bluuebunny/crossref_metadata_2025 huggingface dataset:
Attenuation of Th1 Response in Decoy Receptor 3 Transgenic Mice
Authors: Hsu Tsui-Ling , Wu Ying-Yu , Chang Yung-Chi , Yang Chih-Ya , Lai Ming-Zong , Su Wenlynn B. , Hsieh Shie-Liang
Published: April 2014

The soluble decoy receptor 3 (DcR3) is a member of the TNFR superfamily. Because DcR3 is up-regulated in tumor tissues and is detectable in the sera of cancer patients, it is regarded as an immunosuppressor to down-regulate immune responses. To understand the function of DcR3 in vivo, we generated transgenic mice overexpressing DcR3 systemically. In comparison with HNT-TCR (HNT) transgenic mice, up-regulation of IL-4 and IL-10 and down-regulation of IFN-γ, IL-12, and TNF-α were observed in the influenza hemagglutinin126–138 peptide-stimulated splenocytes of HNT-DcR3 double-transgenic mice. When infected with Listeria monocytogenes, DcR3 transgenic mice show attenuated expression of IFN-γ as well as increased susceptibility to infection. The Th2 cell-biased phenotype in DcR3 transgenic mice is attributed to decreased IL-2 secretion by T cells, resulting in the suppression of IL-2 dependent CD4+ T cell proliferation. This suggests that DcR3 might help tumor growth by attenuating the Th1 response and suppressing cell-mediated immunity.
Functional Characterization of Human Cysteinyl Leukotriene 1 Receptor Gene Structure
Authors: Woszczek Grzegorz , Pawliczak Rafal , Qi Hai-Yan , Nagineni Sahrudaya , Alsaaty Sura , Logun Carolea , Shelhamer James H.
Published: April 2014

The 5-lipoxygenase pathway has been strongly implicated in the pathogenesis of chronic inflammatory disorders, such as bronchial asthma and atherosclerosis. Cysteinyl leukotrienes (cysLTs), 5-lipoxygenase pathway products, are recognized now not only as important factors in asthmatic inflammation, but also as mediators of cell trafficking and innate immune responses. To study a role of cysLTs in inflammatory reactions we have characterized the gene structure of human cysteinyl leukotriene receptor type I (cysLT1R). The cysLT1R gene consists of 5 exons that are variably spliced and a single promoter region with multiple transcription start sites. Four different cysLT1R transcripts were identified. RT-PCR showed dominant and wide expression of the transcript I, containing exons 1, 4, and 5, with the strongest presence in blood leukocytes, spleen, thymus, lung, and heart. The expression of cysLT1R is functionally regulated at the transcriptional level by IL-4 through a STAT6 response element localized to the proximal cysLT1R promoter region. IL-4 stimulation increased cysLT1R mRNA (real-time PCR) and surface protein expression (flow cytometry) in a time-dependent fashion. CysLTs (LTD4 and LTC4) induced an increased production of a potent monocyte chemoattractant CCL2 (MCP-1) in IL-4-primed THP-1 cells in a dose-dependent manner. This effect was effectively inhibited by the cysLT1R-selective antagonist MK571 in a dose-dependent manner and only partially by a nonselective cysLT1R/cysLT2R inhibitor BAY-u9773, implying a cysLT1R-mediated mechanism. Thus, cysLTs signaling through cysLT1R might contribute to inflammatory reactions by cooperating with IL-4 in enhanced CCL2 production in human monocytic cells.
Hypermutation at A-T Base Pairs: The A Nucleotide Replacement Spectrum Is Affected by Adjacent Nucleotides and There Is No Reverse Complementarity of Sequences Flanking Mutated A and T Nucleotides
Authors: Spencer Jo , Dunn-Walters Deborah K.
Published: April 2014

Hypermutation is thought to be a two-phase process. The first phase is via the action of activation-induced cytidine deaminase (AID), which deaminates C nucleotides in WRC motifs. This results in the RGYW/WRCY hot spot motifs for mutation from G and C observed in vivo. The resemblance between the hot spot for C mutations and the reverse complement of that for G mutations implies a process acting equally on both strands of DNA. The second phase of hypermutation generates mutations from A and T and exhibits strand bias, with more mutations from A than T. Although this does not concur with the idea of one mechanism acting equally on both strands, it has been suggested that the AT mutator also has a reversible motif; WA/TW. We show here that the motifs surrounding the different substitutions from A vary significantly; there is no single targeting motif for all A mutations. Sequence preferences associated with mutations from A more likely reflect an influence of adjacent nucleotides over what the A mutates “to.” This influence tends toward “like” replacements: Purines (A or G) in the 5′ position bias toward replacement by another purine (G), whereas replacement with pyrimidines (C or T) is more likely if the preceding base is also a pyrimidine. There is no reverse complementarity in these observations, in that similar influences of nucleotides adjacent to T are not seen. Hence, WA and TW should not be considered as reverse complement hot spot motifs for A and T mutations.
Sequence Motifs in IL-4Rα Mediating Cell-Cycle Progression of Primary Lymphocytes
Authors: Stephenson Linda M. , Park Do-Sim , Mora Ana L. , Goenka Shreevrat , Boothby Mark
Published: April 2014

