bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of Spontaneous Breast Carcinoma by Prophylactic Vaccination with Dendritic/Tumor Fusion Cells
Authors: Xia Jianchuan , Tanaka Yasuhiro , Koido Shigeo , Liu Chunlei , Mukherjee Pinku , Gendler Sandra J. , Gong Jianlin
Published: April 2014

Genetically modified mice with spontaneous development of mammary carcinoma provide a powerful tool to study the efficacy of tumor vaccines, since they mimic breast cancer development in humans. We used a transgenic murine model expressing polyomavirus middle T oncogene and mucin 1 tumor-associated Ag to determine the preventive effect of a dendritic/tumor fusion cell vaccine. The MMT (a transgenic murine model) mice developed mammary carcinoma between the ages of 65–108 days with 100% penetrance. No spontaneous CTL were detected. However, prophylactic vaccination of MMT mice with dendritic/tumor fusion cells induced polyclonal CTL activity against spontaneous mammary carcinoma cells and rendered 57–61% of the mice free of the disease at the end of experiment (180 days). Furthermore, the level of CTL activity was maintained with multiple vaccinations. The antitumor immunity induced by vaccination with dendritic/tumor fusion cells reacted differently to injected tumor cells and autochthonous tumor. Whereas the injected tumor cells were rejected, the autochthonous tumor evaded the attack and was allowed to grow. Collectively these results indicate that prophylactic vaccination with dendritic/tumor fusion cells confers sufficient antitumor immunity to counter the tumorigenesis of potent oncogenic products. The findings in the present study are highly relevant to cancers in humans.
Regression of a Mammary Adenocarcinoma in STAT6−/− Mice Is Dependent on the Presence of STAT6-Reactive T Cells
Authors: Jensen Shawn M. , Meijer Sybren L. , Kurt Robert A. , Urba Walter J. , Hu Hong-Ming , Fox Bernard A.
Published: April 2014

Polarization of the immune response toward a type 1 cytokine profile has been posited to be associated with a therapeutic antitumor immune response. STAT6−/− mice are unable to generate a type 2 immune response, and instead mount an enhanced type 1 response. STAT6−/− mice are significantly more resistant to 4T1, a mammary adenocarinoma cell line, resisting a 10-fold higher tumor dose compared with wild-type (wt) BALB/c mice. An analysis of the T cells from tumor-bearing STAT6−/− mice revealed that they contained a population primed by a peptide (STAT6531–539) of the STAT6 protein expressed in 4T1. The adoptive transfer of T cells from STAT6531–539-vaccinated STAT6−/− mice significantly reduced the number of 4T1 pulmonary metastases in recipient mice. Additionally, the role of these STAT6531–539-reactive T cells against s.c. 4T1 tumor challenge was determined by tumor-challenging wt BALB/c mice reconstituted with STAT6−/− bone marrow, thereby assessing whether a polarized type 1 immune response in the absence of STAT6-reactive T cells was sufficient to reject a 4T1 tumor challenge. T cells from the STAT6−/− bone marrow chimeras failed to recognize the STAT6531–539, and these mice proved to be as susceptible as wt BALB/c mice to 4T1 challenge. This demonstrated that the absence of STAT6531–539-reactive T cells correlated with the inability to reject 4T1 challenge. Additionally, these data emphasize that the enhanced ability to mount a type 1-polarized immune response is inconsequential if a sufficient antitumor immune response is not primed by the tumor.
Memory Inflation: Continuous Accumulation of Antiviral CD8+ T Cells Over Time
Authors: Karrer Urs , Sierro Sophie , Wagner Markus , Oxenius Annette , Hengel Hartmut , Koszinowski Ulrich H. , Phillips Rodney E. , Klenerman Paul
Published: April 2014

CD8+ T lymphocytes play an important role in the control of intracellular pathogens during both acute and persistent infections. This is particularly true in the case of persistent herpesviruses such as human CMV, which are typified by large virus-specific CD8+ T cell populations during viral latency. To understand the origin of these populations and the factors shaping them over time, we investigated the CD8+ T cell response after murine CMV (MCMV) infection. The kinetics of the acute response were characterized by rapid expansion of activated T cells, followed by a contraction phase. Thereafter, we observed a striking pattern, where MCMV-specific memory CD8+ T cells steadily accumulated over time, with 20% of all CD8+ T cells at 1 year specific for one MCMV epitope. Accumulation of MCMV-specific CD8+ T lymphocytes was seen in all organs tested and was associated with continuous activation of specific CD8+ T lymphocytes, primarily within lymph nodes. The pattern of accumulation was observed in only two of five epitopes tested, and was accompanied by a gradual restriction in usage of the variable region of the TCR β-chain over time. This novel pattern of a virus-specific CD8+ T cell response suggests that continuous or repetitive exposure to Ag can slowly mold memory T cell populations over time. This may be relevant for understanding the evolution of the large human CMV-specific CD8+ T cell populations seen in humans.
Role of CD4 T Cell Help and Costimulation in CD8 T Cell Responses During Listeria monocytogenes Infection
Authors: Shedlock Devon J. , Whitmire Jason K. , Tan Joyce , MacDonald Andrew S. , Ahmed Rafi , Shen Hao
Published: April 2014

