bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Hepatic Fibrosis and Extracellular Matrix Genes by the Th Response: New Insight into the Role of Tissue Inhibitors of Matrix Metalloproteinases
Authors: Vaillant Brian , Chiaramonte Monica G. , Cheever Allen W. , Soloway Paul D. , Wynn Thomas A.
Published: April 2014

Hepatic fibrosis is the hallmark of Schistosoma mansoni infection and often results in portal hypertension and bleeding from esophageal varices. The fibrotic process is highly dependent on type 2 cytokines, yet their role in the regulation of extracellular matrix remodeling genes remains largely unknown. Here, we examined the expression of matrix metalloproteases (MMP) -2, -3, -9, -12, and -13 and their inhibitors, tissue inhibitor of metalloproteases (TIMP) -1, -2, and -3, in the livers of infected mice and correlated their expression profiles with fibrosis and type 2 cytokine production. Expression of MMP-2, -3, -9, -12, and -13 and of TIMP-1 and -2 mRNA rapidly increased at the onset of egg laying in infected mice, while TIMP-3 was unchanged. Because TIMP are presumed to be important regulators of the extracellular matrix, and their expression correlated with the development of fibrosis, we studied their role in fibrogenesis by infecting TIMP-1- and TIMP-2-deficient mice. Strikingly, our data revealed no role for TIMP-1 or -2 in the fibrotic pathology induced by S. mansoni eggs. Because of these findings, we infected IL-10/IFN-γ-deficient mice that develop an exaggerated fibrotic response to determine whether changes in type 2 cytokine dominance influence the pattern of MMP and TIMP expression. Fibrosis and type 2 cytokine production correlated with increased MMP-2/MMP-9 vs TIMP-1/TIMP-2 expression. These data, in addition to our knockout studies, demonstrate that TIMP-1/TIMP-2 play no essential role in fibrogenesis in schistosomiasis. Indeed, our findings suggest that inhibiting profibrotic cytokines or specific MMP may be a more effective strategy to ameliorate fibrotic pathology.
Novel Engagement of CD14 and Multiple Toll-Like Receptors by Group B Streptococci
Authors: Henneke Philipp , Takeuchi Osamu , van Strijp Jos A. , Guttormsen Hilde-Kari , Smith Jason A. , Schromm Andra B. , Espevik Terje A. , Akira Shizuo , Nizet Victor , Kasper Dennis L. , Golenbock Douglas T.
Published: April 2014

Group B streptococcus (GBS) imposes a major health threat to newborn infants. Little is known about the molecular basis of GBS-induced sepsis. Both heat-inactivated whole GBS bacteria and a heat-labile soluble factor released by GBS during growth (GBS-F) induce nuclear translocation of NF-κB, the secretion of TNF-α, and the formation of NO in mouse macrophages. Macrophages from mice with a targeted disruption of MyD88 failed to secrete TNF-α in response to both heat-inactivated whole bacteria and GBS-F, suggesting that Toll-like receptors (TLRs) are involved in different aspects of GBS recognition. Immune cell activation by whole bacteria differed profoundly from that by secreted GBS-F. Whole GBS activated macrophages independently of TLR2 and TLR6, whereas a response to the secreted GBS-F was not observed in macrophages from TLR2-deficient animals. In addition to TLR2, TLR6 and CD14 expression were essential for GBS-F responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved. Heat lability distinguished GBS-F from peptidoglycan and lipoproteins. GBS mutants deficient in capsular polysaccharide or β-hemolysin had GBS-F activity comparable to that of wild-type streptococci. We suggest that CD14 and TLR2 and TLR6 function as coreceptors for secreted microbial products derived from GBS and that cell wall components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or 6.
IFN-γ-Inducible Protein-10 (CXCL10) Is Hepatoprotective During Acute Liver Injury Through the Induction of CXCR2 on Hepatocytes
Authors: Bone-Larson Cynthia L. , Hogaboam Cory M. , Evanhoff Holly , Strieter Robert M. , Kunkel Steven L.
Published: April 2014

IFN-γ-inducible protein-10 (IP-10/CXCL10) is a non-ELR-CXC chemokine that is present during various forms of acute and chronic liver injury. The purpose of this study was to explore the role of IP-10 during acute liver injury induced by acetaminophen (APAP). After a 400 mg/kg APAP challenge in fasted CD-1 mice, immunoreactive levels of IP-10 were dramatically elevated in the serum within 8 h. CXCR3, the receptor for IP-10, was up-regulated in the liver. Mice that received an i.v. injection of rIP-10 10 h after APAP challenge exhibited a dramatic reduction in alanine aminotransferase 8 h later. Histologic analysis confirmed that the delayed IP-10 therapy dramatically improved the appearance of the liver when examined 48 h after APAP. The therapeutic effect of IP-10 was associated with a marked increase in CXCR2 expression on hepatocytes. Neutralization of CXCR2 during IP-10 therapy resulted in an abrogation of the hepatoprotective effect of IP-10. Furthermore, IP-10 treatment of cultured hepatocytes stimulated a CXCR2-dependent proliferative response. In conclusion, IP-10 has a hepatoregenerative effect in a murine model of acute liver injury that is dependent on its up-regulation of CXCR2 on hepatocytes.
Disruption of Neutrophil Migration in a Conditional Transgenic Model: Evidence for CXCR2 Desensitization In Vivo
Authors: Wiekowski Maria T. , Chen Shu-Cheng , Zalamea Petronio , Wilburn Brian P. , Kinsley David J. , Sharif Wanda W. , Jensen Kristian K. , Hedrick Joseph A. , Manfra Denise , Lira Sergio A.
Published: April 2014

