bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of H2 Histamine Receptor-Mediated Cation Channel Opening by Protein Kinase C in Human Promyelocytic Cells
Authors: Suh Byung-Chang , Lee Hyun , Jun Dong-Jae , Chun Jang-Soo , Lee Jong-Hee , Kim Kyong-Tai
Published: April 2014

Histamine, through H2 receptors, triggers a prominent rise in intracellular free Ca2+ concentration ([Ca2+]i) in addition to an elevation of cAMP level in HL-60 promyelocytes. Here we show that the histamine-induced [Ca2+]i rise was due to influx of Ca2+ from the extracellular space, probably through nonselective cation channels, as incubation of the cells with SKF 96365 abolished the histamine-induced [Ca2+]i rise, Na+ influx, and membrane depolarization. The Ca2+ influx was specifically inhibited by pretreatment of the cells with PMA or extracellular ATP with 50% inhibitory concentrations of 0.12 ± 0.03 nM and 185 ± 17 μM, respectively. Western blot analysis of protein kinase C (PKC) isoforms revealed that PMA (≤1 nM) and ATP (300 μM) caused selective translocation of PKC-δ to the particulate/membrane fraction. Costimulation of the cells with histamine and SKF 96365 partially reduced histamine-induced granulocytic differentiation, which was evaluated by looking at the extent of fMet-Leu-Phe-induced [Ca2+]i rise and superoxide generation. In conclusion, nonselective cation channels are opened by stimulation of the H2 receptor, and the channels are at least in part involved in the induction of histamine-mediated differentiation processes. Both effects of histamine were selectively inhibited probably by the δ isoform of PKC in HL-60 cells.
The Generation of Both T Killer and Th Cell Clones Specific for the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells
Authors: Meyer zum Büschenfelde Christian , Metzger Jochen , Hermann Christine , Nicklisch Nicole , Peschel Christian , Bernhard Helga
Published: April 2014

Induction of antitumor immunity involves the presence of both CD8+ CTLs and CD4+ Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34+ hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8+ CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4+ Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4+ T cells were cloned that released IFN-γ upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8+ and CD4+ T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.
Apoptosis and Altered Dendritic Cell Homeostasis in Lupus Nephritis Are Limited by Anti-CD154 Treatment
Authors: Kalled Susan L. , Cutler Anne H. , Burkly Linda C.
Published: April 2014

Autoimmunity results from a failure in central and/or peripheral tolerance; however, the events that initiate and maintain this dysfunction remain unclear. To better understand the mediators involved in autoimmunity, we investigated the cellular mechanisms maintaining disease in the (SWR × NZB)F1 (SNF1) mouse model of systemic lupus erythematosus. Previously, we have shown that autoimmunity in this model is dependent on CD40-CD154 interactions. Herein, our studies reveal that the severity of disease in SNF1 mice correlates with a marked increase in the frequency of apoptotic splenocytes, including a higher proportion of apoptotic dendritic cells (DC) in vivo. In addition, we demonstrate a significant disease-related increase in the absolute number of splenic CD11chigh DC. The increased DC number appears to be attributable to DC proliferation and enhanced migration to the spleen, most likely induced by elevated splenic expression of secondary lymphoid chemokine. Importantly, these imbalances in apoptosis, secondary lymphoid chemokine expression, and DC homeostasis were reduced or normalized by anti-CD154 treatment. Thus, our data demonstrate CD154-dependent regulation of apoptosis and DC homeostasis in mice with lupus-like autoimmune disease. We suggest that these mechanisms comprise an autostimulatory loop, maintaining the cascade of autoimmunity by DC presentation of self-Ags derived from apoptotic cells and CD154-mediated costimulation.
IL-13 and IFN-γ: Interactions in Lung Inflammation
Authors: Ford Jean G. , Rennick Donna , Donaldson Debra D. , Venkayya Rajeev , McArthur Cliff , Hansell Elisabeth , Kurup Viswanath P. , Warnock Martha , Grünig Gabriele
Published: April 2014