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4−/−, and MHC class II−/− mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4−/− mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L−/− mice but defective in CD28−/− and CD137L−/− mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.
Suppression of NF-κB Activation and Proinflammatory Cytokine Expression by Shiga Toxin-Producing Escherichia coli
Authors: Hauf Nadja , Chakraborty Trinad
Published: April 2014

The NF-κB family of transcription factors forms one of the first lines of defense against infectious disease by inducing the expression of genes involved in inflammatory and immune responses. In this study, we analyzed the impact of Shiga toxin-producing Escherichia coli (STEC) on the NF-κB DNA-binding activity in HeLa cells. After a period of weak initial activation, DNA binding of NF-κB was actively suppressed by viable, E. coli secreted protein B (EspB)-secreting STEC. Sustained NF-κB activity was observed either using an isogenic mutant lacking EspB or after gentamicin-based killing of STEC after allowing bacterial attachment. These observations indicate that the ability of STEC to cause NF-κB activation is suppressed by a translocated bacterial effector protein, which is either EspB itself or requires EspB for delivery into the host cell. We found that STEC, enterohemorrhagic E. coli, and enteropathogenic E. coli all interfere with NF-κB activation initiated by TNF-α, indicating that suppression of signal-induced NF-κB activity is a property common to several attaching and effacing bacteria. As a consequence of NF-κB suppression, wild-type STEC induces significantly lower mRNA levels of IL-8, IL-6, and IL-1α upon prolonged infection periods compared with bacteria lacking EspB. For IL-8 and IL-6, the suppressive effect was also reflected at the level of cytokine secretion. Suppression of both basal and signal-induced NF-κB DNA binding by attaching and effacing-inducing bacteria appears to be an active strategy to counteract host defense responses, thus favoring intestinal colonization by these pathogens.
Dual Roles of IL-4 in Lung Injury and Fibrosis
Authors: Huaux François , Liu Tianju , McGarry Bridget , Ullenbruch Matt , Phan Sem H.
Published: April 2014

Increased lung IL-4 expression in pulmonary fibrosis suggests a potential pathogenetic role for this cytokine. To dissect this role, bleomycin-induced pulmonary inflammation and fibrosis were analyzed and compared in wild type (IL-4+/+) vs IL-4-deficient (IL-4−/−) mice. Lethal pulmonary injury after bleomycin treatment was higher in IL-4−/− vs IL-4+/+ mice. By administration of anti-CD3 Abs, we demonstrated that this early response was linked to the marked T lymphocyte lung infiltration and to the overproduction of the proinflammatory mediators such as TNF-α, IFN-γ, and NO in IL-4−/− mice. In contrast to this early anti-inflammatory/immunosuppressive role, during later stages of fibrosis, IL-4 played a profibrotic role since IL-4−/− mice developed significantly less pulmonary fibrosis relative to IL-4+/+ mice. However, IL-4 failed to directly stimulate proliferation, α-smooth muscle actin, and type I collagen expression in lung fibroblasts isolated from the wild-type mice. Upon appropriate stimulation with other known fibrogenic cytokines, fibroblasts from IL-4−/− mice were relatively deficient in the studied parameters in comparison to fibroblasts isolated from IL-4+/+ mice. Taken together, these data suggest dual effects of IL-4 in this model of lung fibrosis: 1) limiting early recruitment of T lymphocytes, and 2) stimulation of fibrosis chronically.
Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression
Authors: Park Dae-Won , Kim Jae-Ryong , Kim Seong-Yong , Sonn Jong-Kyung , Bang Ok-Sun , Kang Shin-Sung , Kim Jung-Hye , Baek Suk-Hwan
Published: April 2014

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A2 (PLA2) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA2 up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA2 receptor (sPLA2R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA2 stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA2, Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA2-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA2, and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA2-induced reporter activity of the iNOS promoter. M-type sPLA2R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA2R enhanced group IIA PLA2-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA2 catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA2 induces nitrite production by involving of M-type sPLA2R, which then mediates signal transduction events that lead to PI3K/Akt activation.
IL-17, Produced by Lymphocytes and Neutrophils, Is Necessary for Lipopolysaccharide-Induced Airway Neutrophilia: IL-15 as a Possible Trigger
Authors: Ferretti Stephane , Bonneau Olivier , Dubois Gerald R. , Jones Carol E. , Trifilieff Alexandre
Published: April 2014