IL-4 signaling through the IL-4Rα chain regulates the development and proliferation of the Th2 lineage of effector CD4+ T cells. Analyses of the IL-4R in factor-dependent cell lines led to the development of two apparently conflicting models of the primary structural determinants of IL-4R-mediated proliferative signaling. In one model, proliferation was dependent on the first conserved tyrosine in the cytoplasmic tail (Y1), while in the second, proliferation was independent of cytoplasmic tyrosines. We found that in activated primary T cells, mutation of only the Y1 residue resulted in a modest decrease in IL-4-induced S phase entry, a further decrease in cell-cycle completion, and a complete failure of IL-4 to induce p70S6 kinase phosphorylation. Consistent with a role for the PI3K/mammalian target of rapamycin pathway in mediating cytokine acceleration of G2/M transit, pretreatment of activated T cells with rapamycin resulted in only a modest decrease in IL-4-induced S phase entry, but a total block of cell-cycle completion. Strikingly, IL-4Rα chains that lacked all cytoplasmic tyrosines were competent to signal for STAT5 phosphorylation, mediated efficient S phase entry, and promoted cell-cycle progression. The ability of tyrosine-deficient IL-4Rs to mediate proliferative signaling and STAT phosphorylation was absolutely dependent on the presence of an intact ID-1 region. These findings show that IL-4Rα lacking cytoplasmic tyrosine residues is competent to induce ID-1-dependent proliferation, and indicate that IL-4 can promote G2/M progression via activation of the mammalian target of rapamycin pathway initiated at the Y1 residue.
Allele-Specific Regulation of TCRβ Variable Gene Segment Chromatin Structure
Authors: Jackson Annette M. , Krangel Michael S.
Published: April 2014

Allelic exclusion of the murine Tcrb locus is imposed at the level of recombination and restricts each cell to produce one functional VDJβ rearrangement. Allelic exclusion is achieved through asynchronous Vβ to DJβ recombination as well as feedback inhibition that terminates recombination once a functional rearrangement has occurred. Because the accessibility of Vβ gene segment chromatin is diminished as thymocytes undergo allelic exclusion at the CD4−CD8− (double-negative) to CD4+CD8+ (double-positive) transition, chromatin regulation was thought to be an important component of the feedback inhibition process. However, previous studies of chromatin regulation addressed the status of Tcrb alleles using genetic models in which both alleles remained in a germline configuration. Under physiological conditions, developing thymocytes would undergo Vβ to DJβ recombination on one or both alleles before the enforcement of feedback. On rearranged alleles, Vβ gene segments that in germline configuration are regulated independently of the Tcrb enhancer are now brought into its proximity. We show in this study that in contrast to Vβ segments on a nonrearranged allele, those situated upstream of a functionally rearranged Vβ segment are contained in active chromatin as judged by histone H3 acetylation, histone H3 lysine 4 (K4) methylation, and germline transcription. Nevertheless, these Vβ gene segments remain refractory to recombination in double-positive thymocytes. These results suggest that a unique feedback mechanism may operate independent of chromatin structure to inhibit Vβ to DJβ recombination after the double-negative stage of thymocyte development.
Splenic Marginal Zone Dendritic Cells Mediate the Cholera Toxin Adjuvant Effect: Dependence on the ADP-Ribosyltransferase Activity of the Holotoxin
Authors: Grdic Dubravka , Ekman Lena , Schön Karin , Lindgren Kristina , Mattsson Johan , Magnusson Karl-Eric , Ricciardi-Castagnoli Paola , Lycke Nils
Published: April 2014

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent and mediated by the B subunit of CT (CTB). The targeted MZ DC were quite unique in their phenotype: CD11c+, CD8α−, CD11b−, B220−, and expressing intermediate or low levels of MHC class II and DEC205. Whereas CTB only delivered the Ag to MZ DC, the ADP-ribosyltransferase activity of CT was required for the maturation and migration of DC to the T cell zone, where these cells distinctly up-regulated CD86, but not CD80. This interaction appeared to instruct Ag-specific CD4+ T cells to move into the B cell follicle and strongly support germinal center formations. These events may explain why CT-conjugated Ag is substantially more immunogenic than Ag admixed with soluble CT and why CTB-conjugated Ag can tolerize immune responses when given orally or at other mucosal sites.
Human High Mobility Group Box Transcription Factor 1 Affects Thymocyte Development and Transgene Variegation
Authors: Sekkali Belaïd , Szabat Ewa , Ktistaki Eleni , Tolaini Mauro , Roderick Kathleen , Harker Nicky , Patel Amisha , Williams Keith , Norton Trisha , Kioussis Dimitris
Published: April 2014

