bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction and Expression of β-Calcitonin Gene-Related Peptide in Rat T Lymphocytes and Its Significance
Authors: Xing Liyu , Guo Jingxuan , Wang Xian
Published: April 2014

Our previous data have shown that rat lymphocytes can synthesize calcitonin gene-related peptide (CGRP), a neuropeptide. In this study the type, characteristics, and functional role of lymphocyte-derived CGRP were investigated. The results showed that treatment with Con A (4 μg/ml) and recombinant human IL-2 (rhIL-2; 750 U/ml) for 3–5 days induced CGRP synthesis and secretion by lymphocytes from both thymus and mesenteric lymph nodes in a time-dependent manner. Stimulation of these cells with Con A (1–8 μg/ml) or rhIL-2 (94–1500 U/ml) for 5 days induced a significant increase in CGRP secretion in a concentration-dependent manner. The maximal secretion of CGRP with Con A by thymocytes was elevated from 104 ± 11 to 381 ± 44 pg/108 cells, and that by mesenteric lymph node lymphocytes was elevated from 83 ± 10 to 349 ± 25 pg/108 cells, respectively. The maximal CGRP secretion with rhIL-2 by thymocytes was elevated from 116 ± 3 to 607 ± 23 pg/108, and that by mesenteric lymph node lymphocytes was elevated from 117 ± 9 to 704 ± 37 pg/108 cells, respectively. The nucleotide sequencing study showed that lymphoid cells expressed β-CGRP cDNA only. The levels of β-CGRP mRNA in mitogen-stimulated lymphocytes of both sources were also increased. However, LPS had no such effect on either source of cells. hCGRP8–37 (2.0 μM), a CGRP1 receptor antagonist, enhanced Con A-induced proliferation and IL-2 release of thymocytes by 41.3 and 35.8% over those induced by Con A alone, respectively. The data suggest that T lymphocyte mitogens can induce the production of endogenous β-CGRP from T lymphocytes, which may partially inhibit the proliferation and IL-2 release of rat T lymphocyte under immune challenges.
Distinct Human T Cell Repertoires Mediate Immediate and Delayed-Type Hypersensitivity to theTrichophytonAntigen, Tri r 2
Authors: Woodfolk Judith A. , Sung Sun-Sang J. , Benjamin David C. , Lee Jae K. , Platts-Mills Thomas A. E.
Published: April 2014

The 29-kDa subtilase homologue, Tri r 2, derived from the dermatophyte fungus Trichophyton rubrum, exhibits unique immunologic characteristics in its ability to elicit immediate (IH) and delayed-type (DTH) hypersensitivity skin tests in different individuals. Thus, Tri r 2 provides a model for comparing the T cell repertoire in subjects with distinct immune responses to a single Ag. Recombinant Tri r 2 produced as a GST fusion protein in Escherichia coli stimulated strong in vitro lymphoproliferative responses in 10 IH and 10 DTH responders. Patterns of T cell epitope recognition were compared between skin test groups using 28 overlapping peptides (each in 12 replicate wells) derived from Tri r 2 to stimulate T lymphocyte proliferation in vitro. Peptide 5 (P5; aa 41–60) induced the strongest response in DTH subjects and showed the largest difference between DTH and IH responders in proliferation (mean standardized index, 2.22 and 0.82, respectively; p = 0.0047) and number of positive wells (81 vs 12). Responses to P5 were associated with diverse HLA haplotypes. These results showed that P5 contains an immunodominant epitope specifically associated with DTH and that this peptide is recognized in a permissive manner. Cross-validated linear discriminant analysis using T cell proliferative responses to two regions of Tri r 2 (aa 51–90 and 231–270) gave a 95% predictive accuracy for classification of subjects into IH or DTH groups. We conclude that different immune responses to Trichophyton are mediated by distinct T cell repertoires between individuals with IH and DTH reactions to Tri r 2.
Notch Signaling Enhances Survival and Alters Differentiation of 32D Myeloblasts
Authors: Tan-Pertel Hongying Tina , Walker Liberty , Browning Damaris , Miyamoto Alison , Weinmaster Gerry , Gasson Judith C.
Published: April 2014