IL-17 is a cytokine implicated in the regulation of inflammation. We investigated the role of this cytokine in neutrophil recruitment using a model of LPS-induced lung inflammation in mice. In the bronchoalveolar lavage, LPS induced a first influx of neutrophils peaking at day 1, followed by a second wave, peaking at day 2. IL-17 levels were increased during the late phase neutrophilia (day 2), and this was concomitant with an increased number of T cells and macrophages, together with an increase of KC and macrophage-inflammatory protein-2 levels in the lung tissue. Intranasal treatment with a neutralizing murine anti-IL-17 Ab inhibited the late phase neutrophilia. In the bronchoalveolar lavage cells, IL-17 mRNA was detected at days 1, 2, and 3 postchallenge, with a strong expression at day 2. This expression was associated with CD4+ and CD8+ cells, but also with neutrophils. When challenged with LPS, despite the absence of T cells, SCID mice also developed a neutrophilic response associated with IL-17 production. In BALB/c mice, IL-15 mRNA, associated mainly with neutrophils, was evidenced 1 day after LPS challenge. In vitro, IL-15 was able to induce IL-17 release from purified spleen CD4+ cells, but not spleen CD8+ or airway neutrophils. We have shown that IL-17, produced mainly by CD4+ cells, but also by neutrophils, plays a role in the mobilization of lung neutrophils following bacterial challenge. In addition, our results suggest that IL-15 could represent a physiological trigger that leads to IL-17 production following bacterial infection.
Lipopolysaccharide Down-Regulates the Leukotriene C4 Synthase Gene in the Monocyte-Like Cell Line, THP-1
Authors: Serio Kenneth J. , Johns Scott C. , Luo Linda , Hodulik Craig R. , Bigby Timothy D.
Published: April 2014

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC4 synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC4 release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC4 synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC4 synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC4 synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-α and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-κB activation resulted in inhibition of the LPS effect, while activation of NF-κB and p50/p65 overexpression down-regulated the LTC4 synthase gene. LPS down-regulates cysteinyl LT release and LTC4 synthase gene expression in mononuclear phagocytes by an NF-κB-mediated mechanism.
Synoviocyte-Derived CXCL12 Is Displayed on Endothelium and Induces Angiogenesis in Rheumatoid Arthritis
Authors: Pablos José L. , Santiago Begoña , Galindo María , Torres Carmen , Brehmer María T. , Blanco Franciso J. , García-Lázaro Francisco J.
Published: April 2014

CXCL12 (stromal cell-derived factor-1) is a potent CXC chemokine that is constitutively expressed by stromal resident cells. Although it is considered a homeostatic rather than an inflammatory chemokine, CXCL12 has been immunodetected in different inflammatory diseases, but also in normal tissues, ant its potential functions and regulation in inflammation are not well known. In this study, we examined the cellular sources of CXCL12 gene expression and the mechanism and effects of its interactions with endothelial cells in rheumatoid arthritis synovium. We show that CXCL12 mRNA was not overexpressed nor induced in cultured rheumatoid synoviocytes, but it specifically accumulated in the rheumatoid hyperplastic lining layer and endothelium. CXCL12 gene expression was restricted to fibroblast-like synoviocytes, whereas endothelial cells did not express CXCL12 mRNA, but displayed the protein on heparitinase-sensitive factors. CXCL12 colocalized with the angiogenesis marker αvβ3 integrin in rheumatoid endothelium and induced angiogenesis in s.c. Matrigel plugs in mice. The angiogenic activity of rheumatoid synovial fluid in vivo was abrogated by specific immunodepletion of CXCL12. Our results indicate that synoviocyte-derived CXCL12 accumulates and it is immobilized on heparan sulfate molecules of endothelial cells, where it can promote angiogenesis and inflammatory cell infiltration, supporting a multifaceted function for this chemokine in the pathogenesis of rheumatoid arthritis.
Induction of Experimental Autoimmune Encephalomyelitis in IL-12 Receptor-β2-Deficient Mice: IL-12 Responsiveness Is Not Required in the Pathogenesis of Inflammatory Demyelination in the Central Nervous System
Authors: Zhang Guang-Xian , Gran Bruno , Yu Shuo , Li Jifen , Siglienti Ines , Chen Xiaohan , Kamoun Malek , Rostami Abdolmohamad
Published: April 2014