It has been shown previously that a human CD2 (hCD2) disabled locus control region (LCR) transgene is unable to establish an open chromatin configuration in all the T cells, and this leads to position effect variegation of the transgene. In this study we show that thymus-specific overexpression of human high mobility group box transcription factor 1 (HBP1), a transcription factor that binds a specific sequence within the hCD2 LCR, affects thymus cellularity as well as the number of CD8+ thymocytes in two independent transgenic mouse lines and increases the proportion of T cells that fully activate the transgenic locus in hCD2 variegating mice in a sequence-specific dependent manner. This finding suggests that overexpression of HBP1 can affect lineage commitment and can relieve the suppressive influence of heterochromatin, allowing thymocytes to express the variegating target locus more efficiently. These effects could be the result of direct HBP1 action on LCR activity. Alternatively, the extra HBP1 molecules may sequester repressive elements away from the LCR, thus allowing transcription permissive states to form on the transgene locus.
Unusually High Frequency MHC Class I Alleles in Mauritian Origin Cynomolgus Macaques
Authors: Krebs Kendall C. , Jin ZheYuan , Rudersdorf Richard , Hughes Austin L. , O’Connor David H.
Published: April 2014

Acute shortages of Indian origin Rhesus macaques significantly hinder HIV/AIDS research. Cellular immune responses are particularly difficult to study because only a subset of animals possess MHC class I (MHC I) alleles with defined peptide-binding specificities. To expand the pool of nonhuman primates suitable for studies of cellular immunity, we defined 66 MHC I alleles in Cynomolgus macaques (Macaca fascicularis) of Chinese, Vietnamese, and Mauritian origin. Most MHC I alleles were found only in animals from a single geographic origin, suggesting that Cynomolgus macaques from different origins are not interchangeable in studies of cellular immunity. Animals from Mauritius may be particularly valuable because >50% of these Cynomolgus macaques share the MHC class I allele combination Mafa-B*430101, Mafa-B*440101, and Mafa-B*460101. The increased MHC I allele sharing of Mauritian origin Cynomolgus macaques may dramatically reduce the overall number of animals needed to study cellular immune responses in nonhuman primates while simultaneously reducing the confounding effects of genetic heterogeneity in HIV/AIDS research.
The Murine Family of Gut-Restricted Class Ib MHC Includes Alternatively Spliced Isoforms of the Proposed HLA-G Homolog, “Blastocyst MHC”
Authors: Guidry Paula A. , Stroynowski Iwona
Published: April 2014

The gastrointestinal tract is populated by a multitude of specialized immune cells endowed with receptors for classical (class Ia) and nonclassical (class Ib) MHC proteins. To identify class I products that engage these receptors and impact immunity/tolerance, we studied gut-transcribed class Ib loci and their polymorphism in inbred, outbred, and wild-derived mice. Intestinal tissues enriched in epithelial cells contained abundant transcripts of ubiquitously expressed and preferentially gut-restricted Q and T class I loci. The latter category included the “blastocyst Mhc” gene, H2-Bl, and its putative paralog, Tw5. Expression of H2-Bl was previously detected only at the maternal/fetal interface, where it was proposed to induce immune tolerance via interactions with CD94/NKG2A receptors. Analysis of coding region polymorphism performed here revealed two major alleles of H2-Bl with conserved residues at positions critical for class I protein folding and peptide binding. Both divergent alleles are maintained in outbred and wild mice under selection for fecundity and pathogen resistance. Surprisingly, we found that alternative splicing of H2-Bl mRNA in gut tissues is prevalent and allele-specific. It leads to strain-dependent expression of diverse repertoires of canonical and noncanonical transcripts that may give rise to distinct ligands for intestinal NK cell, T cell, and/or intraepithelial lymphocyte receptors.
Increase in Phagocytosis after Geldanamycin Treatment or Heat Shock: Role of Heat Shock Proteins
Authors: Vega Virginia L. , De Maio Antonio
Published: April 2014