We developed transgenic mice conditionally expressing the neutrophil chemoattracting chemokine KC and the β-galactosidase gene in multiple tissues. In these transgenic mice, doxycycline treatment induced a strong up-regulation in the expression of KC in several tissues, including heart, liver, kidney, skin, and skeletal muscle. Expression of KC within these tissues led to a rapid and substantial increase in the serum levels of KC (serum KC levels were higher than 200 ng/ml 24 h after treatment). Accordingly, β-galactosidase expression was also detected after injection of doxycycline and was highest in skeletal muscle, pancreas, and liver. Surprisingly, despite expression of KC in multiple tissues, no neutrophil infiltration was observed in any of the tissues examined, including skin. Doxycycline treatment of nontransgenic mice grafted with transgenic skin caused dense neutrophilic infiltration of the grafts, but not the surrounding host skin, indicating that the KC produced in transgenic tissues was biologically active. In separate experiments, neutrophil migration toward a localized source of recombinant KC was impaired in animals overexpressing KC but was normal in response to other neutrophil chemoattractants. Analysis of transgenic neutrophils revealed that high concentrations of KC in transgenic blood had no influence on L-selectin cell surface expression but caused desensitization of the receptor for KC, CXCR2. These results confirm the neutrophil chemoattractant properties of KC and provide a mechanistic explanation for the paradoxical lack of leukocyte infiltration observed in the presence of elevated concentrations of this chemokine.
Genetic Contributions of Nonautoimmune SWR Mice Toward Lupus Nephritis
Authors: Xie Shangkui , Chang SoogHee , Yang Ping , Jacob Chryshanthi , Kaliyaperumal Arunan , Datta Syamal K. , Mohan Chandra
Published: April 2014

(SWR × New Zealand Black (NZB))F1 (or SNF1) mice succumb to lupus nephritis. Although several NZB lupus susceptibility loci have been identified in other crosses, the potential genetic contributions of SWR to lupus remain unknown. To ascertain this, a panel of 86 NZB × F1 backcross mice was immunophenotyped and genome scanned. Linkage analysis revealed four dominant SWR susceptibility loci (H2, Swrl-1, Swrl-2, and Swrl-3) and a recessive NZB locus, Nba1. Early mortality was most strongly linked to the H2 locus on chromosome (Chr) 17 (log likelihood of the odds (LOD) = 4.59 − 5.38). Susceptibility to glomerulonephritis was linked to H2 (Chr 17, LOD = 2.37 − 2.70), Swrl-2 (Chr 14, 36 cM, LOD = 2.48 − 2.71), and Nba1 (Chr 4, 75 cM, LOD = 2.15 − 2.23). IgG antinuclear autoantibody development was linked to H2 (Chr 17, LOD = 4.92 − 5.48), Swrl-1 (Chr 1, 86 cM, colocalizing with Sle1 and Nba2, LOD = 2.89 − 2.91), and Swrl-3 (Chr 18, 14 cM, LOD = 2.07 − 2.13). For each phenotype, epistatic interaction of two to three susceptibility loci was required to attain the high penetrance levels seen in the SNF1 strain. Although the SWR contributions H2, Swrl-1, and Swrl-2 map to loci previously mapped in other strains, often linked to very similar phenotypes, Swrl-3 appears to be a novel locus. In conclusion, lupus in the SNF1 strain is truly polygenic, with at least four dominant contributions from the SWR strain. The immunological functions and molecular identities of these loci await elucidation.
Rheumatoid Arthritis (RA)-Associated HLA-DR Alleles Form Less Stable Complexes with Class II-Associated Invariant Chain Peptide Than Non-RA-Associated HLA-DR Alleles
Authors: Patil Namrata S. , Pashine Achal , Belmares Michael P. , Liu Wendy , Kaneshiro Brandy , Rabinowitz Joshua , McConnell Harden , Mellins Elizabeth D.
Published: April 2014

Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.
Genetic Control of T and B Lymphocyte Activation in Nonobese Diabetic Mice
Authors: Chiu Priscilla P. L. , Jevnikar Anthony M. , Danska Jayne S.
Published: April 2014