The Notch transmembrane receptors play important roles in precursor survival and cell fate specification during hematopoiesis. To investigate the function of Notch and the signaling events activated by Notch in myeloid development, we expressed truncated forms of Notch1 or Notch2 proteins that either can or cannot activate the core binding factor 1 (CBF1) in 32D (clone 3) myeloblasts. 32D cells proliferate as blasts in the presence of the cytokines, GM-CSF or IL-3, but they initiate differentiation and undergo granulopoiesis in the presence of granulocyte CSF (G-CSF). 32D cells expressing constitutively active forms of Notch1 or Notch2 proteins that signal through the CBF1 pathway maintained significantly higher numbers of viable cells and exhibited less cell death during G-CSF induction compared with controls. They also displayed enhanced entry into granulopoiesis, and inhibited postmitotic terminal differentiation. In contrast, Notch1 constructs that either lacked sequences necessary for CBF1 binding or that failed to localize to the nucleus had little effect. Elevated numbers of viable cells during G-CSF treatment were also observed in 32D cells overexpressing the basic helix-loop-helix protein (bHLH), HES1, consistent with activation of the CBF1 pathway. Taken together, our data suggest that Notch signaling enhances 32D cell survival, promotes entry into granulopoiesis, and inhibits postmitotic differentiation through a CBF1-dependent pathway.
Identification and Characterization of a β Proteasome Subunit Cluster in the Japanese Pufferfish (Fugu rubripes)
Authors: Clark Melody S. , Pontarotti Pierre , Gilles André , Kelly Alison , Elgar Greg
Published: April 2014

The low molecular mass polypeptide (LMP2, LMP7, and MECL-1) genes code for β-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the MHC class I-mediated Ag-presenting pathway. These gene products are up-regulated in response to infection by IFN-γ and replace the corresponding constitutively expressed subunits (X, Y, and Z) during the immune response. In humans, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), while MECL-1 is located at 16q22.1. In the present study, we have identified all three IFN-γ-regulated β-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC class I region. We show that in this species, LMP7, LMP2, and MECL-1 are linked. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested to date) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes.
Metalloprotease-Mediated Shedding of Enzymatically Active Mouse ecto-ADP-ribosyltransferase ART2.2 Upon T Cell Activation
Authors: Kahl Sarah , Nissen Marion , Girisch Roman , Duffy Ted , Leiter Edward H. , Haag Friedrich , Koch-Nolte Friedrich
Published: April 2014

T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-α-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.
Changing the Antigen Binding Specificity by Single Point Mutations of an Anti-p24 (HIV-1) Antibody
Authors: Winkler Karsten , Kramer Achim , Küttner Gabriele , Seifert Martina , Scholz Christa , Wessner Helga , Schneider-Mergener Jens , Höhne Wolfgang
Published: April 2014

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr32Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe94Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe94Ala up to the level of the e-pep affinity to the wild-type scFv.
Rapid Recruitment of Neutrophils Containing Prestored IL-12 During Microbial Infection
Authors: Bliss Susan K. , Butcher Barbara A. , Denkers Eric Y.
Published: April 2014

Neutrophils are well known to rapidly migrate to foci of infection, where they exert microbicidal functions. We sought to determine whether neutrophils responding to in vivo infection with the protozoan pathogen Toxoplasma gondii were capable of IL-12 production as suggested by recent in vitro studies. Intraperitoneal infection induced a neutrophil influx by 4 h, accompanied by ex vivo IL-12 p40 and p70 release. Approximately 85% of the neutrophils displayed intracellular stores of IL-12, as determined by flow cytometry and confocal fluorescence microscopy. Neutrophils from IFN-γ knockout mice also expressed IL-12, ruling out an IFN-γ-priming requirement. Neither infected nor uninfected peritoneal macrophages displayed intracellular IL-12, but these cells were strongly IL-10+. Infection per se was unnecessary for IL-12 production because peritoneal and peripheral blood neutrophils from uninfected animals contained IL-12+ populations. Expression of the granulocyte maturation marker Gr-1 (Ly-6G) was correlated with IL-12 production. Mice depleted of their granulocytes by mAb administration at the time of infection had decreased serum levels of IL-12 p40. These results suggest a model in which neutrophils with prestored IL-12 are rapidly mobilized to an infection site where they are triggered by the parasite to release cytokine. Our findings place neutrophils prominently in the cascade of early events leading to IL-12-dependent immunity to T. gondii.
Novel Mechanism of Antibody-Independent Complement Neutralization of Herpes Simplex Virus Type 1
Authors: Friedman Harvey M. , Wang Liyang , Pangburn Michael K. , Lambris John D. , Lubinski John
Published: April 2014