The response to injury is activated at the systemic and cellular levels. At the systemic level, phagocytosis plays a key role in controlling infections and clearing necrotic and apoptotic cells. The expression of heat shock proteins (Hsp), which is a well-conserved process, is a major component of cellular response to stress. This study investigated the relationship between Hsps and phagocytosis. An increase in the phagocytosis of opsonized bacteria particles and latex beads was observed upon incubation of murine macrophages with geldanamycin (GA), a specific inhibitor of the Hsp90 family of proteins. The effect of GA on phagocytosis was blocked by coincubation with inhibitors of transcription (actinomycin D) or translation (cycloheximide), suggesting that gene expression was required. Because expression of Hsps has been observed after GA treatment, the effect of heat shock on phagocytosis was investigated. Similar to GA treatment, heat shock resulted in an actinomycin D-sensitive elevation of phagocytosis, which suggests that Hsps are involved. The increase in phagocytosis after GA treatment was not due to increased binding of opsonized particles to their respective receptors on the macrophage surface or to elevated oxidative stress. However, it was correlated with a rapid polymerization of actin in proximity to the plasma membrane. These results suggest that Hsps play a role in the modulation of the phagocytic process, which is part of the stress response.
IL-12 Is Required for Induction but Not Maintenance of Protective, Memory Responses to Blastomyces dermatitidis: Implications for Vaccine Development in Immune-Deficient Hosts
Authors: Wüthrich Marcel , Warner Tom , Klein Bruce S.
Published: April 2014

Cellular immunity mediated by T lymphocytes, in particular CD4+ and CD8+ type 1 (T1) cells, is the main defense against pathogenic fungi. IL-12 initiates T1 cell development and cell-mediated immunity, but it is unclear whether IL-12 contributes to the maintenance of an antifungal T1 response. In this study, we addressed the role of IL-12 for vaccine-induced memory T cell development against experimental pulmonary blastomycosis. CD4+ T cells absolutely required IL-12 to control a live genetically engineered attenuated strain of Blastomyces dermatitidis given s.c. as a vaccine, whereas CD8+ T cells were significantly less dependent on IL-12. Despite differential dependency of T cell subsets on IL-12 during vaccination, neither subset acquired memory immunity in the absence of IL-12. In contrast, adoptive transfer of immune CD4 T cells from wild-type mice into IL-12−/− mice showed that CD4+ T1 memory cells sustained a T1 cytokine profile and remained protective over a period of 6 mo posttransfer. Similarly, memory CD8 cells elicited in IL-12−/− mice with killed yeast and transient rIL-12 treatment (during vaccination) remained durable and protective after animals were rested for 3 mo. In conclusion, these studies demonstrate that once CD4 and CD8 cells have acquired a protective T1 phenotype they no longer require the presence of IL-12 to maintain antifungal protective memory.
Cloning of the Gene Encoding a Protective Mycobacterium tuberculosis Secreted Protein Detected In Vivo during the Initial Phases of the Infectious Process
Authors: Mukherjee Sandeep , Kashino Suely S. , Zhang Yanni , Daifalla Nada , Rodrigues Virmondes , Reed Steven G. , Campos-Neto Antonio
Published: April 2014

The existence of therapeutic agents and the bacille Calmette-Guérin (BCG) vaccine have not significantly affected the current tuberculosis pandemic. BCG vaccine protects against serious pediatric forms of tuberculosis but not against adult pulmonary tuberculosis, the most common and contagious form of the disease. Several vaccine candidates, including Mycobacterium tuberculosis recombinant proteins formulated in newer adjuvants or delivered in bacterial plasmid DNA have recently been described. An attractive source of vaccine candidates has been M. tuberculosis Ags present in culture supernatants of the initial phases of the bacterial growth in vitro. In this study we describe an Ag discovery approach to select for such Ags produced in vivo during the initial phases of the infection. We combined RP-HPLC and mass spectrometry to identify secreted or shed M. tuberculosis proteins eliminated in animal urine within 14 days after the infection. A peptide containing sequence homology with a hypothetical M. tuberculosis protein was identified and the recombinant protein produced in Escherichia coli. The protein was recognized by Ab (IgG2a and IgG1) and T cells (Th1) of mice infected with M. tuberculosis and by lymphoid cells from healthy donors who had a positive purified protein derivative skin test but not from tuberculosis patients. Moreover, this Ag induced protection in mice against M. tuberculosis at levels comparable to protection induced by BCG vaccine. These results validate the Ag discovery approach of M. tuberculosis proteins secreted or shed in vivo during the early phases of the infection and open new possibilities for the development of potential vaccine candidates or of markers of active mycobacterial multiplication and therefore active disease.
Mycobacterium bovis BCG Attenuates Surface Expression of Mature Class II Molecules through IL-10-Dependent Inhibition of Cathepsin S
Authors: Sendide Khalid , Deghmane Ala-Eddine , Pechkovsky Dmitri , Av-Gay Yossef , Talal Amina , Hmama Zakaria
Published: April 2014