Chronic inflammatory diseases of the lungs, such as asthma, are frequently associated with mixed (Th2 and Th1) T cell responses. We examined the impact of critical Th1 and Th2 cytokines, IFN-γ and IL-13, on the responses in the lungs. In a mouse model of airway inflammation induced by mixed T cell responses, the number of Th1 (IFN-γ-positive) cells was found to be negatively correlated with airway hyperreactivity. In these mice, blockade of IL-13 partially inhibited airway hyperreactivity and goblet cell hyperplasia but not inflammation. In contrast, in mice that responded with a polarized Th2 response to the same Ag, blockade of IL-13 inhibited airway hyperreactivity, goblet cell hyperplasia, and airway inflammation. These results indicated that the presence of IFN-γ would modulate the effects of IL-13 in the lungs. To test this hypothesis, wild-type mice were given recombinant cytokines intranasally. IFN-γ inhibited IL-13-induced goblet cell hyperplasia and airway eosinophilia. At the same time, IFN-γ and IL-13 potentiated each other’s effects. In the airways of mice given IL-13 and IFN-γ, levels of IL-6 were increased as well as numbers of NK cells and of CD11c-positive cells expressing MHC class II and high levels of CD86. In conclusion, IFN-γ has double-sided effects (inhibiting some, potentiating others) on IL-13-induced changes in the lungs. This may be the reason for the ambiguous role of Th1 responses on Th2 response-induced lung injury.
Mutation in the Class II trans-Activator Leading to a Mild Immunodeficiency
Authors: Wiszniewski Wojciech , Fondaneche Marie-Claude , Le Deist Françoise , Kanariou Maria , Selz Françoise , Brousse Nicole , Steimle Viktor , Barbieri Giovanna , Alcaide-Loridan Catherine , Charron Dominique , Fischer Alain , Lisowska-Grospierre Barbara
Published: April 2014

The expression of MHC class II molecules is essential for all Ag-dependent immune functions and is regulated at the transcriptional level. Four trans-acting proteins control the coordinate expression of MHC class II molecules: class II trans-activator (CIITA), regulatory factor binding to the X box (RFX)-associated protein; RFX protein containing ankyrin repeats, and RFX5. In humans, defects in these genes result in MHC class II expression deficiency and cause combined immunodeficiency. Most patients with this deficiency suffer from severe recurrent infections that frequently lead to death during early childhood. We investigated three sisters, now ages 21, 22, and 24 years, in whom MHC-II deficiency was detected. Even though the eldest sibling was asymptomatic and the other two had only mild immunodeficiency, none of the three class II isotypes was expressed on T cell blasts, fibroblasts, EBV B cell lines, or epidermal dendritic cells. Residual HLA-II expression was detected in fresh PBMC. Somatic complementation identified the disease as CIITA deficiency. A homozygous T1524C (L469P) substitution was found in the coding region of the CIITA cDNA and was shown to be responsible for the defect in MHC-II expression. This missense mutation prevents the normal functioning of MHC-II but does not lead to the nuclear exclusion of the L469P CIITA. Transfection experiments demonstrated that the CIITA L469P mutant had residual MHC class II trans activation activity, which might explain the unusual clinical course of the patients studied. This study shows that an attenuated clinical phenotype or an asymptomatic clinical course can be observed in patients despite a profound defect in the expression of MHC class II genes. The frequency of the inherited MHC class II deficiency might thus be underestimated.
AChlamydia pneumoniae-Specific Peptide Induces Experimental Autoimmune Encephalomyelitis in Rats
Authors: Lenz Derek C. , Lu Lin , Conant Stephanie B. , Wolf Norbert A. , Gérard Hervé C. , Whittum-Hudson Judith A. , Hudson Alan P. , Swanborg Robert H.
Published: April 2014

It has been reported recently that the bacterial respiratory pathogen Chlamydia pneumoniae is present in the cerebrospinal fluid of a subset of multiple sclerosis (MS) patients. However, it is not known whether this organism is a causative agent of MS, or merely an opportunistic pathogen that takes advantage of a disease process initiated by some other means. We report identification of a 20-mer peptide from a protein specific to C. pneumoniae which shares a 7-aa motif with a critical epitope of myelin basic protein, a major CNS Ag targeted by the autoimmune response in MS. This bacterial peptide induces a Th1 response accompanied by severe clinical and histological experimental autoimmune encephalomyelitis in Lewis rats, a condition closely reflective of many aspects of MS. Studies with peptide analogues suggest that different populations of encephalitogenic T cells are activated by the C. pneumoniae and myelin basic protein Ags. Mild experimental autoimmune encephalomyelitis was also observed when rats were immunized with sonicated C. pneumoniae in CFA.
B7 Interactions with CD28 and CTLA-4 Control Tolerance or Induction of Mucosal Inflammation in Chronic Experimental Colitis
Authors: Liu Zhanju , Geboes Karel , Hellings Peter , Maerten Philippe , Heremans Hubertine , Vandenberghe Peter , Boon Louis , van Kooten Peter , Rutgeerts Paul , Ceuppens Jan L.
Published: April 2014