The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in ≥100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.
Increased Resistance Against Acute Polymicrobial Sepsis in Mice Challenged with Immunostimulatory CpG Oligodeoxynucleotides Is Related to an Enhanced Innate Effector Cell Response
Authors: Weighardt Heike , Feterowski Carolin , Veit Martin , Rump Martina , Wagner Hermann , Holzmann Bernhard
Published: April 2014

Recent reports support the concept that the major defect in polymicrobial sepsis is an impaired immunologic response to infection. Oligodeoxynucleotides containing CpG sequence motifs (CpG-ODN) were previously shown to induce immune protection in models of chronic infection with intracellular bacteria, parasites, and viruses due to their ability to augment IFN-γ-dependent Th1 responses. Here, we demonstrate that challenging mice with CpG-ODN substantially increases the resistance against acute polymicrobial sepsis. Systemic levels of IL-12, IL-18, and IL-10 were not altered in CpG-ODN-treated mice as compared with controls. In contrast, administration of CpG-ODN resulted in a strongly enhanced accumulation of neutrophils at the primary site of infection. Neutrophils of CpG-ODN-treated mice exhibited an up-regulation of phagocytic receptors, an increased phagocytic activity, and an elevated production of reactive oxygen metabolites. These results suggest that the protective effects of CpG-ODNs in acute polymicrobial sepsis are related to an enhanced effector cell response of innate immunity. CpG-ODN may therefore represent potent agents for the treatment of sepsis-associated immunoparalysis.
Memory-Type CD8+ T Cells Protect IL-2 Receptor α-Deficient Mice from Systemic Infection with Herpes Simplex Virus Type 2
Authors: Tsunobuchi Hironaka , Nishimura Hitoshi , Goshima Fumi , Daikoku Tohru , Nishiyama Yukihiro , Yoshikai Yasunobu
Published: April 2014

IL-2Rα-deficient (IL-2Rα−/−) mice exhibit an impaired activation-induced cell death for T cells and develop abnormal T cell activation with age. In our study, we found that IL-2Rα−/− mice at the age of 5 wk contained an increased number of CD44+CD69−CD8+ T cells in lymph nodes, which expressed a high intensity of IL-2Rβ and vigorously proliferated in response to a high dose of IL-15 or IL-2. The T cells produced a large amount of IFN-γ in response to IL-15 plus IL-12 in a TCR-independent bystander manner. When IL-2Rα−/− mice were inoculated i.p. with HSV type 2 (HSV-2) 186 strain, they showed resistance to the infection accompanied by an increased level of serum IL-15. The depletion of CD8+ T cells by in vivo administration of anti-CD8 mAb rendered IL-2Rα−/− mice susceptible to HSV-2-induced lethality. These results suggest that memory-type CD8+ T cells play a novel role in the protection against HSV-2 infection in IL-2Rα−/− mice.
A Role for Parasite-Induced PGE2 in IL-10-Mediated Host Immunoregulation by Skin Stage Schistosomula ofSchistosoma mansoni
Authors: Ramaswamy Kalyanasundaram , Kumar Pawan , He Yi-Xun
Published: April 2014

Significant quantities of PGE2 were produced by cercariae of Schistosoma mansoni following incubation with linoleic acid, a free fatty acid found on the surface of the skin. Cyclooxygenase (COX) 2 inhibitors failed to block this PGE2 production, suggesting that a different biochemical pathway may be involved in the production of PGE2 by the parasite. In addition, the parasites were also able to induce PGE2 and IL-10 from human and mouse keratinocytes. Analysis of mouse skin during skin migratory phases of infection confirmed these in vitro observations. COX2 inhibitors blocked the parasite-induced PGE2 and IL-10 from keratinocytes. Further analysis of the parasite secretions showed that the PGE2/IL-10-inducing effect was associated with a fraction <30 kDa molecular size. Addition of this fraction or parasite-stimulated keratinocyte culture supernatant to Con A-stimulated spleen cells resulted in the suppression of cell proliferation. This effect could be blocked by anti-IL-10 treatment. In sharp contrast, attenuation of the parasites with γ-irradiation significantly abrogated their ability to induce PGE2 or IL-10 from skin cells. Significance of IL-10 in host immunoregulation by skin stage schistosomula of S. mansoni was further confirmed by using IL-10-deficient mice. In these mice the normal subdued cutaneous reaction to the parasite was absent. Instead, a prominent cellular reaction occurred around the parasite, and there was considerable delay in parasitic migration through the skin. Thus these results suggest a key role for parasite-induced PGE2 in IL-10-dependent down-regulation of host immune responses in the skin.
The Rel Family Member P50 Mediates Cytokine-Induced C-Reactive Protein Expression by a Novel Mechanism
Authors: Cha-Molstad Hyunjoo , Agrawal Alok , Zhang Dongxiao , Samols David , Kushner Irving
Published: April 2014