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-γ. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-γ-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of αβ class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4+ T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.
Characterization of a Novel Chemokine-Containing Storage Granule in Endothelial Cells: Evidence for Preferential Exocytosis Mediated by Protein Kinase A and Diacylglycerol
Authors: Øynebråten Inger , Barois Nicolas , Hagelsteen Kathrine , Johansen Finn-Eirik , Bakke Oddmund , Haraldsen Guttorm
Published: April 2014

We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs). Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites. Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GROα/CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present study, we report that GROα and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator, i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GROα/MCP-1-containing granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered distinct dose and time-dependent responses of GROα release. Furthermore, GROα release was more sensitive than IL-8 release to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor, indicating that GROα release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the basis of these findings, we designated the GROα/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs, considering the WPB the type 1 compartment. In conclusion, we propose that the GROα/MCP-1-containing type 2 granule shows preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow differential release of selected chemokines.
TGF-β and Smad3 Signaling Link Inflammation to Chronic Fibrogenesis
Authors: Bonniaud Philippe , Margetts Peter J. , Ask Kjetil , Flanders Kathy , Gauldie Jack , Kolb Martin
Published: April 2014

Transient adenovirus-mediated gene transfer of IL-1β (AdIL-1β), a proinflammatory cytokine, induces marked inflammation and severe and progressive fibrosis in rat lungs. This is associated with an increase in TGF-β1 concentration in bronchoalveolar lavage (BAL) fluid. TGF-β1 is a key cytokine in the process of fibrogenesis, using intracellular signaling pathways involving Smad2 and Smad3. In this study we investigate whether inflammation induced by IL-1β is able to independently induce lung fibrosis in mice deficient in the Smad3 gene. Seven days after AdIL-1β administration, similar levels of IL-1β transgene are seen in BAL in both wild-type (WT) and knockout (KO) mice, and BAL cell profiles demonstrated a similar marked neutrophilic inflammation. Phospho-Smad2 staining was positive in areas of inflammation in both WT and KO mice at day 7. By day 35 after transient IL-1β expression, WT mice showed marked fibrosis in peribronchial areas, quantified by picrosirius red staining and morphometry. However, there was no evidence of fibrosis or collagen accumulation in IL-1β-treated KO mice, and peribronchial areas were not different from KO mice treated with the control adenovector. TGF-β1 and phospho-Smad2 were strongly positive at day 35 in fibrotic areas observed in WT mice, but no such staining was detectable in KO mice. The IL-1β-induced chronic fibrotic response in mouse lungs is dependent on Smad3. KO and WT animals demonstrated a similar inflammatory response to overexpression of IL-1β indicating that inflammation must link to the Smad3 pathway, likely through TGF-β, to induce progressive fibrosis.
Altered CXCR2 Signaling in β-Arrestin-2-Deficient Mouse Models
Authors: Su Yingjun , Raghuwanshi Sandeep K. , Yu Yingchun , Nanney Lillian B. , Richardson Ricardo M. , Richmond Ann
Published: April 2014

CXCR2 is a G-protein-coupled receptor (GPCR) that binds the CXC chemokines, CXCL1–3 and CXCL5–8, and induces intracellular signals associated with chemotaxis. Many adaptor proteins are actively involved in the sequestration, internalization, and trafficking of CXCR2 and transduction of agonist-induced intracellular signaling. We have previously shown that adaptor protein β-arrestin-2 (βarr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of βarr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of βarr2-deficient (βarr2−/−) and their wild-type (βarr2+/+) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca2+ mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of βarr2 resulted in increased Ca2+ mobilization, superoxide anion production, and GTPase activity in neutrophils, but decreased receptor internalization relative to wild-type mice. Two animal models, the dorsal air pouch model and the excisional wound healing model, were used to further study the in vivo effects of βarr2 on CXCR2-mediated neutrophil chemotaxis and on cutaneous wound healing. Surprisingly, the recruitment of neutrophils was increased in response to CXCL1 in the air pouch model and in the excisional wound beds of βarr2−/− mice. Wound re-epithelialization was also significantly faster in βarr2−/− mice than in βarr2+/+ mice. Taken together, the data indicate that βarr2 is a negative regulator for CXCR2 in vivo signaling.
The Chemokines CXCL9, CXCL10, and CXCL11 Differentially Stimulate Gαi-Independent Signaling and Actin Responses in Human Intestinal Myofibroblasts
Authors: Kouroumalis Andreas , Nibbs Robert J. , Aptel Herve , Wright Karen L. , Kolios George , Ward Stephen G.
Published: April 2014