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RBhighCD4+ T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RBhighCD4+ cells from CD28−/− mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RBhighCD4+ cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-γ by lamina propria CD4+ cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25+CD4+ cells from CD28−/− mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RBhigh cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.
Cutting Edge: Selective Usage of Chemokine Receptors by Plasmacytoid Dendritic Cells
Authors: Penna Giuseppe , Sozzani Silvano , Adorini Luciano
Published: April 2014

The existence of dendritic cell (DC) subsets is firmly established, but their trafficking properties are virtually unknown. In this study, we show that myeloid (M-DCs) and plasmacytoid (P-DCs) DCs isolated from human blood differ widely in the capacity to migrate to chemotactic stimuli. The pattern of chemokine receptors expressed by blood M-DCs and P-DCs, with the exception of CCR7, is similar. However, most chemokine receptors of P-DCs, in particular those specific for inflammatory chemokines and classical chemotactic agonists, are not functional in circulating cells. Following maturation induced by CD40 ligation, the receptors for inflammatory chemokines are down-regulated, and CCR7 on P-DCs becomes coupled to migration. The drastically impaired capacity of blood P-DCs to migrate in response to inflammatory chemotactic signals contrasts with the response to lymph node-homing chemokines, indicating a propensity to migrate to secondary lymphoid organs rather than to sites of inflammation.
Essential Role of RelB in Germinal Center and Marginal Zone Formation and Proper Expression of Homing Chemokines
Authors: Weih Debra S. , Yilmaz Z. Buket , Weih Falk
Published: April 2014

High levels of the Rel/NF-κB family member RelB are restricted to specific regions of thymus, lymph nodes, and Peyer’s patches. In spleen, RelB is expressed in periarteriolar lymphatic sheaths, germinal centers (GCs), and the marginal zone (MZ). In this study, we report that RelB-deficient (relB−/−) mice, in contrast to nfkb1−/−, but similar to nfkb2−/− mice, are unable to form GCs and follicular dendritic cell networks upon Ag challenge in the spleen. RelB is also required for normal organization of the MZ and its population by macrophages and B cells. Reciprocal bone marrow transfers demonstrate that RelB expression in radiation-resistant stromal cells, but not in bone marrow-derived hemopoietic cells, is required for proper formation of GCs, follicular dendritic cell networks, and MZ structures. However, the generation of MZ B cells requires RelB in hemopoietic cells. Expression of TNF ligand/receptor family members is only moderately altered in relB−/− splenocytes. In contrast, expression of homing chemokines is strongly reduced in relB−/− spleen with particularly low mRNA levels of the chemokine B lymphocyte chemoattractant. Our data indicate that activation of p52-RelB heterodimers in stromal cells downstream of TNF/lymphotoxin is required for normal expression of homing chemokines and proper development of spleen microarchitecture.
Development of the Thymus Requires Signaling Through the Fibroblast Growth Factor Receptor R2-IIIb
Authors: Revest Jean-Michel , Suniara Ravinder K. , Kerr Karen , Owen John J. T. , Dickson Clive
Published: April 2014

Mice deficient for fibroblast growth factor (Fgf)R2-IIIb show a block in thymic growth after embryonic day 12.5, a stage that just precedes its detection in thymic epithelial cells. Fgf7 and Fgf10, the main ligands for FgfR2-IIIb, are expressed in the mesenchyme surrounding the thymic epithelial primordium, and Fgf10-deficient mice also exhibit impaired thymic growth. Hence, Fgf signaling is essential for thymic epithelial proliferation. In addition to the proliferative block, most thymic epithelial cells fail to progress from an immature cytokeratin 5-positive to a cytokeratin 5-negative phenotype. Nevertheless, sufficient epithelial cell differentiation occurs in the severely hypoplastic thymus to allow the development of CD4/CD8-double-positive thymocytes and a very small number of single-positive thymocytes expressing TCRs.
Human Recent Thymic Emigrants–Identification, Expansion, And Survival Characteristics
Authors: Hassan Jaythoon , Reen Denis J.
Published: April 2014