Type 1 diabetes in nonobese diabetic (NOD) mice is characterized by the infiltration of T and B cells into pancreatic islets. T cells bearing the TCR Vβ3 chain are disproportionately represented in the earliest stages of islet infiltration (insulitis) despite clonal deletion of most Vβ3+ immature thymocytes by the mammary tumor virus-3 (Mtv-3) superantigen (SAg). In this report we showed that a high frequency of NOD Vβ3+ T cells that escape deletion are activated in vivo and that this phenotype is linked to the Mtv-3 locus. One potential mechanism of SAg presentation to peripheral T cells is by activated B cells. Consistent with this idea, we found that NOD mice harbor a significantly higher frequency of activated B cells than nondiabetes-prone strains. These activated NOD B cells expressed cell surface molecules consistent with APC function. At the molecular level, the IgH repertoire of activated B cells in NOD mice was equivalent to resting B cells, suggesting a polyclonal response in vivo. Genetic analysis of the activated B cell phenotype showed linkage to Idd1, the NOD MHC haplotype (H-2g7). Finally, Vβ3+ thymocyte deletion and peripheral T cell activation did not require B cells, suggesting that other APC populations are sufficient to generate both Mtv-3-linked phenotypes. These data provide insight into the genetic regulation of NOD autoreactive lymphocyte activation that may contribute to failure of peripheral tolerance and the pathogenesis of type I diabetes.
Potentiation of Simian Immunodeficiency Virus (SIV)-Specific CD4+ and CD8+ T Cell Responses by a DNA-SIV and NYVAC-SIV Prime/Boost Regimen
Authors: Hel Zdeněk , Tsai Wen-Po , Thornton Arthur , Nacsa Janos , Giuliani Laura , Tryniszewska Elzbieta , Poudyal Monita , Venzon David , Wang Xiaochi , Altman John , Watkins David I. , Lu Wenhong , von Gegerfelt Agneta , Felber Barbara K. , Tartaglia James , Pavlakis George N. , Franchini Genoveffa
Published: April 2014

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8+ and CD4+ T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4+ and CD8+ T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8+ T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.
Cutting Edge: Neosynthesis Is Required for the Presentation of a T Cell Epitope from a Long-Lived Viral Protein
Authors: Khan Selina , de Giuli Rita , Schmidtke Gunter , Bruns Michael , Buchmeier Michael , van den Broek Maries , Groettrup Marcus
Published: April 2014

CTLs recognize peptide epitopes which are proteolytically generated by the proteasome and presented on MHC class I molecules. According to the defective ribosomal product (DRiP) hypothesis, epitopes originate from newly synthesized polypeptides which are degraded shortly after their translation. The DRiP hypothesis would explain how epitopes can be generated from long-lived proteins. We examined whether neosynthesis is required for presentation of the immunodominant epitope NP118 of the lymphocytic choriomeningitis virus nucleoprotein, which has a half-life of >3 days. Two days after nucleoprotein biosynthesis was terminated in a tetracycline-regulated transfectant, the presentation of the NP118 epitope ceased. This indicates that NP118 epitopes are generated from newly synthesized nucleoproteins rather than from the long-lived pool of nucleoproteins in the cell. Therefore, the lymphocytic choriomeningitis virus nucleoprotein is the first substrate for which a major prediction of the DRiP hypothesis, namely the requirement for neosynthesis, is shown to hold true.
Murine Renal Allografts: Spontaneous Acceptance Is Associated with Regulated T Cell-Mediated Immunity
Authors: Bickerstaff Alice A. , Wang Jiao-Jing , Pelletier Ronald P. , Orosz Charles G.
Published: April 2014

It was shown >20 yr ago that mice will spontaneously accept renal allografts in the absence of immunosuppression, but the mechanism responsible for this is not understood. We transplanted DBA/2 (H-2d) kidneys into nephrectomized C57BL/6 (H-2b) mice, and the allografts were spontaneously accepted for >60 days without immunosuppression. In contrast, nonimmunosuppressed cardiac and skin allografts in the same strain combination are rejected within approximately 10 days. The accepted renal allografts have a prominent leukocytic infiltrate, suggesting an ongoing, local immune response. At 60 days post-transplant, the recipients of accepted renal allografts display DBA/2-reactive alloantibodies. They also display DBA/2-reactive delayed-type hypersensitivity responses that are actively counter-regulated by DBA/2-induced TGF-β production, but not by IL-10 production. These data suggest that a donor-reactive, cell-mediated immune mechanism involving TGF-β is associated with the spontaneous acceptance of renal allografts in mice.
Expression of the Ryanodine Receptor Isoforms in Immune Cells
Authors: Hosoi Eiji , Nishizaki Chiharu , Gallagher Kathleen L. , Wyre Hadley W. , Matsuo Yoshinobu , Sei Yoshitatsu
Published: April 2014

