bluuebunny/crossref_metadata_2025 huggingface dataset:
NK Cells Respond to Pulmonary Infection with Mycobacterium tuberculosis, but Play a Minimal Role in Protection
Authors: Junqueira-Kipnis Ana Paula , Kipnis Andre , Jamieson Amanda , Juarrero Mercedes Gonzalez , Diefenbach Andreas , Raulet David H. , Turner Joanne , Orme Ian M.
Published: April 2014

Both innate and adaptive immune systems contribute to host defense against infection with Mycobacterium tuberculosis. NK cells have been associated with early resistance against intracellular pathogens and are known to be potent producers of the cytokine IFN-γ. In C57BL/6 mice infected by aerosol exposure with M. tuberculosis, NK cells increased in the lungs over the first 21 days of infection. Expansion of the NK cell subset was associated with increased expression of activation and maturation markers. In addition, NK cells isolated from the infected lungs were capable of producing IFN-γ and became positive for perforin. In vivo depletion of NK cells using a lytic Ab had no influence on bacterial load within the lungs. These findings indicate that NK cells can become activated during the early response to pulmonary tuberculosis in the mouse model and are a source of IFN-γ, but their removal does not substantially alter the expression of host resistance.
Human C-Reactive Protein Does Not Protect against Acute Lipopolysaccharide Challenge in Mice
Authors: Hirschfield Gideon M. , Herbert Jeff , Kahan Melvyn C. , Pepys Mark B.
Published: April 2014

The physiological and pathophysiological functions of C-reactive protein (CRP), the classical acute-phase protein, are not well established, despite many reports of biological effects of CRP in vitro and in model systems in vivo. Limited, small scale experiments have suggested that rabbit and human CRP may both protect mice against lethal toxicity of Gram-negative bacterial LPS. However, in substantial well-controlled studies in C57BL/6 mice challenged with Escherichia coli O111:B4 LPS, we show in this work that significant protection against lethality was conferred neither by an autologous acute-phase response to sterile inflammatory stimuli given to wild-type mice 24 h before LPS challenge, nor by human CRP, whether passively administered or expressed transgenically. Male mice transgenic for human CRP, which mount a major acute-phase response of human CRP after LPS injection, were also not protected against the lethality of LPS from either E. coli O55:B5 or Salmonella typhimurium. Even when the acute-phase human CRP response was actively stimulated in transgenic mice before LPS challenge, no protection against LPS toxicity was observed. Indeed, male mice transgenic for human CRP that were pretreated with casein to stimulate an acute-phase response 24 h before LPS challenge suffered significantly greater mortality than unstimulated human CRP transgenic controls. Rather than being protective in this situation, human CRP may thus have pathogenic proinflammatory effects in vivo.
IL-1R-Associated Kinase 4 Is Required for Lipopolysaccharide- Induced Activation of APC
Authors: Suzuki Nobutaka , Suzuki Shinobu , Eriksson Urs , Hara Hiromitsu , Mirtosis Christine , Chen Nien-Jung , Wada Teiji , Bouchard Denis , Hwang Irene , Takeda Kiyoshi , Fujita Takashi , Der Sandy , Penninger Josef M. , Akira Shizuo , Saito Takashi , Yeh Wen-Chen
Published: April 2014

The bacterial product LPS is a critical stimulus for the host immune system in the response against the corresponding bacterial infection. LPS provides an activation stimulus for macrophages and a maturation signal for dendritic cells to set up innate and adaptive immune responses, respectively. The signaling cascade of myeloid differentiation factor 88→IL-1R-associated kinase (IRAK)→TNFR-associated factor 6 has been implicated in mediating LPS signaling. In this report, we studied the function of IRAK-4 in various LPS-induced signals. We found that IRAK-4-deficient cells were severely impaired in producing some IFN-regulated genes as well as inflammatory cytokines in response to LPS. Among the critical downstream signaling pathways induced by LPS, NF-κB activation but not IFN regulatory factor 3 or STAT1 activation was defective in cells lacking IRAK-4. IRAK-4 was also required for the proper maturation of dendritic cells by LPS stimulation, particularly in terms of cytokine production and the ability to stimulate Th cell differentiation. Our results demonstrate that IRAK-4 is critical for the LPS-induced activations of APCs.
FcγRIIIb Allele-Sensitive Release of α-Defensins: Anti-Neutrophil Cytoplasmic Antibody-Induced Release of Chemotaxins
Authors: Tanaka Sumiaki , Edberg Jeffrey C. , Chatham Winn , Fassina Giorgio , Kimberly Robert P.
Published: April 2014