This study shows that, in humans at birth, circulating T cells represent recent thymic emigrants (RTEs) as reflected in their high level of expression of TCR excision circles. RTEs express “thymocyte-like” characteristics with regard to rapid rate of apoptosis. In the presence of common γ-chain cytokines, in particular IL-7, they show enhanced potential to survive, entry into cell cycle, and proliferation. Although common γ-chain cytokines were also potent antiapoptotic stimuli for mature adult-derived naive CD4+CD45RA+ T cells, these cells were refractory to IL-7-induced expansion in vitro. RTEs cultured with IL-7 could not reinduce recombination-activating gene-2 gene expression in vitro. These data suggest that postthymic naive T cells in the periphery during early life are at a unique stage in ontogeny as RTEs, during which they can undergo homeostatic regulation including expansion and survival in an Ag-independent manner while maintaining their preselected TCR repertoire.
Adaptive Tolerance of CD4+ T Cells In Vivo: Multiple Thresholds in Response to a Constant Level of Antigen Presentation
Authors: Tanchot Corinne , Barber Daniel L. , Chiodetti Lynda , Schwartz Ronald H.
Published: April 2014

The in vivo T cell response to persistent Ag contains a hyporesponsive phase following an initial expansion and subsequent partial deletion of the responding cells. The mechanism(s) responsible for this tolerance process is poorly understood. In this study, we describe a new paired transgenic model (TCR and Ag), which within 7–14 days produces 20–40 million hyporesponsive T cells. This state is characterized by an 85–95% reduction in all cytokine production, an impairment of re-expression of CD25 and CD69, and a desensitization of the proliferative response to Ag. TCR levels were normal, and in vivo mixing experiments showed no evidence for active suppression. The hyporesponsiveness partially dissipated without proliferation when the cells were transferred into a non-Ag-bearing host. If the second host expressed Ag, the T cells initially regained responsiveness, but then slowly entered an even deeper state of tolerance characterized by an additional 7- to 10-fold lowering of cytokine production and a greater desensitization of proliferation. Surprisingly, this readaptation took place with the same level of Ag presentation, suggesting that other parameters can influence the tolerance threshold. Both the readjustment in sensitivity and the reversal without Ag convincingly demonstrate for the first time a truly adaptive tolerance process in CD4+ T cells in vivo.
Signaling Alterations in Activation-Induced Nonresponsive CD8 T Cells
Authors: Tham Ee Loon , Mescher Matthew F.
Published: April 2014

Costimulation-dependent production and autocrine use of IL-2 by activated CD8 T cells results in initial clonal expansion, but this is transient. The cells quickly become anergic, unable to produce IL-2 in response to Ag and costimulation, irrespective of the form of costimulation. This activation-induced non-responsiveness (AINR) differs from “classical” anergy in that it results despite the cells receiving both signal 1 and signal 2. AINR cells can still proliferate in response to exogenous IL-2, but can no longer produce it. Other TCR-mediated events including cytolytic function and IFN-γ production are not affected in the AINR state. To characterize the mechanism(s) responsible for lack of IL-2 production in CD8 T cells in the AINR state, microspheres bearing immobilized anti-TCR Abs or peptide-MHC complexes, B7-1, and ICAM-1 were used to provide well-defined stimuli to the cells. Comparison of normal and AINR cells revealed that in AINR cells extracellular signal-regulated kinase (ERK) is upregulated more transiently, Janus kinase activation is substantially reduced, and activation of p38 is eliminated. PMA and ionomycin restored proliferation and IL-2 production in AINR cells, indicating a signaling defect upstream of Ras and protein kinase C. Inhibitors of ERK (PD98059) and of p38 kinase (SB202190) blocked IL-2 mRNA expression and proliferation of both peptide-MHC/B7-1/ICAM-1-stimulated normal cells and PMA/ionomycin-stimulated AINR cells. Together these results demonstrate that activation of at least ERK and p38 is essential for IL-2 production by CD8 T cells and that up-regulation of these mitogen-activated protein kinases, along with Janus kinase, is defective in AINR cells.
Nicotinamide Adenine Dinucleotide (NAD) and Its Metabolites Inhibit T Lymphocyte Proliferation: Role of Cell Surface NAD Glycohydrolase and Pyrophosphatase Activities
Authors: Bortell Rita , Moss Joel , McKenna Robert C. , Rigby Mark R. , Niedzwiecki Dena , Stevens Linda A. , Patton Walter A. , Mordes John P. , Greiner Dale L. , Rossini Aldo A.
Published: April 2014

