Troy A. , Ostergaard Hanne L.
Published: April 2014

Glucosidase II (GII) stably interacts with the external domain of CD45 in a carbohydrate-dependent manner. We have found that the association occurs in immature cells, but is significantly reduced in mature T cells. Using mannose-binding protein (MBP), in both FACS analysis and pull-down assays, we find that MBP can specifically recognize cell surface CD45 from immature, but not mature T cells. Analysis of thymocytes reveals increased MBP binding and GII association with CD45 in double-positive thymocytes compared with either double-negative or single-positive thymocytes. As well, the same pool of CD45 recognized by MBP can also associate with GII. Initial analysis of the basis of the interaction between CD45 and MBP suggests MBP binds two different glycoforms of CD45 based on the differential competition with glucose. Finally, inhibition of GII activity in cells that do not normally express MBP ligands results in significant increases in cell surface MBP ligands, including CD45. Taken together, these data suggest that the glucose content of the cell surface CD45 changes as thymocytes undergo maturation to mature T cells, and may be regulated by GII interactions. Such changes in the cell surface carbohydrate on CD45 may affect the development of thymocytes, perhaps via binding of CD45 on thymocytes to lectins on stromal cells.
Intestinal Trefoil Factor Induces Decay-Accelerating Factor Expression and Enhances the Protective Activities Against Complement Activation in Intestinal Epithelial Cells
Authors: Andoh Akira , Kinoshita Koichi , Rosenberg Ian , Podolsky Daniel K.
Published: April 2014

Mucosal damage induces a massive influx of serum complement components into the lumen. The epithelium produces a number of factors that can potentially ameliorate injury including intestinal trefoil factor (ITF), a small protease-resistant peptide produced and secreted onto the mucosal surface by goblet cells, and decay-accelerating factor (DAF), a protein produced by columnar epithelium which protects the host tissue from autologous complement injury. However, coordination of these intrinsic defensive products has not been delineated. DAF protein and mRNA expression were evaluated by immunoblotting and Northern blotting, respectively. NF-κB-DNA binding activity and DAF promoter activity were assessed by an electrophoretic gel mobility shift assay and a reporter gene luciferase assay, respectively. ITF induced a dose- and time-dependent increase in DAF protein and mRNA expression in human (HT-29 and T84) and rat (IEC-6) intestinal epithelial cells. In differentiated T84 cells grown on cell culture inserts, basolateral stimulation with ITF strongly enhanced DAF expression, but apical stimulation had no effects. The C3 deposition induced by complement activation was significantly blocked by the treatment with ITF. In HT-29 cells, ITF increased the stability of DAF mRNA. ITF also enhanced the promoter activity of the DAF gene via NF-κB motif and induced activation of NF-κB-DNA binding activity. ITF promotes protection of epithelial cells from complement activation via up-regulation of DAF expression, contributing to a robust mucosal defense.
Passively Acquired Antibodies Suppress Humoral But Not Cell-Mediated Immunity in Mice Immunized with Live Attenuated Respiratory Syncytial Virus Vaccines
Authors: Crowe James E. , Firestone Cai-Yen , Murphy Brian R.
Published: April 2014

A respiratory syncytial virus (RSV) vaccine will need to be administered by 1 mo of age to protect young infants; therefore, it will need to be effective in the presence of maternally acquired RSV Abs. In the present study, the immunogenicity and efficacy of two live attenuated RSV vaccine candidates of different level of attenuation were evaluated in mice passively immunized with varying quantities of RSV Abs. The replication of the RSV vaccines was suppressed in the lower, but not the upper, respiratory tract of the passively immunized mice. Immunization with either vaccine candidate was highly efficacious against challenge with wild-type RSV in both passively immunized and control mice. Nonetheless, a high level of immunity was seen even in passively/actively immunized animals that failed to develop a humoral immune response, suggesting that T cells mediated the immunity. Depletion of CD4+ and CD8+ T cells in passively/actively immunized and control animals at the time of challenge with wild-type RSV demonstrated that CD4+ and CD8+ T cells made significant independent contributions to the restriction of replication of RSV challenge virus in both the upper and lower respiratory tracts. Although passively acquired serum RSV Abs suppressed the primary systemic and mucosal Ab responses of IgM, IgG, and IgA isotypes, B lymphocytes were nevertheless primed for robust secondary Ab responses. Thus, immunity mediated by CD4+ and CD8+ T cells and Abs can be readily induced in mice by live RSV vaccine candidates in the presence of physiologic levels of RSV neutralizing Abs.
Chronic Inflammation and Protection from Acute Hepatitis in Transgenic Mice Expressing TNF in Endothelial Cells
Authors: Willuweit Antje , Sass Gabriele , Schöneberg Annette , Eisel Ulrich , Tiegs Gisa , Clauss Matthias
Published: April 2014