Intestinal myofibroblasts have been implicated in the pathogenesis of chronic inflammatory conditions such as Crohn’s disease via interactions with an elaborate network of cytokines, growth factors, and other inflammatory mediators. CXCR3 is a Gαi protein-coupled receptor that binds the proinflammatory chemokines CXCL9, CXCL10, and CXCL11, which are released from the intestinal epithelium. The three CXCR3 ligands shared the ability to activate biochemical (e.g., PI3K and MAPK activation) and functional events (actin reorganization) in intestinal myofibroblasts. However, CXCL11 is unique in its ability to elevate intracellular calcium. Surprisingly, although CXCR3 mRNA is detectable in these myofibroblasts, there is no detectable surface expression of CXCR3. Furthermore, the biochemical responses and actin reorganization stimulated by the CXCR3 ligands in intestinal myofibroblasts are insensitive to the Gαi inhibitor, pertussis toxin. This suggests either the existence of differential receptor coupling mechanisms in myofibroblasts for CXCR3 that are distinct from those observed in PBLs and/or that these cells express a modified or variant CXCR3 compared with the CXCR3 expressed on PBLs.
Carboxylated Glycans Mediate Colitis through Activation of NF-κB
Authors: Srikrishna Geetha , Turovskaya Olga , Shaikh Raziya , Newlin Robbin , Foell Dirk , Murch Simon , Kronenberg Mitchell , Freeze Hudson H.
Published: April 2014

The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic lamina propria, and in inflammatory infiltrates in colon tissues from Crohn’s disease patients. We assessed the contribution of these glycans to the development of colitis induced by CD4+CD45RBhigh T cell transfer to Rag1−/− mice. Administration of an anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab treatment reduced accumulation of CD4+ T cells in colon. This was accompanied by reduction in inflammatory cells, reduced expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked activation of NF-κB p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-glycan-dependent pathways contribute to the early onset of colitis.
Oxidative Stress Augments the Production of Matrix Metalloproteinase-1, Cyclooxygenase-2, and Prostaglandin E2 through Enhancement of NF-κB Activity in Lipopolysaccharide-Activated Human Primary Monocytes
Authors: Lu Yunbiao , Wahl Larry M.
Published: April 2014

The excessive production of reactive oxidative species (ROS) associated with inflammation leads to a condition of oxidative stress. Cyclooxygenase-2 (COX-2), PGE2, and matrix metalloproteinases (MMPs) are important mediators during the process of inflammation. In this paper we report on studies examining how the ROS hydrogen peroxide (H2O2) affects the production of MMP-1, COX-2, and PGE2. Addition of H2O2 to LPS-activated monocytes, but not naive monocytes, caused a significant enhancement of the LPS-induced production of MMP-1, COX-2, and PGE2. The mechanism by which H2O2 increased these mediators was through enhancement of IκBα degradation, with subsequent increases in NF-κB activation and NF-κB p50 translocation to the nucleus. The effects of H2O2 on IκBα degradation, NF-κB activation, and NF-κB p50 localization to the nucleus were demonstrated through studies of coimmunoprecipitation of IκBα with p50, ELISA of NF-κB p65 activity, and Western blot analysis of the nuclear fraction extract for p50. The key role for NF-κB in this process was demonstrated by the ability of MG-132 or lactacystin (proteasome inhibitors) to block the enhanced production of MMP-1, COX-2, and PGE2. In contrast, indomethacin, which inhibited PGE2 production, partially blocked the enhanced MMP-1 production. Moreover, although PGE2 restored MMP-1 production in indomethacin-treated monocyte cultures; it failed to significantly restore MMP-1 production in proteasome inhibitor-treated cultures. Thus, in the presence of LPS and H2O2, NF-κB plays a dominate role in the regulation of MMP-1, COX-2, and PGE2 expression.
Nonpathogenic, Environmental Fungi Induce Activation and Degranulation of Human Eosinophils
Authors: Inoue Yoshinari , Matsuwaki Yoshinori , Shin Seung-Heon , Ponikau Jens U. , Kita Hirohito
Published: April 2014

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an Mr of ∼60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.
Vγ9Vδ2 T Cell Response to Colon Carcinoma Cells
Authors: Corvaisier Murielle , Moreau-Aubry Agnès , Diez Elisabeth , Bennouna Jaafar , Mosnier Jean-Francois , Scotet Emmanuel , Bonneville Marc , Jotereau Francine
Published: April 2014

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vγ9Vδ2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-α and IFN-γ secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vγ9Vδ2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vγ9Vδ2 T cells of various origins, and Vγ9Vδ2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vγ9Vδ2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vγ9Vδ2 T cell agonists in immunotherapies targeting colon tumors.
Defined Blocks in Terminal Plasma Cell Differentiation of Common Variable Immunodeficiency Patients
Authors: Taubenheim Nadine , von Hornung Marcus , Durandy Anne , Warnatz Klaus , Corcoran Lynn , Peter Hans-Hartmut , Eibel Hermann
Published: April 2014