The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2− T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.
Testosterone Acts Directly on CD4+ T Lymphocytes to Increase IL-10 Production
Authors: Liva Stephanie M. , Voskuhl Rhonda R.
Published: April 2014

Males are less susceptible than females to experimental autoimmune encephalomyelitis and many other autoimmune diseases. Gender differences in cytokine production have been observed in splenocytes of experimental autoimmune encephalomyelitis mice stimulated with myelin proteins and may underlie gender differences in susceptibility. As these differences should not be limited to responses specific for myelin proteins, gender differences in cytokine production upon stimulation with Ab to CD3 were examined, and the mechanisms were delineated. Splenocytes from male mice stimulated with Ab to CD3 produced more IL-10 and IL-4 and less IL-12 than those from female mice. Furthermore, splenocytes from dihydrotestosterone (DHT)-treated female mice produced more IL-10 and less IL-12 than those from placebo-treated female mice, whereas there was no difference in IL-4. IL-12 knockout mice were then used to determine whether changes in IL-10 production were mediated directly by testosterone vs indirectly by changes in IL-12. The results of these experiments favored the first hypothesis, because DHT treatment of female IL-12 knockout mice increased IL-10 production. To begin to delineate the mechanism by which DHT may be acting, the cellular source of IL-10 was determined. At both the RNA and protein levels, IL-10 was produced primarily by CD4+ T lymphocytes. CD4+ T lymphocytes were then shown to express the androgen receptor, raising the possibility that testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production. In vitro experiments demonstrated increased IL-10 production following treatment of CD4+ T lymphocytes with DHT. Thus, testosterone can act directly via androgen receptors on CD4+ T lymphocytes to increase IL-10 gene expression.
Adenosine 3′,5′-Cyclic Monophosphate (cAMP)-Dependent Inhibition of IL-5 from Human T Lymphocytes Is Not Mediated by the cAMP-Dependent Protein Kinase A
Authors: Staples Karl J. , Bergmann Martin , Tomita Katsuyuki , Houslay Miles D. , McPhee Ian , Barnes Peter J. , Giembycz Mark A. , Newton Robert
Published: April 2014

IL-5 is implicated in the pathogenesis of asthma and is predominantly released from T lymphocytes of the Th2 phenotype. In anti-CD3 plus anti-CD28-stimulated PBMC, albuterol, isoproterenol, rolipram, PGE2, forskolin, cholera toxin, and the cAMP analog, 8-bromoadenosine cAMP (8-Br-cAMP) all inhibited the release of IL-5 and lymphocyte proliferation. Although all of the above compounds share the ability to increase intracellular cAMP levels and activate protein kinase (PK) A, the PKA inhibitor H-89 failed to ablate the inhibition of IL-5 production mediated by 8-Br-cAMP, rolipram, forskolin, or PGE2. Similarly, H-89 had no effect on the cAMP-mediated inhibition of lymphocyte proliferation. Significantly, these observations occurred at a concentration of H-89 (3 μM) that inhibited both PKA activity and CREB phosphorylation in intact cells. Additional studies showed that the PKA inhibitors H-8, 8-(4-chlorophenylthio) adenosine-3′,5′-cyclic monophosphorothioate Rp isomer, and a myristolated PKA inhibitor peptide also failed to block the 8-Br-cAMP-mediated inhibition of IL-5 release from PBMC. Likewise, a role for PKG was considered unlikely because both activators and inhibitors of this enzyme had no effect on IL-5 release. Western blotting identified Rap1, a downstream target of the cAMP-binding proteins, exchange protein directly activated by cAMP/cAMP-guanine nucleotide exchange factors 1 and 2, in PBMC. However, Rap1 activation assays revealed that this pathway is also unlikely to be involved in the cAMP-mediated inhibition of IL-5. Taken together, these results indicate that cAMP-elevating agents inhibit IL-5 release from PBMC by a novel cAMP-dependent mechanism that does not involve the activation of PKA.
Distinct Control of the Frequency and Allelic Exclusion of the Vβ Gene Rearrangement at the TCRβ Locus
Authors: Sieh Ping , Chen Jianzhu
Published: April 2014