Antineutrophil cytoplasmic Abs (ANCA) can activate neutrophils in an FcγR-dependent manner, but the link between this ANCA-induced effect and mononuclear cell activation with the characteristic granuloma formation of Wegener’s granulomatosis is unclear. Human α-defensins, small cationic antimicrobial peptides, are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and monocytes. In this study, we quantitated the release of α-defensins (human neutrophil peptides 1–3) from human neutrophils after targeted FcγR cross-linking (XL). Homotypic XL of FcγRIIa, FcγRIIIb, or heterotypic XL of both receptors resulted in significant release of α-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-proteinase 3). This release of α-defensins, as well as of other granule constituents (ANCA targets anti-proteinase 3 and myeloperoxidase and elastase), was significantly greater in donors homozygous for the NA1 allele of FcγRIIIb than in donors homozygous for NA2. Interestingly, the ANCA-induced release was completely inhibited by the IgG Fc-binding peptide TG19320, which blocks the IgG-Fc region from binding to FcγR. Based on their chemotactic properties, α-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma formation. The greater activity of the FcγRIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele.
Activation of Endothelial Extracellular Signal-Regulated Kinase Is Essential for Neutrophil Transmigration: Potential Involvement of a Soluble Neutrophil Factor in Endothelial Activation
Authors: Stein Brian N. , Gamble Jennifer R. , Pitson Stuart M. , Vadas Mathew A. , Khew-Goodall Yeesim
Published: April 2014

During an inflammatory response induced by infection or injury, leukocytes traverse the endothelial barrier into the tissue space. Extravasation of leukocytes is a multistep process involving rolling, tethering, firm adhesion to the endothelium, and finally, transendothelial migration, the least characterized step in the process. The resting endothelium is normally impermeable to leukocytes; thus, during inflammation, intracellular signals that modulate endothelial permeability are activated to facilitate the paracellular passage of leukocytes. Using a static in vitro assay of neutrophil transmigration across human umbilical vein endothelium, a panel of inhibitors of intracellular signaling was screened for their ability to inhibit transmigration. PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK) 1/2 activation, inhibited both transmigration across TNF-α-activated endothelium and transmigration induced by the chemoattractant fMLP in a dose-dependent manner. PD98059 did not inhibit neutrophil chemotaxis in the absence of an endothelial barrier nor neutrophil adhesion to the endothelium, suggesting that its effect was on the endothelium, and furthermore, that endothelial ERK activation may be important for transmigration. We demonstrate in this study that endothelial ERK is indeed activated during neutrophil transmigration and that its activation is dependent on the addition of neutrophils to the endothelium. Further characterization showed that the trigger for endothelial ERK activation is a soluble protein of molecular mass ∼30 kDa released from neutrophils after activation.
Endothelial Cells Proactively Form Microvilli-Like Membrane Projections upon Intercellular Adhesion Molecule 1 Engagement of Leukocyte LFA-1
Authors: Carman Christopher V. , Jun Chang-Duk , Salas Azucena , Springer Timothy A.
Published: April 2014

Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants. ICAM-1 was rapidly relocalized to newly formed microvilli-like membrane projections in response to binding LFA-1 on leukocytes. These ICAM-1-enriched projections encircled the leukocytes extending up their sides and clustered LFA-1 underneath into linear tracks. Projections formed independently of VCAM-1/very late Ag 4 interactions, shear, and proactive contributions from the LFA-1-bearing cells. In the ICAM-1-bearing endothelial cells, projections were enriched in actin but not microtubules, required intracellular calcium, and intact microfilament and microtubule cytoskeletons and were independent of Rho/Rho kinase signaling. Disruption of these projections with cytochalasin D, colchicine, or BAPTA-AM had no affect on firm adhesion. These data show that in response to LFA-1 engagement the endothelium proactively forms an ICAM-1-enriched cup-like structure that surrounds adherent leukocytes but is not important for firm adhesion. This finding leaves open a possible role in leukocyte transendothelial migration, which would be consistent with the geometry and kinetics of formation of the cup-like structure.
Toll-Like Receptor 2 Pathway Drives Streptococcal Cell Wall-Induced Joint Inflammation: Critical Role of Myeloid Differentiation Factor 88
Authors: Joosten Leo A. B. , Koenders Marije I. , Smeets Ruben L. , Heuvelmans-Jacobs Marleen , Helsen Monique M. A. , Takeda Kiyoshi , Akira Shizuo , Lubberts Erik , van de Loo Fons A. J. , van den Berg Wim B.
Published: April 2014

The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.
Apoptosis-Associated Speck-Like Protein Containing a Caspase Recruitment Domain Is a Regulator of Procaspase-1 Activation
Authors: Stehlik Christian , Lee Sug Hyung , Dorfleutner Andrea , Stassinopoulos Angela , Sagara Junji , Reed John C.
Published: April 2014

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1β activation by this protease in THP-1 monocytes. ASC enhances IL-1β secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1β secretion but that coexpression of ASC with these proteins results in enhanced IL-1β secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1β secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.
Alterations in Granule Matrix and Cell Surface of Focal Adhesion Kinase-Deficient Mast Cells
Authors: Vial Daniel , Oliver Constance , Jamur Maria Célia , Pastor Maria Verônica Dávila , da Silva Trindade Edvaldo , Berenstein Elsa , Zhang Juan , Siraganian Reuben P.
Published: April 2014