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by defective Ab production and recurrent bacterial infections. The largely unknown causes are likely to comprise a diverse set of genetic or acquired defects. In this study, we investigated terminal B cell differentiation in lymph nodes from CVID patients. Up to the germinal center B cell stage, B cell differentiation was normal but terminal plasma cell development was found to be impaired. Using differential Blimp-1 and Syndecan-1 expression in controls, we defined three different plasma cell subsets that correspond to progressive developmental stages locating to different sites in the lymph node. In the CVID patients, we could only detect one or two of these subsets indicating a defective differentiation. Thus, terminal plasma cell differentiation was found to be impaired despite normal expression of Blimp-1. B cells reaching only the first stage of plasma cell differentiation were further unable to undergo isotype switching and to up-regulate activation markers on B cells stimulated in vitro.
Requirement for Neutralizing Antibodies to Control Bone Marrow Transplantation-Associated Persistent Viral Infection and to Reduce Immunopathology
Authors: Lang Karl S. , Recher Mike , Navarini Alexander A. , Freigang Stefan , Harris Nicola L. , van den Broek Maries , Odermatt Bernhard , Hengartner Hans , Zinkernagel Rolf M.
Published: April 2014

Bone marrow transplantation (BMT) is commonly used in the treatment of leukemia, however its therapeutic application is partly limited by the high incidence of associated opportunistic infections. We modeled this clinical situation by infecting mice that underwent BMT with lymphocytic choriomeningitis virus (LCMV) and investigated the potential of immunotherapeutic strategies to counter such infections. All mice that received BMT survived LCMV infection and developed a virus carrier status. Immunotherapy by adoptive transfer of naive splenocytes protected against low (200 PFU), but not high (2 × 106 PFU), doses of LCMV. Attempts to control infection of high viral titers using strongly elevated frequencies of activated LCMV-specific T cells failed to control virus and resulted in immunopathology and death. In contrast, virus neutralizing Abs combined with naive splenocytes were able to efficiently control high-dose LCMV infection without associated side effects. Thus, cell transfer combined with neutralizing Abs represented the most effective means of controlling BMT-associated opportunistic viral infection in our in vivo model. These data underscore the in vivo efficacy and immunopathological “safety” of neutralizing antibodies.
Cutting Edge: TRAIL Deficiency Accelerates Hematological Malignancies
Authors: Zerafa Nadeen , Westwood Jennifer A. , Cretney Erika , Mitchell Sally , Waring Paul , Iezzi Manuela , Smyth Mark J.
Published: April 2014

TNF apoptosis-inducing ligand is attracting considerable interest as a potential extrinsic tumor suppressor mechanism, although previous reports have conveyed somewhat contrasting views regarding the likely importance of this pathway. In this study, we provide the first evaluation of spontaneous tumor formation over the life span of TRAIL-deficient mice. Interestingly, >25% of these mice do develop lymphoid malignancies after 500 days of life. TRAIL suppressed the initiation and development of both tumors of lymphoid and stromal origin in the context of the loss of at least one p53 allele. Specific examination of the role of TRAIL in Her2/neu oncogene-driven mammary epithelial cancer revealed no critical role for TRAIL despite the inherent TRAIL sensitivity of such mammary carcinomas. Overall, the data indicate an important function of TRAIL in controlling carcinogenesis, but suggest that further examination of this pathway in epithelial malignancies is warranted.
Cutting Edge: CD4+CD25+ Regulatory T Cells Contribute to Gender Differences in Susceptibility to Experimental Autoimmune Encephalomyelitis
Authors: Reddy Jay , Waldner Hanspeter , Zhang Xingmin , Illes Zsolt , Wucherpfennig Kai W. , Sobel Raymond A. , Kuchroo Vijay K.
Published: April 2014

Female B10.S mice are highly resistant to proteolipid protein (PLP) 139–151-induced experimental autoimmune encephalomyelitis (EAE) and depletion of PLP 139–151-reactive CD4+CD25+ regulatory T (Treg) cells can slightly increase their EAE susceptibility. Although male B10.S mice are moderately susceptible to EAE, we report that depletion of Treg cells in male B10.S mice before immunization with PLP 139–151 renders them highly susceptible to severe EAE with more CNS neutrophil infiltrates than nondepleted controls. Increased susceptibility is associated with an enhanced PLP 139–151-specific T cell response and greater production of IFN-γ, IL-6, and IL-17. Male CD4+CD25− effector cells depleted of Treg cells proliferate to a greater degree than those from females in response to either anti-CD3 or PLP 139–151. These data suggest that because of their capacity to regulate potent autoaggressive effector cells, Treg cells partly contribute to the resistance to autoimmunity in the male mice.
Cutting Edge: A Critical Role for Gene Silencing in Preventing Excessive Type 1 Immunity
Authors: Hutchins Anne S. , Artis David , Hendrich Brian D. , Bird Adrian P. , Scott Phillip , Reiner Steven L.
Published: April 2014