Ryanodine receptor (RYR) is a Ca2+ channel that mediates Ca2+ release from intracellular stores. We have used RT-PCR analysis and examined its expression in primary peripheral mononuclear cells (PBMCs) and in 164 hemopoietic cell lines. In PBMCs, type 1 RYR (RYR1) was expressed in CD19+ B lymphocytes, but less frequently in CD3+ T lymphocytes and in CD14+ monocytes. Type 2 RYR (RYR2) was mainly detected in CD3+ T cells. Induction of RYR1 and/or RYR2 mRNA was found after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1α (MIP1α) or TGF-β. Type 3 RYR (RYR3) was not detected in PBMCs. Many hemopoietic cell lines expressed not only RYR1 or RYR2 but also RYR3. The expression of the isoforms was not associated with specific cell lineage. We showed that the RYR-stimulating agent 4-chloro-m-cresol (4CmC) induced Ca2+ release and thereby confirmed functional expression of the RYR in the cell lines expressing RYR mRNA. Moreover, concordant induction of RYR mRNA with Ca2+ channel function was found in Jurkat T cells. In untreated Jurkat T cells, 4CmC (>1 mM) had no effect on Ca2+ release, whereas 4CmC (<400 μM) caused Ca2+ release after the induction of RYR2 and RYR3 that occurred after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1α, or TGF-β. Our results demonstrate expression of all three isoforms of RYR mRNA in hemopoietic cells. Induction of RYRs in response to chemokines and TGF-β suggests roles in regulating Ca2+-mediated cellular responses during the immune response.
Melphalan-Induced Expression of IFN-β in MOPC-315 Tumor-Bearing Mice and Its Importance for the Up-Regulation of TNF-α Expression
Authors: Jovasevic Vladimir M. , Mokyr Margalit B.
Published: April 2014

We have previously shown that administration of a low-dose of melphalan (l-phenylalanine mustard; l-PAM) to mice bearing a large s.c. MOPC-315 tumor leads to up-regulation of TNF-α expression, which is first evident at the mRNA level at 24 h after the chemotherapy. In this study, we show accumulation of IFN-β mRNA in the spleen and tumor nodule of such mice as early as 1 h after the chemotherapy followed by elevated production of IFN-β protein. IFN-β protein in turn was found to be important for the l-PAM-induced up-regulation of TNF-α expression, as neutralization of IFN-β inhibited the l-PAM-induced up-regulation of TNF-α mRNA expression in MOPC-315 tumor cells. In addition, l-PAM failed to up-regulate TNF-α expression in spleen cells from mice in which signaling by IFN-β is deficient. Studies into the mechanism through which l-PAM leads to rapid accumulation of IFN-β mRNA revealed that it requires de novo RNA synthesis, indicating that the regulation is at the transcriptional level. However, it did not require de novo protein synthesis, indicating that activation of pre-existing transcription factors is sufficient for IFN-β gene expression. The l-PAM-induced accumulation of IFN-β mRNA was mimicked with H2O2 and was prevented with the antioxidant N-acetyl-l-cysteine, indicating that reactive oxygen species are involved in the transcriptional regulation of l-PAM-induced IFN-β gene expression. Thus, the IFN-β gene is an early response gene that is activated in response to l-PAM via a pathway that involves reactive oxygen species, and IFN-β in turn plays an important role in l-PAM-induced TNF-α up-regulation.
Broad Distribution of Colony-Forming Cells with Erythroid, Myeloid, Dendritic Cell, and NK Cell Potential Among CD34++ Fetal Liver Cells
Authors: Muench Marcus O. , Bárcena Alicia
Published: April 2014

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38− and CD38+ progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56+ NK cells were detected in 21.9 and 9.9% of colonies grown from CD38− and CD38+ progenitors, respectively. NK cells were detected in mostly large CD14+/CD15+ myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a+ dendritic cells were also present in three-quarters of the colonies grown from CD38− and CD38+ progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38− and CD38+ progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38+ fraction compared with the CD38− fraction because of the higher frequency of CD38+ cells among CD34++ cells. The broad distribution of multipotent CFCs among CD38− and CD38+ progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38+ cells.
Phosphorylation by the Protein Kinase CK2 Promotes Calpain-Mediated Degradation of IκBα
Authors: Shen Jian , Channavajhala Padmalatha , Seldin David C. , Sonenshein Gail E.
Published: April 2014

Rapid IκBα turnover has been implicated in the high basal NF-κB activity in WEHI 231 B immature IgM+ B cells. Here we show that treatment of WEHI 231 cells with apigenin, a selective inhibitor of the protein kinase CK2, decreased the rate of IκBα turnover and nuclear levels of NF-κB. Turnover of IκBα in these cells is mediated in part by the protease calpain. Since both CK2 and calpain target the proline-glutamic acid-serine-threonine (PEST) domain, we investigated the role of CK2 in the degradation of IκBα by calpain using an in vitro phosphorylation/degradation assay. CK2 phosphorylation enhanced μ-calpain-mediated degradation of wild-type IκBα, but not of mutant 3CIκBα, with S283A, T291A, and T299A mutations in phosphorylation sites within the PEST domain. Roles for CK2 and calpain in IκBα turnover were similarly shown in CH31 immature and CH12 mature IgM+ B cells, but not in A20 and M12 IgG+ B cells. These findings demonstrate for the first time that CK2 phosphorylation of serine/threonine residues in the PEST domain promotes calpain-mediated degradation of IκBα and thereby increases basal NF-κB levels in IgM+ B cells.
Microglial Phagocytosis of Apoptotic Inflammatory T Cells Leads to Down-Regulation of Microglial Immune Activation
Authors: Magnus Tim , Chan Andrew , Grauer Oliver , Toyka Klaus V. , Gold Ralf
Published: April 2014