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-γ were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.
Anaphylatoxin C5a Actions in Rat Liver: Synergistic Enhancement by C5a of Lipopolysaccharide-Dependent α2-Macroglobulin Gene Expression in Hepatocytes Via IL-6 Release from Kupffer Cells
Authors: Mäck Claudia , Jungermann Kurt , Götze Otto , Schieferdecker Henrike L.
Published: April 2014

The effects of the anaphylatoxins C5a and C3a on the liver are only poorly characterized in contrast to their well known systemic actions. Recently, it has been demonstrated that the anaphylatoxin C5a enhanced glucose output from hepatocytes (HC) indirectly via prostanoid release from Kupffer cells (KC). In the present study, it is shown that recombinant rat C5a (rrC5a), together with LPS, activated the gene of the acute phase protein α2-macroglobulin (α2MG) in HC also indirectly via IL-6 release from KC. RrC5a alone increased neither IL-6 mRNA in nor IL-6 release from KC, whereas LPS alone did so. However, rrC5a synergistically enhanced the LPS-dependent increase in IL-6 mRNA and IL-6 release. Only rIL-6, but not TNF-α or IL-1β, enhanced α2MG mRNA in HC. In line with the actions of rrC5a and LPS on KC, conditioned medium of KC stimulated only with rrC5a did not increase α2MG mRNA in HC. However, medium of KC stimulated with rrC5a plus LPS induced α2MG mRNA expression in HC more strongly than medium from cells stimulated only with LPS; thus, C5a acted synergistically with LPS. The stimulatory effects of KC-conditioned medium could partially be inhibited by a neutralizing anti-IL-6 Ab, indicating that KC-derived IL-6 was a major mediator in C5a- plus LPS-elicited α2MG gene expression. These results suggest that C5a, besides enhancing glucose output via prostanoids, is involved in the initiation of the acute phase response in HC via proinflammatory cytokines from KC. This provides evidence for another important function of C5a in the regulation of hepatocellular defense reactions.
Inflammatory Cytokines Regulate Function and Expression of Adenosine A2A Receptors in Human Monocytic THP-1 Cells
Authors: Khoa Nguyen D. , Montesinos M. Carmen , Reiss Allison B. , Delano David , Awadallah Nahel , Cronstein Bruce N.
Published: April 2014

Adenosine, acting at its receptors, particularly A2A receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A2A receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680), a selective A2A receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A2A receptor antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-α (100 U/ml) but markedly attenuated in cells pretreated with IFN-γ (100 U/ml). Similarly, IL-1 and TNF-α treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-γ treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-α-treated cells but not in control or IFN-γ-treated cells. Both IL-1 and TNF-α increased A2A receptor mRNA and protein. In parallel with its effect on A2A receptor function, IFN-γ down-regulated A2A receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.
Reconstitution of the Complement Function in C1q-Deficient (C1qa−/−) Mice with Wild-Type Bone Marrow Cells
Authors: Petry Franz , Botto Marina , Holtappels Rafaela , Walport Mark J. , Loos Michael
Published: April 2014