Ag receptor gene loci contain many V gene segments, each of which is recombined and expressed at a different frequency and is subject to allelic exclusion. To probe the parameters that mediate the different levels of regulation of V gene rearrangement, a Vβ gene segment together with 3.6-kb 5′ and 0.7-kb 3′ flanking sequences was inserted 6.8 kb upstream of the Dβ1 gene segment in the murine TCRβ locus. Despite its proximity to the Dβ gene segments and the Eβ enhancer, the inserted Vβ segment underwent VDJ recombination at the same frequency as the natural copy located 470 kb upstream. However, the inserted Vβ segment was no longer under allelic exclusion control as it recombined at a similar frequency in the presence of a TCRβ transgene. These results suggest that while the inserted fragment contains the necessary cis-regulatory elements for determining the frequency of Vβ rearrangement, additional cis-regulatory elements are required for mediating Vβ allelic exclusion. Interestingly, most of the inserted Vβ rearrangements were not transcribed and expressed in the presence of a TCRβ transgene, suggesting that TCRβ allelic exclusion can also be achieved by blocking the transcription of the rearranged gene segments. These findings provide strong evidence for distinct control of the frequency and allelic exclusion of Vβ gene rearrangement.
SCID in Jack Russell Terriers: A New Animal Model of DNA-PKcs Deficiency
Authors: Meek Katheryn , Kienker Laura , Dallas Clarissa , Wang Wei , Dark Michael J. , Venta Patrick J. , Huie Maryann L. , Hirschhorn Rochelle , Bell Tom
Published: April 2014

We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in “leaky” coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.
Characterization of the Active Sites in Decay-Accelerating Factor
Authors: Kuttner-Kondo Lisa A. , Mitchell Lynne , Hourcade Dennis E. , Medof M. Edward
Published: April 2014

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2–4). Previous modeling on the nuclear magnetic resonance structure of CCP15–16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2–3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2–3 of DAF were oriented according to the crystal structure of CCP1–2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.
Human Cathepsin W, a Cysteine Protease Predominantly Expressed in NK Cells, Is Mainly Localized in the Endoplasmic Reticulum
Authors: Wex Thomas , Bühling Frank , Wex Heike , Günther Dagmar , Malfertheiner Peter , Weber Ekkehard , Brömme Dieter
Published: April 2014

Human cathepsin W (also called lymphopain) is a recently described papain-like cysteine protease of unknown function whose gene expression was found to be restricted to cytotoxic cells. Here we demonstrate that cathepsin W is expressed predominantly in NK cells and, to a lesser extent, in CTLs. Quantitative RT-PCR revealed that NK cells contained ∼21 times more cathepsin W transcript than CTLs. The predominant expression of cathepsin W in NK cells was further confirmed by Western blot analysis and immunohistochemistry. IL-2-mediated stimulation of NK cells and CTLs revealed a stronger up-regulation of the cathepsin W gene and protein expression in NK cells (7-fold) than in CTLs (2-fold). Transfection experiments of HeLa cells and biochemical analyses revealed that cathepsin W is exclusively “high mannose-type” glycosylated and is mainly targeted to the endoplasmic reticulum (ER). Interestingly, the ER localization of cathepsin W was also found in NK cells, in which colocalization studies revealed an overlapping staining of cathepsin W and Con A, an ER-specific lectin. Furthermore, subcellular fractionation of cathepsin W-expressing cells confirmed the ER localization and showed that cathepsin W is membrane associated. Based on the results of this study, cathepsin W might represent a putative component of the ER-resident proteolytic machinery. The constitutive expression in NK cells and the stronger up-regulation of cathepsin W by IL-2 in NK cells than CTLs suggest that cathepsin W is not just a marker of cytotoxic cells but is, rather, specifically expressed in NK cells.
Mutation of a Single Conserved Residue in VH Complementarity-Determining Region 2 Results in a Severe Ig Secretion Defect
Authors: Wiens Gregory D. , Lekkerkerker Annemarie , Veltman Imke , Rittenberg Marvin B.
Published: April 2014

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51→Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1–3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51→Arg or Ile51→Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51→Arg or Ile51→Lys mutants were partially rescued when expressed with the J558L λ1 L chain. These results represent the first demonstration that single somatic mutations in VH residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.
Functional Transitions in Macrophages During In Vivo Infection withMycobacterium bovisBacillus Calmette-Guérin
Authors: Hamerman Jessica A. , Aderem Alan
Published: April 2014