Apoptotic cell death is an established mechanism to terminate an inflammatory response in rodent or human brains. Microglia, as the resident phagocyte, is a strong candidate for the clearance of apoptotic lymphocytes. Apoptosis was induced in cultured autologous thymocytes and in myelin basic protein (MBP)-specific, encephalitogenic T cells from Lewis rats by the addition of 0.1 μg/ml methylprednisolone. The amount of phagocytosis of apoptotic cells was assessed using an in vitro phagocytosis assay. Supernatants were collected to measure microglial cytokine secretion. The state of immune activation in microglia was investigated by a T cell proliferation assay and by flow cytometric analysis of microglial surface expression of immune molecules. Microglia ingested specifically apoptotic cells (apoptotic thymocytes as well as MBP-specific T cells) in contrast to nonapoptotic control cells (p < 0.0001). Subsequent secretion of the proinflammatory cytokines TNF-α and IL-12 was significantly decreased, while the secretion of IL-10 and TGF-β was not affected. Furthermore, ingestion of apoptotic cells led to increased microglial MHC class II expression without concomitant increase in MHC class I, costimulatory molecules, and ICAM expression. The Ag-specific activation of MBP-specific T cells in cocultures with microglia that had ingested apoptotic cells was significantly less than that of identical T cells that interacted with nonphagocytosing microglia. Together with negative results obtained in a trans-well system, this is in support of a cell contact-mediated effect. Microglia might play an important role in the clearance of apoptotic cells. The uptake of apoptotic cells by microglia is tolerogenic and results in a reduced proinflammatory cytokine production and a reduced activation of encephalitogenic T cells. This might help to restrict an autoimmune inflammation and minimize damage in the inflamed brain.
CD8+ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization and Lytic Granule Exocytosis
Authors: Radoja Sasa , Saio Masanao , Schaer David , Koneru Mythili , Vukmanovic Stanislav , Frey Alan B.
Published: April 2014

Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8+ TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mRNAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-γ is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction.
C57BL/6 and BALB/c Bronchoalveolar Macrophages Respond Differently to Exercise
Authors: Su Shu-Hui , Chen Hsiun-ing , Jen Chauying J.
Published: April 2014

Macrophages from prototypical Th1 strains (e.g., C57BL/6) and Th2 strains (e.g., BALB/c) are classified as M-1 and M-2 phenotypes. We investigated the different phagocytic responses between M-1 and M-2 bronchoalveolar macrophages (BAMs) under resting and two various exercise conditions. At rest, M-1 BAMs showed higher phagocytic capacity of unopsonized particles, higher expression of MARCO (macrophage receptor with collagenous structure), and higher generation of NO than M-2 BAMs. Severe exercise, but not moderate exercise, significantly enhanced both phagocytosis of unopsonized particles and expression of MARCO in M-2 BAMs. In contrast, M-1 BAMs were unaffected by either exercise protocol. The phagocytosis of unopsonized particles was largely mediated by MARCO, especially in M-1 BAMs. Secreted products from cultured M-2 BAMs isolated after severe exercise, but not those from M-1 BAMs, enhanced BAM phagocytosis. The cultured M-1 BAMs secreted phagocytosis inhibitors, and this effect could be blocked by NO antagonists. Moreover, the extent of phagocytosis suppression induced by M-1 BAM-secreted products correlated with their production of nitrite/nitrate. Exogenous NO donors as well as NO derivatives, nitrite and nitrate, suppressed the BAM phagocytosis. We propose that while the severe exercise-enhanced phagocytosis in M-2 BAMs was largely mediated by MARCO up-regulation and secretion of stimulators, the lack of exercise effect in M-1 BAMs could be partially due to the constitutive secretion of NO-related suppressors. In conclusion, genetically different mice use different strategies in regulating BAM activity under resting conditions and in response to various exercise paradigms.
The Critical Role of LIGHT, a TNF Family Member, in T Cell Development
Authors: Wang Jing , Chun Taehoon , Lo James C. , Wu Qiang , Wang Yang , Foster Amy , Roca Karin , Chen Min , Tamada Koji , Chen Lieping , Wang Chyung-Ru , Fu Yang-Xin
Published: April 2014

Negative selection refers to the selective deletion of autoreactive thymocytes but its molecular events have not been well defined. In this study, we demonstrate that a cellular ligand for herpes virus entry mediator and lymphotoxin receptor (LIGHT), a newly identified member of the TNF superfamily, may play a critical role in negative selection. Using TCR transgenic mice, we find that the blockade of LIGHT signaling in vitro and in vivo prevents negative selection induced by peptide and intrathymically expressed Ags, resulting in the rescue of thymocytes from apoptosis. Furthermore, the thymi of LIGHT transgenic mice show severe atrophy with remarkably reduced CD4+CD8+ double-positive cells caused by increased apoptosis, suggesting that LIGHT can delete immature T cells in vivo. Taken together, these results demonstrate a critical role of LIGHT in thymic negative selection of the T cell repertoire.
The J Chain Is Essential for Polymeric Ig Receptor-Mediated Epithelial Transport of IgA
Authors: Johansen Finn-Eirik , Braathen Ranveig , Brandtzaeg Per
Published: April 2014