Transcription of C-reactive protein (CRP) in Hep 3B cells is induced by IL-6, acting through C/EBP isoforms and STAT3. IL-1β, which alone has no effect, greatly enhances IL-6-induced transcription by unknown mechanisms. Because IL-1β activates the NF-κB system, we explored the effects of overexpressed Rel family members on CRP expression. Unexpectedly, transactivation assays in transiently transfected Hep 3B cells showed p50 overexpression to markedly induce CRP transcription, acting in a region 3′ to −86. In the presence of overexpressed p50, IL-1β induced a 3-fold increase in CRP expression, and responses to IL-6 and to IL-6 plus IL-1β were 4-fold greater than seen in cells without p50 overexpression. In contrast, overexpressed p65 abolished CRP induction by p50 and by cytokines. EMSA studies demonstrated that recombinant p50 bound to a nonconsensus κB site overlapping the proximal C/EBP binding site on the CRP promoter. Mutation of a polypyrimidine tract in the p50-binding site inhibited the transactivating effect of cytokines. P50- but not p65-containing dimers were found in nuclei of Hep 3B cells 18 h after stimulation with IL-1β, when C/EBPβ is greatly activated, in the presence or absence of IL-6. These findings suggest that IL-1β induces nuclear translocation of p50-containing dimers and that p50 interacts with C/EBPβ activated by both IL-6 and IL-1β to induce CRP expression.
ATP Acts as an Agonist to Promote Stimulus-Induced Secretion of IL-1β and IL-18 in Human Blood
Authors: Perregaux David G. , McNiff Pat , Laliberte Ron , Conklyn Maryrose , Gabel Christopher A.
Published: April 2014

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1β, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1β, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X7 receptor involvement. Quantities of IL-1β generated by an individual donor’s blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-α levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1β and IL-18, and suggest that circulating blood monocytes constitutively express a P2X7-like receptor.
Protein Kinase C ζ Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages
Authors: Monick Martha M. , Carter A. Brent , Flaherty Dawn M. , Peterson Michael W. , Hunninghake Gary W.
Published: April 2014

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC ζ, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC ζ, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC ζ partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC ζ to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC ζ. PKC ζ has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC ζ/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC ζ activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC ζ.
In Situ Amplification of 5-Lipoxygenase and 5-Lipoxygenase-Activating Protein in Allergic Airway Inflammation and Inhibition by Leukotriene Blockade
Authors: Chu Shi-Jye , Tang Li-Ou , Watney Elizabeth , Chi Emil Y. , Henderson William R.
Published: April 2014

Leukotrienes are important mediators of the eosinophilic influx and mucus hypersecretion in the lungs in a murine model of asthma. We used in situ PCR in this model of human asthma to detect lung mRNA for 5-lipoxygenase (5-LO) and 5-LO-activating protein (FLAP), key proteins necessary for leukotriene synthesis. Lung tissue was obtained on day 28 from mice treated with i.p. (days 0 and 14) and intranasal (days 14, 25, 26, and 27) OVA or saline. After fixation, the tissue sections underwent protease- and RNase-free DNase digestion, before in situ RT-PCR using target-specific cDNA amplification. 5-LO and FLAP-specific mRNA was visualized by a digoxigenin detection system, and positive cells were analyzed by morphometry. 5-LO and FLAP-specific mRNA and protein were associated primarily with eosinophils and alveolar macrophages in the airways and pulmonary blood vessels in OVA-sensitized/challenged mice. 5-LO and FLAP protein expression increased on a per-cell basis in alveolar macrophages of OVA-treated mice compared with saline controls. Pulmonary blood vessel endothelial cells were also positive for 5-LO, FLAP mRNA, and protein. 5-LO inhibition significantly decreased 5-LO and FLAP-specific mRNA and protein expression in the lung inflammatory cells and endothelial cells. These studies demonstrate a marked increase in key 5-LO pathway proteins in the allergic lung inflammatory response and an important immunomodulatory effect of leukotriene blockade to decrease 5-LO and FLAP gene expression.
ICAM-1-Induced Expression of Proinflammatory Cytokines in Astrocytes: Involvement of Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways
Authors: Lee Sung Joong , Drabik Kathryn , Van Wagoner Nicholas J. , Lee Soojin , Choi Chulhee , Dong Yuanshu , Benveniste Etty N.
Published: April 2014

ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer’s disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1β and IL-1α protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1α and IL-1β expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.
In Vivo Roles of Integrins During Leukocyte Development and Traffic: Insights from the Analysis of Mice Chimeric for α5, αv, and α4 Integrins
Authors: Arroyo Alicia G. , Taverna Daniela , Whittaker Charles A. , Strauch Ulrike G. , Bader Bernhard L. , Rayburn Helen , Crowley Denise , Parker Christina M. , Hynes Richard O.
Published: April 2014

Mice chimeric for integrins α5, αV, or α4 were used to dissect the in vivo roles of these adhesion receptors during leukocyte development and traffic. No major defects were observed in the development of lymphocytes, monocytes, or granulocytes or in the traffic of lymphocytes to different lymphoid organs in the absence of α5 or αV integrins. However, in agreement with previous reports, the absence of α4 integrins produced major defects in development of lymphoid and myeloid lineages and a specific defect in homing of lymphocytes to Peyer’s patches. In contrast, the α4 integrin subunit is not essential for localization of T lymphocytes into intraepithelial and lamina propria compartments in the gut, whereas one of the partners of α4, the β7 chain, has been shown to be essential. However, α4-deficient T lymphocytes cannot migrate properly during the inflammatory response induced by thioglycolate injection into the peritoneum. Finally, in vitro proliferation and activation of lymphocytes deficient for α5, αV, or α4 integrins upon stimulation with different stimuli were similar to those seen in controls. These results show that integrins play distinct roles during in vivo leukocyte development and traffic.
Role of IL-18 in CD4+ T Lymphocyte Activation in Sarcoidosis
Authors: Greene Catherine M. , Meachery Gerard , Taggart Clifford C. , Rooney Cyril P. , Coakley Raymond , O’Neill Shane J. , McElvaney Noel G.
Published: April 2014

Sarcoidosis is a granulomatous disease of unknown etiology associated with the expansion of IL-2-producing activated CD4+ T lymphocytes. A number of factors including the recently described IL-18 have been implicated in IL-2 expression in vitro. We investigated the role of IL-18 in IL-2 expression in sarcoidosis. Eighteen individuals with sarcoidosis and 15 normal controls were studied. IL-18R expression and epithelial lining fluid (ELF) concentrations of IL-18 were significantly elevated in the sarcoid group (p = 0.0143 and 0.0024, respectively). Both AP1 and NF-κB, transcription factors that regulate IL-2 gene expression, were activated in vivo in sarcoid pulmonary CD4+ T lymphocytes. Transcription factor activity was not detected in pulmonary CD4+ T lymphocytes from normal controls or from peripheral blood CD4+ T lymphocytes from individuals with sarcoidosis, further evidence of compartmentalization of the lymphoproliferative process in this condition. We examined the effects of IL-18 on AP1 and NF-κB in Jurkat T cells in vitro. These effects were both time and dose dependent. Examination of transcription factor activation and IL-2 gene expression in Jurkat T cells revealed that sarcoid but not normal ELF activated AP1 and NF-κB, induced IL-2 gene transcription, and up-regulated IL-2 protein production. Addition of IL-18 to normal ELF also induced IL-2 mRNA accumulation, whereas correspondent depletion of IL-18 from sarcoid ELF using neutralizing Abs abrogated all of the effects. These data strongly implicate IL-18 in the pathogenesis of sarcoidosis via activation of AP1 and NF-κB, leading to enhanced IL-2 gene expression and IL-2 protein production and concomitant T cell activation.
Impaired Antibody Affinity Maturation Process Characterizes a Subset of Patients with Common Variable Immunodeficiency
Authors: Bonhomme D. , Hammarström L. , Webster D. , Chapel H. , Hermine O. , Le Deist F. , Lepage E. , Romeo P. H. , Levy Yves
Published: April 2014