Besides Ab-independent and Ab-dependent activation of the complement classical pathway in host defense, C1q plays a key role in the processing of immune complexes and in the clearance of apoptotic cells. In humans, C1q deficiency leads to systemic lupus erythematosus-like symptoms in over 90% of the cases, thus making this defect a strong disease susceptibility factor. Similarly, C1q-deficient mice (C1qa−/−) develop systemic lupus erythematosus-like symptoms, such as autoantibodies and glomerulonephritis. We have previously provided evidence that C1q is produced by cells of the monocyte-macrophage lineage. In this study, we have tested whether transplantation of bone marrow cells would be sufficient to reconstitute C1q levels in C1qa−/− mice. C1qa−/− mice received a single graft of 107 bone marrow cells from wild-type (wt) donors after irradiation doses of 6, 7, 8, or 9 Gy. Engraftment was monitored by a Y chromosome-specific PCR and a PCR that differentiated wt from C1qa−/− genotype. Serum levels of C1q Ag and C1 function increased rapidly in the recipient mice, and titers reached normal levels within 6 wk after bone marrow transplantation. In wt mice that received C1qa−/− bone marrow, serum levels of C1q decreased constantly over time and became C1q deficient within 55 wk. These data clearly demonstrate that bone marrow-derived cells are the source of serum C1q and are competent to reconstitute normal C1q serum levels in C1q-deficient mice. Therefore, stem cell transplantation could be a therapy for patients with hereditary C1q deficiency.
Clonal Biases of Peripheral CD8 T Cell Repertoire Directly Reflect Local Inflammation in Polymyositis
Authors: Nishio Junko , Suzuki Mihoko , Miyasaka Nobuyuki , Kohsaka Hitoshi
Published: April 2014

Polymyositis (PM) involves destruction of striated muscles by autoaggressive CD8 T cells, which accumulate and secrete cytotoxic effector molecules in the affected muscles. Previous studies of peripheral T cell repertoires from normal individuals and patients with viral infections have shown that primed CD8 T cells, unlike CD4 T cells, are prone to expand clonally and persist as large populations in the peripheral blood. These facts made us assume that autoaggressive myocytotoxic CD8 T cells would expand clonally in the peripheral blood from patients with PM. By clonal analyses of peripheral T cells from patients and age-matched controls, we show here that clonal expansion of CD8 T cells was more frequent in patients. This was not significant in CD4 T cells. In analogy to virus-specific T cells, the expanded T cells persisted as large populations over time. Analysis of the muscle biopsy specimens revealed that some of the expanded clones were infiltrating in the affected muscles from the same patients. These results provide the first evidence that local autoimmune reaction directly elicits significant biases in peripheral T cell repertoire. The expanded cells, which should be candidate autoaggressive T cells, were readily isolated from the peripheral blood for analysis of expressed genes including perforin. Thus, our findings should give us an immediate clue to analysis of the pathogenic T cells in PM.
IL-10 Improves Skin Disease and Modulates Endothelial Activation and Leukocyte Effector Function in Patients with Psoriatic Arthritis
Authors: McInnes Iain B. , Illei Gabor G. , Danning Carol L. , Yarboro Cheryl H. , Crane Marianne , Kuroiwa Takashi , Schlimgen Ryan , Lee Eric , Foster Barbara , Flemming Donald , Prussin Calman , Fleisher Thomas A. , Boumpas Dimitrios T.
Published: April 2014

Psoriatic arthritis (PsA) provides an ideal disease model in which to investigate the bioactivities of potentially therapeutic cytokines at multiple sites of tissue inflammation. We investigated the effects of IL-10, an antiinflammatory cytokine, given s.c. for 28 days in a double-blind, placebo-controlled study in PsA patients. Synovial/skin biopsies, peripheral blood leukocytes, articular magnetic resonance images, and clinical disease activity scores were obtained sequentially. Modest, but significant clinical improvement in skin, but not articular disease activity scores with only minor adverse effects was observed. Type 1, but not type 2 T cell cytokine production in vitro was suppressed in human rIL-10 compared with placebo recipients. Similarly, monokine production in vitro was reduced, whereas serum soluble TNFRII levels were elevated, indicating suppression of monocyte function. Decreased T cell and macrophage infiltration in synovial tissues was accompanied by reduced P-selectin expression. Moreover, suppressed synovial enhancement on magnetic resonance imaging and reduced αvβ3 integrin expression on von Willebrand factor+ vessels were observed. Together these data demonstrate that a short course of IL-10 modulates immune responses in vivo via diverse effects on endothelial activation, and leukocyte recruitment and effector function. Such biological changes may result in clinically meaningful improvement in disease activity.