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys13:Cys101 and Cys109:Cys134) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys72:Cys92) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys15 or Cys69 was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.
Molecular Mechanisms of IL-2 Gene Regulation Following Costimulation Through LFA-1
Authors: Abraham Clara , Miller Jim
Published: April 2014

The integrin LFA-1 serves as an accessory molecule in T cell activation. In addition to its well-known role as an adhesion molecule, LFA-1 can contribute to T cell activation and up-regulation of IL-2 gene expression. However, the specific mechanisms by which LFA-1 influences T cell activation have not been elucidated. Therefore, we examined the impact of LFA-1:ICAM-1 interactions on transcriptional and posttranscriptional IL-2 gene regulation, using a costimulation-negative cell line transfected with MHC class II alone, or in combination with ICAM-1 or B7-1. IL-2 transcription was assessed utilizing transgenic mice expressing an IL-2 promoter luciferase reporter construct crossed to DO11.10 TCR-transgenic mice, and IL-2 mRNA stability was evaluated by real-time RT-PCR. Comparison of naive and previously activated T cells demonstrates a dramatic increase in IL-2-luciferase transcription in activated T cells that can, in part, be attributed to downstream signaling events. Costimulation through LFA-1 enhances transcription of the transgenic reporter construct across a wide Ag dose range, but does not affect IL-2 mRNA stability. In contrast, CD28 costimulation is clearly mediated through up-regulation of IL-2 transcription and through enhancement of mRNA stability. These results indicate that the primary pathway whereby engagement of LFA-1 through its ligand ICAM-1 up-regulates IL-2 gene expression is through enhanced IL-2 transcription, in the absence of any effect on IL-2 mRNA stabilization.
Direct Ex Vivo Analysis of Antigen-Specific IFN-γ-Secreting CD4 T Cells in Mycobacterium tuberculosis-Infected Individuals: Associations with Clinical Disease State and Effect of Treatment
Authors: Pathan Ansar A. , Wilkinson Katalin A. , Klenerman Paul , McShane Helen , Davidson Robert N. , Pasvol Geoffrey , Hill Adrian V. S. , Lalvani Ajit
Published: April 2014

The wide spectrum of clinical outcomes following infection with Mycobacterium tuberculosis is largely determined by the host immune response; therefore, we studied several clinically defined groups of individuals (n = 120) that differ in their ability to contain the bacillus. To quantitate M. tuberculosis-specific T cells directly ex vivo, we enumerated IFN-γ-secreting CD4 T cells specific for ESAT-6, a secreted Ag that is highly specific for M. tuberculosis, and a target of protective immune responses in animal models. We found that frequencies of circulating ESAT-6 peptide-specific IFN-γ-secreting CD4 T cells were higher in latently infected healthy contacts and subjects with minimal disease and low bacterial burdens than in patients with culture-positive active pulmonary tuberculosis (p = 0.009 and p = 0.002, respectively). Importantly, the frequency of these Ag-specific CD4 T cells fell progressively in all groups with treatment (p = 0.005), suggesting that the lower responses in patients with more extensive disease were not due to tuberculosis-induced immune suppression. This population of M. tuberculosis Ag-specific Th1-type CD4 T cells appears to correlate with clinical phenotype and declines during successful therapy; these features are consistent with a role for these T cells in the containment of M. tuberculosis in vivo. Such findings may assist in the design and evaluation of novel tuberculosis vaccine candidates.
TNF-α Compensates for the Impaired Host Defense of IL-1 Type I Receptor-Deficient Mice During Pneumococcal Pneumonia
Authors: Rijneveld Anita W. , Florquin Sandrine , Branger Judith , Speelman Peter , Van Deventer Sander J. H. , van der Poll Tom
Published: April 2014

To determine the role of IL-1 in the host defense against pneumonia, IL-1R type I-deficient (IL-1R−/−) and wild-type (Wt) mice were intranasally inoculated with Streptococcus pneumoniae. Pneumonia resulted in elevated IL-1α and IL-1β mRNA and protein levels in the lungs. Survival rates did not differ between IL-1R−/− and Wt mice after inoculation with 5 × 104 or 2 × 105 CFU. At early time points (24 and 48 h) IL-1R−/− mice had 2-log more S. pneumoniae CFU in lungs than Wt mice; at 72 h bacterial outgrowth in lungs was similar in both groups. Upon histopathologic examination IL-1R−/− mice displayed a reduced capacity to form inflammatory infiltrates at 24 h after the induction of pneumonia. IL-1R−/− mice also had significantly less granulocyte influx in bronchoalveolar lavage fluid at 24 h after inoculation. Since TNF is known to enhance host defense during pneumonia, we determined the role of endogenous TNF in the early impairment and subsequent recovery of defense mechanisms in IL-1R−/− mice. All IL-1R−/− mice treated with anti-TNF rapidly died (no survivors (of 14 mice) after 4 days), while 10-day survival in IL-1R−/− mice (control Ab), Wt mice (anti-TNF), and Wt mice (control Ab) was 7 of 13, 3 of 14, and 12 of 13, respectively. These data suggest that TNF is more important for host defense against pneumococcal pneumonia than IL-1, and that the impaired early host defense in IL-1R−/− mice is compensated for by TNF at a later phase.
NK- and CD8+ T Cell-Mediated Eradication of Established Tumors by Peritumoral Injection of CpG-Containing Oligodeoxynucleotides
Authors: Kawarada You , Ganss Ruth , Garbi Natalio , Sacher Torsten , Arnold Bernd , Hämmerling Günter J.
Published: April 2014

Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODN) are known to act as adjuvants and powerful activators of the innate immune system. We investigated the therapeutic effect of CpG-ODN on a variety of established mouse tumors including AG104A, IE7 fibrosarcoma, B16 melanoma, and 3LL lung carcinoma. These tumors are only weakly immunogenic and notoriously difficult to treat. Repeated peritumoral injection of CpG-ODN resulted in complete rejection or strong inhibition of tumor growth, whereas systemic application had only partial effects. The CpG-ODN-induced tumor rejection was found to be mediated by both NK and tumor-specific CD8+ T cells. Comparison of parental tumors and variants rendered more antigenic by transfection with tumor Ags suggested that the efficiency of the CpG-ODN therapy correlated with the antigenicity of the tumors. Peritumoral CpG-ODN treatment was even effective in a situation where the immune system was tolerant for the tumor Ag, as shown by breakage of tolerance and tumor elimination. These results suggest that peritumoral application of CpG-ODN acts locally by inducing NK cells, and also leads to efficient presentation of tumor Ags and stimulation of CD8+ effector and memory T cells, thus providing a powerful antitumor therapy that can be also applied without knowledge of the tumor Ag.
Differential Induction of Endotoxin Tolerance by Lipopolysaccharides Derived from Porphyromonas gingivalis and Escherichia coli
Authors: Martin Michael , Katz Jannet , Vogel Stefanie N. , Michalek Suzanne M.
Published: April 2014

Exposure of mononuclear phagocytes to enterobacterial LPS induces a state of transient hyporesponsiveness to subsequent LPS exposure, termed endotoxin tolerance. In the present study, LPS derived from the oral periodontal pathogen, Porphyromonas gingivalis, was compared with that derived from the enterobacterium, Escherichia coli, for the ability to induce endotoxin tolerance. Pretreatment of the human macrophage cell line, THP-1, with E. coli LPS resulted in a severe reduction in the levels of IL-1β, IL-6, and TNF-α upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a mitigation of IL-1β, but not IL-6 and TNF-α production upon subsequent exposure to P. gingivalis LPS: primary or secondary stimulation with ≤100 ng/ml P. gingivalis LPS resulted in comparable levels of IL-6 and TNF-α, while stimulation of THP-1 cells with ≥1 μg/ml P. gingivalis LPS induced a significant enhancement in IL-6 and TNF-α levels upon secondary exposure. To identify possible mechanisms for these differences, changes in the expression of molecules involved in the LPS-signaling pathway were assessed. Pretreatment of THP-1 cells with E. coli LPS resulted in a significant reduction in surface Toll-like receptor 4 (TLR4) expression and an inability to degrade Ι-κB-α or Ι-κB-β proteins upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a significant enhancement of both CD14 and TLR2, while maintaining the ability to degrade Ι-κB-β only upon secondary stimulation. Thus, E. coli and P. gingivalis LPS differentially affect CD14 and TLR expression as well as secondary LPS-associated responses.
Analysis of In Situ NK Cell Responses During Viral Infection
Authors: Dokun Ayotunde O. , Chu Dortha T. , Yang Liping , Bendelac Albert S. , Yokoyama Wayne M.
Published: April 2014

NK cells are required for early control of murine CMV (MCMV) infection, but the distribution of murine NK cells in situ has not been clearly defined. We tested the reactivity of all available NK cell receptor-specific mAbs by immunohistochemistry. Only one mAb, 4D11 (anti-Ly-49G2), was reactive with C57BL/6 tissue sections. mAb 4D11-reactive cells expressed the nuclear morphology and flow cytometric profile of NK cells. In lymphoid organs, NK cells were distributed primarily in the splenic red pulp, between adjacent lobes in lymph node and randomly in the cortex and medulla of the thymus. No NK cells were detected in normal liver sections. Two days following MCMV infection, most splenic NK cells were associated with the lymphoid follicles and marginal zone. By day 3 following infection, the number of liver NK cells had increased significantly and the cells were detected within inflammatory foci. These changes were independent of IL-12, IFN-γ, and TNF-α, as assessed in mice with targeted mutations. Concurrent immunostaining for NK cells and viral Ags revealed close association of NK cells and MCMV-infected cells in the spleen and liver. Similar results were obtained in CD1−/− and recombination activation gene-1−/− mice lacking NK T or T and B cells, respectively, indicating specificity of staining for NK cells. Thus, following MCMV infection, NK cells accumulate at sites of viral replication in an IL-12-, IFN-γ-, and TNF-α-independent manner.
IL-1 Plays a Critical Role in Oral, But Not Dermal, Wound Healing
Authors: Graves Dana T. , Nooh Nasser , Gillen Thomas , Davey Michael , Patel Shilpa , Cottrell David , Amar Salomon
Published: April 2014