IL-12 is thought to be involved in the susceptibility to experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. IL-12 signals through a heterodimeric receptor (IL-12Rβ1/IL-12Rβ2), whose β2-chain is up-regulated on activated, autoreactive Th1 cells. Contrary to the expectation that the absence of IL-12Rβ2 would protect from EAE, we found that IL-12Rβ2-deficient mice developed earlier and more severe disease, with extensive demyelination and CNS inflammation. The inflammatory cells were mainly comprised of CD4+ T cells, monocyte/macrophages, and dendritic cells. Compared to wild-type mice, IL-12Rβ2-deficient mice exhibited significantly increased autoantigen-induced proliferative response and increased production of TNF-α, GM-CSF, IL-17, IL-18/IL-18Rα, and NO. In addition, we found significantly increased levels of IL-23p19 mRNA expression in spleen cells from immunized IL-12Rβ2−/− mice compared with wild-type mice. These findings indicate that IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS, and that, in the absence of IL-12Rβ2, increased IL-23 and other inflammatory molecules may be responsible for increased severity of EAE.
Peptide Fine Specificity of Anti-Glycoprotein 100 CTL Is Preserved Following Transfer of Engineered TCRαβ Genes Into Primary Human T Lymphocytes
Authors: Schaft Niels , Willemsen Ralph A. , de Vries Jolanda , Lankiewicz Birgit , Essers Bram W. L. , Gratama Jan-Willem , Figdor Carl G. , Bolhuis Reinier L. H. , Debets Reno , Adema Gosse J.
Published: April 2014

TCR with known antitumor reactivity can be genetically introduced into primary human T lymphocytes and provide promising tools for immunogene therapy of tumors. We molecularly characterized two distinct TCRs specific for the same HLA-A2-restricted peptide derived from the melanocyte differentiation Ag gp100, yet exhibiting different stringencies in peptide requirements. The existence of these two distinct gp100-specific TCRs allowed us to study the preservation of peptide fine specificity of native TCRαβ when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCRαβ constructs enabled T lymphocytes to specifically kill and produce TNF-α when triggered by native gp100pos/HLA-A2pos tumor target cells as well as gp100 peptide-loaded HLA-A2pos tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy.
Absence of IL-4, and Not Suppression of the Th2 Response, Prevents Development of Experimental Autoimmune Graves’ Disease
Authors: Dogan Rukiye-Nazan E. , Vasu Chenthamarakshan , Holterman Mark J. , Prabhakar Bellur S.
Published: April 2014

In autoimmune Graves’ disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) and cause hyperthyroidism. We studied the effects of fms-like tyrosine kinase receptor 3 ligand (Flt3-L) or GM-CSF treatment on the development of experimental autoimmune GD (EAGD) in mice, a slowly progressing Ab-mediated organ-specific autoimmune disease of the thyroid induced by immunization with syngeneic cells expressing TSHR. Flt3-L and GM-CSF treatment resulted in up-regulation of CD8a+ and CD8a− dendritic cells, and skewing of cytokine and immune responses to TSHR in favor of Th1 and Th2, respectively. However, this skewing did not persist until the later stages, and thus failed to affect the course or severity of the disease. To determine whether the total absence of either IL-4 or IFN-γ could affect the development of EAGD, we immunized wild-type, IFN-γ−/− and IL-4−/− BALB/c mice with TSHR. Nearly 100% of the wild-type and IFN-γ−/− mice developed EAGD with optimal TSHR-specific immune responses, while IL-4−/− mice completely resisted disease and showed delayed and suboptimal pathogenic Ab response. These data demonstrated that skewing immune responses to TSHR, using either Flt3-L or GM-CSF, in favor of Th1 or Th2, respectively, may not be sufficient to alter the course of the disease, while the complete absence of IL-4, but not IFN-γ, can prevent the development of EAGD.
In Vivo Efficacy of Anti-Glycoprotein 41, But Not Anti-Glycoprotein 120, Immunotoxins in a Mouse Model of HIV Infection
Authors: Pincus Seth H. , Fang Hua , Wilkinson Royce A. , Marcotte Tamera K. , Robinson James E. , Olson William C.
Published: April 2014

Immunotoxins (ITs) targeting the HIV envelope protein are among the most efficacious antiviral therapies when tested in vitro. Yet a first-generation IT targeted to gp120, CD4-PE40 (chimeric immunotoxin using CD4 and the translocation and enzymatic domains of Pseudomonas exotoxin A), showed limited promise in initial clinical testing, highlighting the need for improved ITs. We have used a new mouse model of HIV infection to test the comparative efficacy of anti-HIV ITs targeted to gp120 or to gp41. Irradiated SCID/nonobese diabetic mice are injected with a tumor of human CD4+ cells susceptible to infection and at a separate site persistently HIV-infected cells. The spread of infection from infected to susceptible tumor is monitored by plasma p24 and the presence of HIV-infected cells in the spleen. Anti-gp41 ITs in combination with tetrameric CD4-human Ig fusion protein have pronounced anti-HIV effects. Little if any anti-HIV efficacy was found with either CD4-PE40 or an Ab-targeted anti-gp120 IT. These data support continued exploration of the utility of ITs for HIV infection, particularly the use of anti-gp41 ITs in combination with soluble CD4 derivatives.
Combined Autoimmune Models of Arthritis Reveal Shared and Independent Qualitative (Binary) and Quantitative Trait Loci
Authors: Adarichev Vyacheslav A. , Valdez Juan C. , Bárdos Tamás , Finnegan Alison , Mikecz Katalin , Glant Tibor T.
Published: April 2014

Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are murine models for rheumatoid arthritis both in terms of their pathology and genetics. Using the F2 hybrids of the CIA-susceptible, but PGIA-resistant DBA/1 mice, and the CIA-resistant, but PGIA-susceptible BALB/c mice, our goals were to 1) identify both model-specific and shared loci that confer disease susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze whether any immune subtraits showed colocalization with arthritis-related loci. To identify chromosomal loci, we performed a genome scan on 939 F2 hybrid mice. For pathophysiological analyses, we measured pro- and anti-inflammatory cytokines (IL-1, IL-6, TNF-α, IFN-γ, IL-4, IL-10, IL-12), Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. In addition to multiple CIA- and PGIA-related loci identified in previous studies, we have identified nine new CIA- and eight new PGIA-linked loci. Comprehensive statistical analysis demonstrated that IL-2 production, T cell proliferation, and IFN-γ levels differed significantly between arthritic and nonarthritic animals in both CIA and PGIA populations. High levels of TNF-α, IFN-γ, IL-2, and Ab production were detected in F2 hybrids with CIA, whereas T cell proliferation, IL-2 and IFN-γ production, and a shift to IgG2a isotype were more characteristic of PGIA. Quantitative trait loci analysis demonstrated colocalization of numerous immune subtraits with arthritis-related traits. Quantitative trait loci on chromosomes 5, 10, 17, 18, and X were found to control arthritis in both models.
Heterogeneity of Dendritic Cells in the Mouse Liver: Identification and Characterization of Four Distinct Populations
Authors: Lian Zhe-Xiong , Okada Tomoyuki , He Xiao-Song , Kita Hiroto , Liu Yong-Jun , Ansari Aftab A. , Kikuchi Kentaro , Ikehara Susumu , Gershwin M. Eric
Published: April 2014

Liver dendritic cells (DC) are believed to play important roles in liver immunity, autoimmunity, and in the regulation of hepatic allograft acceptance. However, limited information is available on the phenotypes and functions of DC in the liver. To address this issue, we isolated DC from murine liver using procedures that do not involve collagenase, and characterized the freshly isolated DC population that had not been subjected to in vitro expansion. Thence, based on the expression of CD4, B220, and CD11b, four subsets or groups of hepatic NK1.1−CD11c+ DC were identified with the following phenotypes: B220+CD4+, B220+CD4−, B220−CD11b+, and B220−CD11b−. Each subset was further characterized both phenotypically and functionally. In addition to unique phenotypic expression, each subset displayed different allostimulation capability in mixed lymphocyte reaction assays. All four groups developed DC morphology following in vitro culture with activation agents and synthesized distinct patterns of cytokines in response to different stimuli. Taken together, our results suggest that groups I and II are IFN-α-producing plasmacytoid DC, group III cells are myeloid-related DC, while group IV is a heterogenous population containing both myeloid- and lymphoid-related DC. Our results demonstrate the highly heterogeneous nature of hepatic DC, which is in agreement with the unique requirements for APC in the complex liver environment.
Expression of Complement 3 and Complement 5 in Newt Limb and Lens Regeneration
Authors: Kimura Yuko , Madhavan Mayur , Call Mindy K. , Santiago William , Tsonis Panagiotis A. , Lambris John D. , Del Rio-Tsonis Katia
Published: April 2014

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.
Growth Factor Independence-1B Expression Leads to Defects in T Cell Activation, IL-7 Receptor α Expression, and T Cell Lineage Commitment
Authors: Doan Loretta L. , Kitay Mary Kate , Yu Qing , Singer Alfred , Herblot Sabine , Hoang Trang , Bear Susan E. , Morse Herbert C. , Tsichlis Philip N. , Grimes H. Leighton
Published: April 2014