Common variable immunodeficiency (CVID) is an heterogeneous syndrome characterized by decreased levels of serum Ig and recurrent bacterial infection. Here, we were interested to study whether a qualitative defect of the affinity Ab maturation process could be combined to the low level of serum Ig in a cohort of 38 CVID patients. For this, we designed a novel and rapid screening test for the detection of hypomutated V gene expressed by memory B cells. This test delineated a subset of 9/38 (23%) CVID patients with an abnormal pattern of Ig V gene mutation. The mean frequency of V gene mutation of this subset was significantly lower (1.74%) compared with other CVID patients (5.46%) and normal donors (6.5%) (p < 0.0001). The mean age of this subgroup was significantly higher than other hypogammaglobulinemic patients with normal levels of V gene mutation (p < 0.02), whereas no difference in the duration of symptoms was noted between the two groups. This suggests that hypomutation characterizes patients who began CVID late in life. Recently, it was shown that non-Ig sequences, such as the intronic BCL-6 gene, could be the target of the somatic hypermutation process in normal memory B cells. Our finding of a normal mutation frequency of the BCL-6 gene in two hypomutated CVID point to a defect of the Ig targeting of hypermutation machinery in these cases.
Telomerase Activity Is Increased and Telomere Length Shortened in T Cells from Blood of Patients with Atopic Dermatitis and Psoriasis
Authors: Wu Kaida , Higashi Naoyuki , Hansen Erik René , Lund Marianne , Bang Karen , Thestrup-Pedersen Kristian
Published: April 2014

We studied telomerase activity and telomere length in PBMC and purified CD4+ and CD8+ T cells from blood obtained from a total of 32 patients with atopic dermatitis, 16 patients with psoriasis, and 30 normal controls. The telomerase activity was significantly increased in PBMC from the patients compared with PBMC from normal donors. This increase was most pronounced in the subpopulation of CD4+ T cells, which were significantly above the activity of the CD8+ T cells in atopic dermatitis, psoriasis patients, and control persons. The telomere length was significantly reduced in all T cell subsets from both atopic dermatitis and psoriasis patients compared with normal individuals. Furthermore, the telomere length was found to be significantly shorter in CD4+ memory T cells compared with the CD4+ naive T cells, and both of the cell subsets from diseases were shown to be of significantly shorter telomere length than the same cell subsets from normal controls. No significant difference was observed between CD8+CD28− and CD8+CD28+ T cell populations in both diseases. However, the telomere length of CD8+CD28+ T cells from both diseases was significantly shorter than CD8+CD28+ T cell subsets from normal donors. In conclusion, the increased telomerase activity and shortened telomere length indicates that T lymphocytes in atopic dermatitis and psoriasis are chronically stimulated and have an increased cellular turnover in vivo.
Cutting Edge: Selective IL-18 Requirements for Induction of Compartmental IFN-γ Responses During Viral Infection
Authors: Pien Gary C. , Satoskar Abhay R. , Takeda Kiyoshi , Akira Shizuo , Biron Christine A.
Published: April 2014

Optimal protective effects for defense against infection require orchestration of immune responses spanning multiple host compartments and divergent local regulation at particular sites. During murine cytomegalovirus infections known to target spleen and liver, IL-12-induced IFN-γ from NK cells is crucial for resistance. However, the roles for IL-18 and/or IL-12 in regulating hepatic IFN-γ responses, as compared with systemic or splenic responses, have not been defined. In this report, mice genetically deficient in either IL-18 or IL-12p35 exhibited up to 95% reductions in systemic and splenic IFN-γ responses. Surprisingly, IFN-γ responses were preserved in the livers of IL-18-deficient, but not IL-12p35-deficient, mice. Cytokine requirements for host survival also differed. Under conditions where mice lacking IL-12p35 exhibited 100% mortality, those lacking IL-18 survived. Taken together, our results delineate contrasting compartmental requirements for IL-18 and suggest that preservation of local, hepatic IFN-γ production is critical for host defense during murine cytomegalovirus challenge.
T Regulatory Cells 1 Inhibit a Th2-Specific Response In Vivo
Authors: Cottrez Françoise , Hurst Steven D. , Coffman Robert L. , Groux Hervé
Published: April 2014

We recently described a new population of CD4+ regulatory T cells (Tr1) that inhibits proliferative responses of bystander T cells and prevents colitis induction in vivo through the secretion of IL-10. IL-10, which had been primarily described as a Th2-specific cytokine inhibiting Th1 responses, has displayed in several models a more general immune suppression on both types of effector T cell responses. Using an immediate hypersensitivity model in which BALB/c mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of OVA-specific Tr1 T cell clones to inhibit OVA-specific cytokines and Ab responses. In contrast to Th2 or Th1 T cell clones, transfer of Tr1 T cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses, whereas IgG1 and IgG2a synthesis were not affected. This specific inhibition was mediated in part through IL-10 secretion as anti-IL-10 receptor Abs treatment reverted the inhibitory effect of Tr1 T cell clones. Although specifically targeted to IgE responses, Tr1 clones’ inhibitory effects were more profound as they affected Ag-specific Th2 cell priming both in term of proliferative responses and cytokine secretion. These results suggest that regulatory T cells may play a fundamental role in maintaining the balance of the immune system to prevent allergic disorders.
The NFAT-Related Protein NFATL1 (TonEBP/NFAT5) Is Induced Upon T Cell Activation in a Calcineurin-Dependent Manner
Authors: Trama Jason , Lu Qingjun , Hawley Robert G. , Ho Steffan N.
Published: April 2014