Macrophage activation during the immune response to intracellular bacteria is critical for resolution of the infection. We have investigated the pathway of macrophage activation during murine Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Three distinct phenotypes of macrophages were identified and compared: resident peritoneal macrophages, day 2 postinfection macrophages, and 12-day postinfection macrophages. Compared with resident peritoneal macrophages, day 2 BCG macrophages expressed intermediate levels of the cell surface receptors Mac1 and F4/80 and low levels of MHC class II molecules. These cells were highly phagocytic and produced large amounts of mRNA encoding the chemokine IP-10. In addition, day 2 BCG macrophages did not generate reactive nitrogen intermediates, though they were primed to do so, and did not have increased levels of TNF-α mRNA. Blockade of monocyte influx into the peritoneal cavity using Abs to platelet endothelial cell adhesion molecule 1 had no effect on the appearance of day 2 BCG macrophages, suggesting this cell can differentiate from resident peritoneal macrophages. In contrast to day 2 BCG macrophages, day 12 BCG macrophages were poorly phagocytic, but produced high levels of reactive nitrogen intermediates, IP-10 and TNF-α mRNA, and class II MHC molecules. We propose that day 2 BCG macrophages are specialized for phagocytic uptake of pathogens from the extracellular space, whereas day 12 BCG macrophages are specialized for killing of the internalized pathogens. This functional transition during activation is reminiscent of that seen during maturation/activation of the related dendritic cell lineage induced by bacterial or inflammatory stimuli.
Targeting of Pseudomonas aeruginosa in the Bloodstream with Bispecific Monoclonal Antibodies
Authors: Lindorfer Margaret A. , Nardin Alessandra , Foley Patricia L. , Solga Michael D. , Bankovich Alexander J. , Martin Edward N. , Henderson Andrea L. , Price Carol W. , Gyimesi Edit , Wozencraft Colin P. , Goldberg Joanna B. , Sutherland William M. , Taylor Ronald P.
Published: April 2014

We examined the ability of a bispecific mAb reagent, consisting of a mAb specific for the primate erythrocyte complement receptor cross-linked with an anti-bacterial mAb, to target bacteria in the bloodstream in an acute infusion model in monkeys. In vitro studies demonstrated a variable level of complement-mediated binding (immune adherence) of Pseudomonas aeruginosa (strain PAO1) to primate E in serum. In vivo experiments in animals depleted of complement revealed that binding of bacteria to E was <1% before administration of the bispecific reagent, but within 5 min of its infusion, >99% of the bacteria bound to E. In complement-replete monkeys, a variable fraction of infused bacteria bound to E. This finding may have significant implications in the interpretation of animal models and in the understanding of bacteremias in humans. Treatment of these complement-replete monkeys with the bispecific reagent led to >99% binding of bacteria to E. Twenty-four-hour survival studies were conducted; several clinical parameters, including the degree of lung damage, cytokine levels, and liver enzymes in the circulation, indicate that the bispecific mAb reagent provides a degree of protection against the bacterial challenge.
Induction of Tolerance to Lipopolysaccharide and Mycobacterial Components in Chinese Hamster Ovary/CD14 Cells Is Not Affected by Overexpression of Toll-Like Receptors 2 or 4
Authors: Medvedev Andrei E. , Henneke Philipp , Schromm Andra , Lien Egil , Ingalls Robin , Fenton Matthew J. , Golenbock Douglas T. , Vogel Stefanie N.
Published: April 2014

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-κB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-κB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-κB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-κB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.
Differences in Functional Consequences and Signal Transduction Induced by IL-3, IL-5, and Nerve Growth Factor in Human Basophils
Authors: Miura Katsushi , Saini Sarbjit S. , Gauvreau Gail , MacGlashan Donald W.
Published: April 2014