T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Although recent studies have characterized the effects of proximal TCR signaling on T cell differentiation, the downstream integration of these signals remains largely unknown. The growth factor independence-1 (GFI1) and GFI1B transcriptional repressors may regulate cytokine signaling pathways to affect lymphocyte growth and survival. In this study, we show that Gfi1 expression is induced upon induction of the T cell program. Gfi1B expression is low and dynamic during T cell development, but is terminated in mature thymocytes. Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional consequences of constitutive expression. GFI1 potentiates response to TCR stimulation and IL-2, whereas GFI1B-transgenic T cells are defective in T cell activation. Moreover, GFI1B-transgenic thymocytes display reduced expression of the late-activation marker IL-7Rα, and a decrease in CD4−8+ single-positive T cells that can be mitigated by transgenic expression of BCL2 or GFI1. These data show that GFI1 and GFI1B are functionally unique, and implicate a role for GFI1 in the integration of activation and survival signals.
Inhibition of TCR-Induced CD8 T Cell Death by IL-12: Regulation of Fas Ligand and Cellular FLIP Expression and Caspase Activation by IL-12
Authors: Lee Seung Woo , Park Yunji , Yoo Jae Kwang , Choi So Young , Sung Young Chul
Published: April 2014

In this study we demonstrate the anti-apoptotic effect of IL-12 and its underlying mechanism in CD8 T cells. The prolonged stimulation of CD8 T cells with anti-CD3 alone caused apoptosis mediated by Fas and the caspase signaling pathway. However, costimulation with IL-12 significantly prevented anti-CD3-induced apoptosis of CD8 T cells. IL-12 decreased the number of Fas ligand-positive CD8 T cells and inhibited the activation of caspase-8 and caspase-3. In addition, IL-12 up-regulated cellular FLIPs but not Bcl-2 family proteins or cellular inhibitor of apoptosis proteins. These data suggest that IL-12 provides survival signals to CD8 T cells by down-regulating Fas ligand and up-regulating cellular FLIPs, followed by inhibiting caspase activation, which implies a role for IL-12 in peripheral responses of CD8 T cells in vivo.
IL-13 Is Necessary, Not Simply Sufficient, for Epicutaneously Induced Th2 Responses to Soluble Protein Antigen
Authors: Herrick Christina A. , Xu Lan , McKenzie Andrew N. J. , Tigelaar Robert E. , Bottomly Kim
Published: April 2014

Th2 responses are clearly involved in the pathogenesis of atopic disease. Thus, understanding the factors responsible for Th2 sensitization at sites of allergen exposure, such as airway and skin, is crucial for directing therapeutic or preventive strategies. Contrary to other models of Th2 sensitization to proteins, we have reported that Th2 responses induced by epicutaneous exposure to OVA are IL-4 independent. Combined deficiency of both IL-4 and IL-13 signaling did prevent Th2 generation, suggesting that IL-13 was mediating these IL-4-independent responses. It was not clear, however, whether IL-13 was simply replacing the need for IL-4 in genetically deficient mice or if IL-13 played a unique role. In the present study, we show that Th2 responses induced by epicutaneous OVA exposure (including lung inflammatory responses after inhaled Ag challenge, OVA-specific IgG1, and draining lymph node IL-5 production) are impaired in IL-13-deficient (IL-13−/−) mice compared with wild type. In contrast, i.p. sensitization of IL-13−/− mice resulted in responses equivalent to wild type. Generation of contact hypersensitivity to dinitrofluorobenzene, which involves Th1 and CD8+ effector cells, was also intact in IL-13−/− mice. Taken together, the data indicate that IL-13 is the major inducer of Th2 generation in the cutaneous microenvironment, being required independently of IL-4. This fact, in combination with the known abundance of IL-13 in atopic dermatitis skin lesions, emphasizes the potentially important role of the skin as a site for Th2 sensitization to environmental allergens, particularly in atopic individuals.
CD4+CD25− T Cells That Express Latency-Associated Peptide on the Surface Suppress CD4+CD45RBhigh-Induced Colitis by a TGF-β-Dependent Mechanism
Authors: Oida Takatoku , Zhang Xingmin , Goto Masao , Hachimura Satoshi , Totsuka Mamoru , Kaminogawa Shuichi , Weiner Howard L.
Published: April 2014

Murine CD4+CD25+ regulatory cells have been reported to express latency-associated peptide (LAP) and TGF-β on the surface after activation, and exert regulatory function by the membrane-bound TGF-β in vitro. We have now found that a small population of CD4+ T cells, both CD25+ and CD25−, can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP+ cells are also positive for thrombospondin, which has the ability to convert latent TGF-β to the active form. In the CD4+CD45RBhigh-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25+LAP+ and CD25+LAP− cells, but also by CD25−LAP+ cells. CD4+CD25−LAP+ T cells were part of the CD45RBlow cell fraction. CD4+CD25−LAP−CD45RBlow cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25−LAP+ cells was abrogated in vivo by anti-TGF-β mAb. These results identify a new TGF-β-dependent regulatory CD4+ T cell phenotype that is CD25− and LAP+.
Role of the Polymeric Ig Receptor in Mucosal B Cell Homeostasis
Authors: Uren Tania K. , Johansen Finn-Eirik , Wijburg Odilia L. C. , Koentgen Frank , Brandtzaeg Per , Strugnell Richard A.
Published: April 2014