Immunity often depends on proper cell fate choice by helper T lymphocytes. A naive cell, with minimal expression of IFN-γ and IL-4, must give rise to progeny expressing high levels of either one, but not both, of those cytokines to defend against protozoan and helminthic pathogens, respectively. In the present study, we show that inactivation of the Mbd2 gene, which links DNA methylation and repressed chromatin, results in enhanced resistance to the protozoan parasite Leishmania major but impaired immunity to the intestinal helminth Trichuris muris. Helper T cells from methyl CpG-binding domain protein-2-deficient mice exhibit exuberant patterns of cytokine expression despite appropriate silencing of genes encoding the lineage-specifying factors T-bet and GATA-3. These results suggest that gene silencing can facilitate the ability of a progenitor cell to give rise to appropriately differentiated daughter cells in vivo. These findings also point to novel pathways that could participate in genetic control of resistance to infection and autoimmunity.
Cutting Edge: Macrophage Migration Inhibitory Factor Is Necessary for Progression of Experimental Autoimmune Encephalomyelitis
Authors: Powell Nicole D. , Papenfuss Tracey L. , McClain Melanie A. , Gienapp Ingrid E. , Shawler Todd M. , Satoskar Abhay R. , Whitacre Caroline C.
Published: April 2014

Macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of inflammatory and autoimmune diseases. The role of MIF in the progression of experimental autoimmune encephalomyelitis (EAE) was explored using MIF−/− mice. Wild-type mice showed a progressive disease course, whereas MIF−/− mice exhibited acute signs but no further progression of clinical disease. MIF−/− mice displayed markedly elevated corticosterone levels and significant decreases in the inflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-6 before, during, and after EAE onset. Taken together, these findings support that MIF is an important mediator of EAE progression through glucocorticoid antagonism and up-regulation of the inflammatory response.
Cutting Edge: Double-Stranded DNA Breaks in the IgV Region Gene Were Detected at Lower Frequency in Affinity-Maturation Impeded GANP−/− Mice
Authors: Kawatani Yousuke , Igarashi Hideya , Matsui Takeshi , Kuwahara Kazuhiko , Fujimura Satoru , Okamoto Nobukazu , Takagi Katsumasa , Sakaguchi Nobuo
Published: April 2014

Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken γ-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP−/− B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.
Cutting Edge: Memory CD8 T Cell Maturation Occurs Independently of CD8αα
Authors: Chandele Anmol , Kaech Susan M.
Published: April 2014

As memory CD8 T cells form during acute viral infection, several changes in gene expression and function occur, but little is known about the control of this process. It was reported previously that the homodimer CD8αα was involved in generating IL-7Rαhigh memory CD8 T cell precursors, and consequently, protective memory CD8 T cells did not form in animals significantly impaired in CD8αα expression (E8I−/− mice). However, the precise contribution of CD8αα to sustained IL-7Rα expression and other memory CD8 T cell-associated changes has not been investigated. We found that IL-7Rα expression and generation of memory CD8 T cells that protect against secondary viral infection was considerably normal in E8I−/− animals. Interestingly, virus-specific CD4 T cell responses were elevated, and the relative surface levels of CD8αβ in activated T cells were reduced in E8I−/− mice compared with wild-type animals. Our results indicate that memory CD8 T cell development can occur independently of CD8αα.
CD4-Dependent Signaling Is Required for a Late Checkpoint during Th2 Development Associated with Resistance to Activation-Induced Cell Death
Authors: Tatari-Calderone Zohreh , Brogdon Jennifer L. , Tinsley Kevin W. , Ramezani Anahita , Leitenberg David
Published: April 2014

Previous studies have found that class II-restricted T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene have a specific defect in the development of Th2 effector cells; however, the reason for this defect was not clear. Following stimulation with a high potency peptide and exogenous IL-4, CD4-dependent signaling is required for optimal generation of a Th2 effector population. However, initial IL-4 and GATA-3 transcription is appropriately induced, suggesting that the initial stages of Th2 development are intact and independent of CD4 after priming with a strong agonist peptide. In addition to the defect in Th2 development, CD4 mutant T cells are also relatively resistant to activation-induced cell death (AICD). Furthermore, inhibition of AICD in wild-type T cells causes a defect in Th2 development similar to that seen in the CD4 mutant T cells. These data support the hypothesis that CD4-dependent signaling pathways regulate a distinct checkpoint in the expansion and commitment phase of Th2 development, which is related to dysregulation of AICD.