Previous studies have indicated a redundancy in the effects of the cytokines, IL-3, IL-5, and nerve growth factor (NGF) on acute priming of human basophils. In the current study, we have examined the effects of these three cytokines on 18-h priming for leukotriene C4 generation, their ability to induce FcεRIβ mRNA expression, or their ability to sustain basophil viability in culture. We also examine a variety of the signaling steps that accompany activation with these cytokines. In contrast with the ability of IL-3 to alter secretagogue-mediated cytosolic calcium responses following 18-h cultures, 18-h treatment with IL-5 or NGF did not affect C5a-induced leukotriene C4 generation or alter C5a-induced intracellular Ca2+ concentration elevations. IL-3 and IL-5, but not NGF, induced FcεRIβ mRNA expression and all three improved basophil viability in culture with a ranking of IL-3 > IL-5 ≥ NGF. All three cytokines acutely activated the extracellular signal-regulated kinase pathway and the signaling elements that preceded extracellular signal-regulated kinase and cytosolic phospholipase A2 phosphorylation, consistent with their redundant ability to acutely prime basophils. However, only IL-3 and IL-5 induced Janus kinase 2 and STAT5 phosphorylation. This pattern of signal element activation among the three cytokines most closely matched their ability to induce expression of FcεRIβ mRNA. Induction of the sustained calcium signaling that follows overnight priming with IL-3 appeared to be related to the strength of the early signals activated by these cytokines but the relevant pathway required was not identified. None of the signaling patterns matched the ability of the cytokines to promote basophil survival.
Eosinophil Tissue Recruitment to Sites of Allergic Inflammation in the Lung Is Platelet Endothelial Cell Adhesion Molecule Independent
Authors: Miller Marina , Sung K.-L. Paul , Muller William A. , Cho Jae Youn , Roman Mark , Castaneda Diego , Nayar Jyothi , Condon Thomas , Kim John , Sriramarao P. , Broide David H.
Published: April 2014

Platelet endothelial cell adhesion molecule (PECAM or CD31) is a cell adhesion molecule expressed on circulating leukocytes and endothelial cells that plays an important role in mediating neutrophil and monocyte transendothelial migration in vivo. In this study, we investigated whether eosinophils, like neutrophils and monocytes, utilize PECAM for tissue recruitment to sites of allergic inflammation in vivo. Eosinophils express similar levels of PECAM as neutrophils as assessed by FACS analysis. RT-PCR studies demonstrate that eosinophils like neutrophils express the six extracellular domains of PECAM. Eosinophils exhibit homophilic binding to recombinant PECAM as assessed in a single-cell micropipette adhesion assay able to measure the biophysical strength of adhesion of eosinophils to recombinant PECAM. The strength of eosinophil adhesion to recombinant PECAM is the same as that of neutrophil binding to recombinant PECAM and can be inhibited with an anti-PECAM Ab. Although eosinophils express functional PECAM, anti-PECAM Abs did not inhibit bronchoalveolar lavage eosinophilia, lung eosinophilia, and airway hyperreactivity to methacholine in a mouse model of OVA-induced asthma in vivo. Thus, in contrast to studies that have demonstrated that neutrophil and monocyte tissue recruitment is PECAM dependent, these studies demonstrate that eosinophil tissue recruitment in vivo in this model is PECAM independent.
C5b-9 Terminal Complement Complex Protects Oligodendrocytes from Death by Regulating Bad Through Phosphatidylinositol 3-Kinase/Akt Pathway
Authors: Soane Lucian , Cho Hyun-Jun , Niculescu Florin , Rus Horea , Shin Moon L.
Published: April 2014

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a Giα-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.
Real-Time Imaging of Vascular Endothelial-Cadherin During Leukocyte Transmigration Across Endothelium
Authors: Shaw Sunil K. , Bamba Paul S. , Perkins Brandy N. , Luscinskas Francis W.
Published: April 2014

Vascular endothelial-cadherin (VE-cadherin) is a component of the adherens junctions of endothelial cells whose role in endothelial transmigration of leukocytes has been controversial. Using a VE-cadherin/green fluorescent protein fusion construct (VEcadGFP) that mimics the native molecule, we visualized alterations in endothelial junctional structure in real time during transmigration of human neutrophils and monocytes in an in vitro flow model. We observed abundant transmigration occurring exclusively at the cell borders (paracellularly). Surprisingly, transmigration occurred both through de novo formation of transient gaps in VEcadGFP junctional distribution, and also through preexisting gaps. De novo gaps 4–6 μm in size were formed after a leukocyte arrived at a junction, whereas preexisting gaps were present even before the leukocyte had interacted with the endothelial cells contributing to a junction. Gaps rapidly resealed within 5 min after leukocyte transmigration. Migrating leukocytes appeared to push aside VEcadGFP in the plane of the junction, and this displaced material subsequently diffused back to refill the junction. To our knowledge, this is the first example where molecular events at the lateral junction have been tracked in real time during transmigration.