Secretory IgA (SIgA) is the most characteristic component of the mucosal immune system and has long been considered the major protective factor that prevents pathogens from invading hosts through the mucosae. Recent studies, however, have suggested that complete immunity against a range of mucosal bacterial and viral pathogens can be achieved in the absence of IgA. Therefore, to further dissect the role of SIgA, we generated mice deficient in the polymeric Ig receptor (pIgR−/− mice). As a result of an inability to transport dimeric IgA to the secretions, pIgR−/− mice are deficient in SIgA and accumulate circulating dimeric IgA, with serum levels 100-fold greater than those observed in normal mice. Examination of lamina propria mononuclear cells showed that pIgR−/− mice had ∼3 times as many IgA-secreting cells as C57BL/6 mice. Further analysis showed that these cells displayed the differentiated IgA+ B220− phenotype and accounted for a 2-fold increase in the number of lamina propria blast cells in the pIgR−/− mice. Subsequent experiments showed that OVA-specific CD4+ T cell expansion following OVA feeding was not elevated in pIgR−/− mice. Furthermore, no differences in CD8+ T cell tolerance or induction of influenza virus-specific CD8+ T cells were detected in pIgR−/− mice compared with controls. Therefore, while SIgA is clearly involved in maintaining some parameters of mucosal homeostasis in the intestine, the mechanisms associated with its barrier function and the clinical consequences of its deficiency are yet to be identified.
Unique Monoclonal Antibodies Define Expression of FcγRI on Macrophages and Mast Cell Lines and Demonstrate Heterogeneity Among Subcutaneous and Other Dendritic Cells
Authors: Tan Peck S. , Gavin Amanda L. , Barnes Nadine , Sears Duane W. , Vremec David , Shortman Ken , Amigorena Sebastian , Mottram Patricia L. , Hogarth P. Mark
Published: April 2014

The mouse FcγRI is one of the most fundamentally important FcRs. It participates in different stages of immunity, being a low affinity receptor for T-independent IgG3 and yet a high affinity receptor for IgG2a, the product of a Th1 immune response. However, analysis of this receptor has been difficult due largely to the failure to generate specific Abs to this FcR. We have made use of the polymorphic differences between BALB/c and NOD/Lt mice to generate mAb specific for the FcγRI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected FcγRI encoded by the more common Fcgr1a and Fcgr1b alleles, and although they identified different epitopes, none inhibited the binding of IgG to FcγRI. When bound to FcγRI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of FcγRI. Resting and IFN-γ-induced macrophages expressed FcγRI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed FcγRI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of FcγRI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8α, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, FcγRI on APC may act as a link between innate and adaptive immunities by binding and mediating the uptake of T-independent immune complexes for presentation, thereby assisting in the development of T-dependent immune responses.
Bone Morphogenetic Protein-7 Inhibits Constitutive and Interleukin-1β-Induced Monocyte Chemoattractant Protein-1 Expression in Human Mesangial Cells: Role for JNK/AP-1 Pathway
Authors: Lee Myung-Ja , Yang Chul Woo , Jin Dong Chan , Chang Yoon Sik , Bang Byung Kee , Kim Yong-Soo
Published: April 2014

Bone morphogenetic protein-7 (BMP-7), which belongs to the TGF-β superfamily, has been shown to reduce macrophage infiltration and tissue injury in animal models of inflammatory renal disease. To explore the mechanism involved in the anti-inflammatory effect, we investigated the effect of BMP-7 on monocyte chemoattractant protein-1 (MCP-1) expression in cultured human mesangial cells. BMP- 7 significantly inhibited constitutive and IL-1β-induced MCP-1 protein production and MCP-1 mRNA expression by mesangial cells in a time- and concentration-dependent manner. BMP-7 also inhibited IL-1β-induced monocyte chemotactic activity released from the mesangial cells. We examined the role of transcription factors NF-κB and AP-1 in BMP-7 inhibition of IL-1β-induced MCP-1 expression. IL-1β increased NF-κB and AP-1 activity and both transcription factors mediated IL-1β-induced MCP-1 expression in mesangial cells. BMP-7 inhibited IL-1β-induced AP-1 activity in a concentration-dependent manner. In contrast, IL-1β-induced NF-κB activity and IκBα degradation were not affected by BMP-7. Furthermore, IL-1β-induced phosphorylation of c-Jun N-terminal kinase was inhibited by BMP-7. These data suggest that BMP-7 inhibits constitutive and IL-1β-induced MCP-1 expression in human mesangial cells partly by inhibiting c-Jun N-terminal kinase activity and subsequent AP-1 activity, and provide new insight into the therapeutic potential of BMP-7 in the inflammatory renal diseases.
CD8-Independent Tumor Cell Recognition Is a Property of the T Cell Receptor and Not the T Cell
Authors: Roszkowski Jeffrey J. , Yu David C. , Rubinstein Mark P. , McKee Mark D. , Cole David J. , Nishimura Michael I.
Published: April 2014