NFAT DNA binding complexes regulate programs of cellular activation and differentiation by translating receptor-dependent signaling events into specific transcriptional responses. NFAT proteins, originally defined as calcium/calcineurin-dependent regulators of cytokine gene transcription in T lymphocytes, are expressed in many different cell types and represent critical signaling intermediates that mediate an increasingly wide spectrum of biologic responses. Recent studies have identified a novel protein containing a region of similarity to the NFAT DNA binding domain. Here we demonstrate that this protein, designated NFATL1 (also known as tonicity enhancer binding protein and NFAT5) is expressed at high levels in the thymus but is undetectable in mature lymphocytes. However, NFATL1 can be induced in both primary quiescent T lymphocytes and differentiated Th1 and Th2 cell populations upon mitogen- or Ag receptor-dependent activation. The induction of NFATL1 protein, as well as NFATL1-dependent transcription, is inhibited by cyclosporin A and FK506, and expression of constitutively active calcineurin induces NFATL1-dependent transcription. Overexpression of NFATc1 and inhibition of NFATc activity through the use of a dominant negative NFATc1 protein have no affect on NFATL1-dependent transcription, indicating that NFATc proteins do not play a role in the calcineurin-dependent induction of NFATL1. Interestingly, induction of NFATL1 by a hyperosmotic stimulus is not blocked by the inhibition of calcineurin. Moreover, osmotic stress response genes such as aldose reductase are not induced upon T cell activation. Thus inducible expression of NFATL1 represents a mechanism by which receptor-dependent signals as well as osmotic stress signals are translated into transcriptional responses that regulate cell function.
Host T Cells Resist Graft-Versus-Host Disease Mediated by Donor Leukocyte Infusions
Authors: Blazar Bruce R. , Lees Christopher J. , Martin Paul J. , Noelle Randolph J. , Kwon Byoung , Murphy William , Taylor Patricia A.
Published: April 2014

Delayed lymphocyte infusions (DLIs) are used to treat relapse occurring post bone marrow transplantation (BMT) and to increase the donor chimerism in recipients receiving nonmyeloablative conditioning. As compared with donor lymphocytes given early post-BMT, DLIs are associated with a reduced risk of graft-vs-host disease (GVHD). The mechanism(s) responsible for such resistance have remained incompletely defined. We now have observed that host T cells present 3 wk after lethal total body irradiation, at the time of DLI, contribute to DLI-GVHD resistance. The infusion of donor splenocytes on day 0, a time when host bone marrow (BM)-derived T cells are absent, results in greater expansion than later post-BMT when host and donor BM-derived T cells coexist. Selective depletion of host T cells with anti-Thy1 allelic mAb increased the GVHD risk of DLI, indicating that a Thy1+ host T cell regulated DLI-GVHD lethality. The conditions by which host T cells are required for optimal DLI resistance were determined. Recipients unable to express CD28 or 4-1BB were as susceptible to DLI-GVHD as anti-Thy1 allelic mAb-treated recipients, indicating that CD28 and 4-1BB are critical to DLI-GVHD resistance. Recipients deficient in both perforin and Fas ligand but not individually were highly susceptible to DLI-GVHD. Recipients that cannot produce IFN-γ were more susceptible to DLI-GVHD, whereas those deficient in IL-12 or p55 TNFRI were not. Collectively, these data indicate that host T cells, which are capable of generating antidonor CTL effector cells, are responsible for the impaired ability of DLI to induce GVHD. These same mechanisms may limit the efficacy of DLI in cancer therapy under some conditions.
IL-18 Receptors, Their Role in Ligand Binding and Function: Anti-IL-1RAcPL Antibody, a Potent Antagonist of IL-18
Authors: Debets Reno , Timans Jackie C. , Churakowa Tatyana , Zurawski Sandra , de Waal Malefyt Rene , Moore Kevin W. , Abrams John S. , O’Garra Anne , Bazan J. Fernando , Kastelein Robert A.
Published: April 2014