Wound healing is a well-orchestrated complex process leading to the repair of injured tissues. After injury, proinflammatory cytokines act as important modulators of the inflammatory process. IL-1 expression has been regarded as necessary for healing; however, its effects have also been implicated in delayed wound repair. Currently, there is no consensus or direct evidence that IL-1 activity plays a central role in the healing process. The present investigation was undertaken to define the role of IL-1R signaling in the healing outcome of an excisional wound in the palate or scalp of mice that had targeted deletions of the IL-1R type 1 (IL-1R1−/−) compared with matched wild-type mice. Histomorphometric analysis was undertaken to assess the degree of healing and the recruitment of polymorphonuclear and mononuclear phagocytes. After 14 days, wild-type mice exhibited complete closure of intraoral wounds, while IL-1R1−/− animals had only partial closure (50%). In the IL-1R1−/− mice, healing tissues exhibited a persistent inflammatory cell infiltrate, which did not occur in wild-type animals. Treatment with antibiotics significantly diminished the persistent inflammatory infiltrate and improved healing in the experimental animals. In contrast to oral wounds, the rate of healing and recruitment of polymorphonuclear cells in scalp wounds was similar in IL-1R1−/− and wild-type mice. The present data underscore the importance of IL-1 in wound healing in a challenging environment and identify its principal role in facilitating the healing process by protecting an open wound from bacterial insult. In a less challenging environment, the production of new connective tissue and its coverage by migrating epithelium are minimally affected by the absence of IL-1 activity.
Intact Intestinal mRNAs and Intestinal Epithelial Cell Esterase, But Not Cryptosporidium parvum, Reach Mesenteric Lymph Nodes of Infected Mice
Authors: Ponnuraj Esther M. , Hayward Anthony R.
Published: April 2014

Dendritic cells from the mesenteric lymph nodes (MLN) contain dense esterase-positive inclusions that may originate in effete intestinal epithelial cells and reach MLN without degradation. The MLN esterases have the electrophoretic mobilities of both intestinal and mononuclear cells. Cryptosporidium parvum (CP)-infected mice have CP Ag-positive cells in MLN and also increased numbers of dense esterase-positive cells, but the CP Ag-positive cells do not stain for esterase. To characterize the handling of epithelial cell products by dendritic cells, we analyzed mRNAs in the MLN of control and CP-infected recombination-activating gene−/−DO11.10 mice by oligoarrays. mRNAs for 115 proteins were increased in MLN after CP infection, of which the principal increases in trypsin and chymotrypsin approximated to 250-fold. Colipase, reg-1, C-reactive protein-ductin, and amyloid were also up-regulated >10-fold and all returned to baseline by 28 days after infection. mRNAs for the same proteins were detected in intestinal epithelial cells of infected mice by oligoarrays and RT-PCR after infection. mRNA for CP β-tubulin was detectable in intestinal epithelial cells between 5 and 18 days after infection but was not detected in the MLN throughout the observation period. It appears that host response to CP infection includes expression of mRNA for some pancreatic enzymes by intestinal epithelial cells and their subsequent transport to the MLN. The esterase and trypsin, and mRNAs for chymotrypsin, colipase, and others that may derive from uninfected epithelial cells, appear to be transported to the MLN intact, while mRNA for CP β-tubulin that is derived from infected cells is degraded.
Novel Anti-inflammatory Effects of the Inhaled Corticosteroid Fluticasone Propionate During Lung Myofibroblastic Differentiation
Authors: Cazes Eric , Giron-Michel Julien , Baouz Soria , Doucet Christelle , Cagnoni Francesca , Oddera Susanna , Körner Marie , Dasic Gorana , Testi Renato , Azzarone Bruno , Canonica Giorgio Walter
Published: April 2014

Asthma is characterized by an irreversible subepithelial fibrosis with the appearance of myofibroblasts, which can be now considered important early participants in inflammatory responses as well as potential targets for anti-inflammatory drugs. In this study, we show that fluticasone propionate (FP), a powerful inhaled corticosteroid (ICS), displays novel anti-inflammatory effects on human lung fibroblasts during their myofibroblastic differentiation. Indeed, FP inhibits in lung myofibroblasts, at a very early stage of differentiation, the activation of Janus kinase/STAT pathways induced by IL-13 (tyrosine kinase 2, STAT1, STAT3, STAT6, mitogen-activated protein kinase). Contrarily, in mildly or fully differentiated myofibroblastic cultures, FP still displays a potential anti-inflammatory activity even if it only inhibits tyrosine kinase 2 phosphorylation. Moreover, FP inhibits constitutive and TGF-β-induced expression of α-smooth muscle actin, the main marker of myofibroblastic differentiation, both in very early and in mild differentiated myofibroblasts. Finally, FP displays an additional powerful anti-inflammatory effect, decreasing nuclear translocation of NF-κB independent of the degree of myofibroblastic differentiation. These data 1) suggest that myofibroblasts are priority targets for ICS, which is able to revert them to a normal phenotype even if they appear to be already engaged in their differentiation, and 2) may help to explain why asthma is improved by an early ICS treatment, whereas advanced asthma is more resistant to these drugs.