The CD8 coreceptor enhances T cell function by stabilizing the TCR/peptide/MHC complex and/or increasing T cell avidity via interactions with the intracellular kinases Lck and LAT. We previously reported a CD4+ T cell (TIL 1383I), which recognizes the tumor-associated Ag tyrosinase in the context of HLA-A2. To determine whether CD8 independent tumor cell recognition is a property of the TCR, we used retroviral transduction to express the TIL 1383I TCR in the CD8− murine lymphoma, 58 α−/β−. Immunofluorescent staining of TCR-transduced cells with human TCR Vβ subfamily-specific and mouse CD3-specific Abs confirmed surface expression of the transferred TCR and coexpression of mouse CD3. Transduced effector cells secreted significant amounts of IL-2 following Ag presentation by tyrosinase peptide-pulsed T2 cells as well as stimulation with HLA-A2+ melanoma lines compared with T2 cells alone or HLA-A2− melanoma cells. Further analysis of TCR-transduced clones demonstrated a correlation between T cell avidity and cell surface expression of the TCR. Therefore, the TIL 1383I TCR has sufficient affinity to mediate recognition of the physiologic levels of Ag expressed by tumor cells in the absence of CD8 expression.
Activation of TGF-β by Leishmania chagasi: Importance for Parasite Survival in Macrophages
Authors: Gantt Kira R. , Schultz-Cherry Stacey , Rodriguez Nilda , Jeronimo Selma M. B. , Nascimento Eliana T. , Goldman Todd L. , Recker Thomas J. , Miller Melissa A. , Wilson Mary E.
Published: April 2014

TGF-β is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-γ, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-β is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-β levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-β was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-β in human macrophage cultures without changing the total TGF-β. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-β. Incubation of latent TGF-β with Leishmania sp. promastigotes caused active TGF-β to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-β. TGF-β activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-β inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-β had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-β stores at extracellular sites during infection, and the ability of the parasite to activate TGF-β in its local environment, leads to high levels of active TGF-β in the vicinity of the infected macrophage. Locally activated TGF-β could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.
HIV-1 Tat-Mediated Apoptosis in Human Brain Microvascular Endothelial Cells
Authors: Kim Tae-Aug , Avraham Hava Karsenty , Koh Young-Ho , Jiang Shuxian , Park In-Woo , Avraham Shalom
Published: April 2014

The integrity of the blood-brain barrier (BBB) is critical for normal brain function. Neuropathological abnormalities in AIDS patients have been associated with perivascular HIV-infected macrophages, gliosis, and abnormalities in the permeability of the BBB. The processes by which HIV causes these pathological conditions are not well understood. To characterize the mechanism by which HIV-1 Tat protein modulates human brain microvascular endothelial cell (HBMEC) functions, we studied the effects of HIV-1 Tat in modulating HBMEC apoptosis and permeability. Treatment of HBMEC with HIV-1 Tat led to Flk-1/KDR and Flt-4 receptor activation and the release of NO. The protein levels of endothelial NO synthase (NOS) and inducible NOS were increased by HIV-1 Tat stimulation. Importantly, HIV-1 Tat caused apoptosis of HBMEC, as evidenced by changes in the cleavage of poly(A)DP-ribose polymerase, DNA laddering, and incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay). HIV-1 Tat-mediated apoptosis in HBMEC was significantly inhibited in the presence of N-nitro-l-arginine methyl ester (an inhibitor of NOS) and wortmannin (a phosphoinositol 3-kinase inhibitor). Furthermore, HIV-1 Tat treatment significantly increased HBMEC permeability, and pretreatment with both N-nitro-l-arginine methyl ester and wortmannin inhibited the Tat-induced permeability. Taken together, these results indicate that dysregulated production of NO by HIV-1 Tat plays a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB.
Essential Role of Phosphoinositide 3-Kinase δ in Neutrophil Directional Movement
Authors: Sadhu Chanchal , Masinovsky Boris , Dick Ken , Sowell C. Gregory , Staunton Donald E.
Published: April 2014

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3Kδ in neutrophil chemotaxis, we have developed a potent and selective PI3Kδ inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3Kδ activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3Kδ, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3Kδ can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.