IL-18 is critical in eliciting IFN-γ production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (Kd of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-κB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-γ by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.
Potent Induction of α(1,3)-Fucosyltransferase VII in Activated CD4+ T Cells by TGF-β1 Through a p38 Mitogen-Activated Protein Kinase-Dependent Pathway
Authors: Wagers Amy J. , Kansas Geoffrey S.
Published: April 2014

Homing of effector T cells to sites of inflammation, particularly in the skin, is dependent on T cell expression of ligands for the endothelial selectins. Underlying expression of these ligands is the expression of α(1,3)-fucosyltransferase VII (FucT-VII), a FucT essential for biosynthesis of selectin ligands. FucT-VII is sharply induced in activated T cells by IL-12, but cytokines other than IL-12 that induce FucT-VII and functional selectin ligands have not been identified, and are likely to be important in homing of T cells to other selectin-dependent sites. Screening of a number of cytokines known to be active on T cells identified only TGF-β1 as able to up-regulate FucT-VII mRNA levels and selectin ligands on activated CD4 T cells. The sharp increase in FucT-VII induced by TGF-β1 in activated T cells was completely blocked by pharmacologic inhibition of p38 mitogen-activated protein kinase, but was unaffected by mitogen-activated protein/extracellular signal-related kinase kinase inhibitors. The selective ability of TGF-β1 to induce selectin ligands on activated T cells is likely important for T cell homing to the gut, which is a strongly selectin-dependent site, and correlates with the ability of TGF-β1 to coordinately induce other gut-associated homing pathways.
Regulation of Cell Surface Expression of CTLA-4 by Secretion of CTLA-4-Containing Lysosomes Upon Activation of CD4+ T Cells
Authors: Iida Tomohiko , Ohno Hiroshi , Nakaseko Chiaki , Sakuma Machie , Takeda-Ezaki Mitsue , Arase Hisashi , Kominami Eiki , Fujisawa Takehiko , Saito Takashi
Published: April 2014

CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of CTLA-4 with the clathrin-associated adaptor complex AP-2 induces internalization of CTLA-4 and keeps the surface expression low. However, the mechanism to induce high expression on the cell surface upon stimulation has not yet been clarified. To address this, we investigated the intracellular dynamics of CTLA-4 by analyzing its localization and trafficking in wild-type and mutant CTLA-4-transfected Th1 clones. CTLA-4 is accumulated in intracellular granules, which we identified as lysosomes. CTLA-4 is degraded in lysosomes in a short period, and the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression. Upon TCR stimulation, CTLA-4-containing lysosomes are secreted as proven by the secretion of cathepsin D and β-hexosaminidase in parallel with the increase of surface expression of CTLA-4 and lysosomal glycoprotein 85, a lysosomal marker. These results suggest that the cell surface expression of CTLA-4 is up-regulated upon stimulation by utilizing a mechanism of secretory lysosomes in CD4+T cells.
The Role of Thymus-Expressed Chemokine and Its Receptor CCR9 on Lymphocytes in the Regional Specialization of the Mucosal Immune System
Authors: Papadakis Konstantinos A. , Prehn John , Nelson Viera , Cheng Lorna , Binder Scott W. , Ponath Paul D. , Andrew David P. , Targan Stephan R.
Published: April 2014

Chemokines play an important role in the migration of leukocytes at sites of inflammation, and some constitutively expressed chemokines may direct lymphocyte trafficking within lymphoid organs and peripheral tissues. Thymus-expressed chemokine (TECK or Ckβ-15/CCL25), which signals through the chemokine receptor CCR9, is constitutively expressed in the thymus and small intestine but not colon, and chemoattracts a small fraction of PBLs that coexpress the integrin α4β7. Here we show that TECK is expressed in the human small bowel but not colon by endothelial cells and a subset of cells in intestinal crypts and lamina propria. CCR9 is expressed in the majority of freshly isolated small bowel lamina propria mononuclear cells (LPMC) and at significantly higher levels compared with colonic LPMC or PBL. TECK was selectively chemotactic for small bowel but not colonic LPMC in vitro. The TECK-induced chemotaxis was sensitive to pertussis toxin and partially inhibited by Abs to CCR9. TECK attracts predominantly the T cell fraction of small bowel LPMC, whereas sorted CD3+CCR9+ and CD3+CCR9− lymphocytes produce similar Th1 or Th2 cytokines at the single cell level. Collectively, our data suggest that the selective expression of TECK in the small bowel underlie the homing of CCR9+ intestinal memory T cells to the small bowel rather than to the colon. This regional specialization implies a segregation of small intestinal from colonic immune responses.