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcεRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK−/− embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing β7 integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcεRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.
B-1 B Cells Mediate Required Early T Cell Recruitment to Elicit Protein-Induced Delayed-Type Hypersensitivity
Authors: Szczepanik Marian , Akahira-Azuma Moe , Bryniarski Krzysztof , Tsuji Ryohei F. , Kawikova Ivana , Ptak Wlodzimierz , Kiener Claudia , Campos Regis A. , Askenase Philip W.
Published: April 2014

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5+, CD19+, CD22+, B220+, Thy1+, and Mac1+, suggesting that they are B-1 B cells. DTH initiation is absent in μMT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.
Mechanisms of Spontaneous Resolution versus Fibrosis in Granulomatous Experimental Autoimmune Thyroiditis
Authors: Chen Kemin , Wei Yongzhong , Sharp Gordon C. , Braley-Mullen Helen
Published: April 2014

When granulomatous experimental autoimmune thyroiditis (G-EAT) was induced in CBA/J or DBA/1 mice, thyroid lesions resolved in less severe (3+) G-EAT in wild-type mice or severe (5+) G-EAT in IFN-γ−/− mice, but progressed to fibrosis in 5+ G-EAT in wild-type mice. To define the mechanisms leading to these distinct outcomes, the expression of inflammatory and apoptotic molecules and infiltrating cells was evaluated using immunohistochemistry, RT-PCR, and confocal microscopy. The ratio of CD4+/CD8+ T cells in thyroid infiltrates was one factor that predicted G-EAT outcome. CD4+ T cells outnumbered CD8+ T cells when lesions progressed to fibrosis, while CD8+ T cells outnumbered CD4+ T cells in thyroids that resolved. Fas, Fas ligand, FLIP, TNF-α, inducible NO synthase, TGF-β, and IFN-γ were highly expressed by infiltrating cells when G-EAT progressed to fibrosis. The expression of active caspase-3 was low, possibly contributing to the persistence of CD4+ T cells in fibrosis. In contrast, FLIP was mainly expressed by thyrocytes in resolving G-EAT, the expression of active caspase-3 was high, and resolution correlated with apoptosis of infiltrating cells. There was also relatively less expression of TGF-β, IFN-γ, TNF-α, and inducible NO synthase and higher expression of IL-10 in resolving G-EAT than in G-EAT that progressed to fibrosis. These differences were particularly striking when comparing IFN-γ−/− vs wild-type mice. These results suggest that several opposing biological mechanisms contribute to the outcome of an ongoing autoimmune response. These include differential expression of pro- and antiapoptotic molecules, cytokines, and the ratio of CD4+ vs CD8+ T cells.
Injection of Immature Dendritic Cells into Adjuvant-Treated Skin Obviates the Need for Ex Vivo Maturation
Authors: Nair Smita , McLaughlin Catherine , Weizer Alon , Su Zhen , Boczkowski David , Dannull Jens , Vieweg Johannes , Gilboa Eli
Published: April 2014

A key and limiting step in the process of generating human monocyte-derived dendritic cells (DC) for clinical applications is maturation. In the setting of immunotherapy, DC are matured ex vivo by culturing them with various agents that mimic the conditions encountered at a site of inflammation. This study examined whether the ex vivo DC maturation step could be replaced by maturing DC in situ by injecting immature DC into sites pre-exposed to agents that induce a microenvironment conducive to in situ maturation of the injected DC. The hypothesis was that recapitulation of the physiological conditions occurring during pathogen infection would lead to optimal conditions for DC maturation, migration, and function. Murine immature DC injected into adjuvant (Adjuprime, poly-arginine, or Imiquimod)-pretreated skin exhibited lymph node migratory capacity comparable to and immunostimulatory capacity equal to or exceeding that of ex vivo matured DC. Acquisition of migratory capacity did not always correlate with enhanced immunostimulatory capacity. Immunostimulatory capacity was not enhanced when mature DC were injected into adjuvant-pretreated sites and remained below that seen with immature DC matured in situ. Immature DC injected into adjuvant-pretreated sites were more effective than mature DC in stimulating antitumor immunity in mice. 111Indium-labeled human monocyte-derived immature DC injected into adjuvant (Imiquimod)-pretreated sites in cancer patients acquired lymph node migratory capacity comparable to ex vivo matured DC. This study shows that in situ maturation offers a simpler and potentially superior method to generate potent immunostimulatory DC for clinical immunotherapy.
Cutting Edge: CIAS1/Cryopyrin/PYPAF1/NALP3/ CATERPILLER 1.1 Is an Inducible Inflammatory Mediator with NF-κB Suppressive Properties
Authors: O’Connor William , Harton Jonathan A. , Zhu Xinsheng , Linhoff Michael W. , Ting Jenny P.-Y.
Published: April 2014

Mutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene have been recently linked to three chronic autoinflammatory disorders. These observations point to an important role for CIAS1 in regulating inflammatory processes. We report that TNF-α and ligands recognized by multiple Toll-like receptors rapidly induce CIAS1 gene expression in primary human monocytes. Transfection of full-length CIAS1 or either of two shorter, naturally occurring isoforms dramatically inhibited TNF-α-induced activation of NF-κB reporter activity. Furthermore, CIAS1 suppressed TNF-α-induced nuclear translocation of endogenous p65. Transcriptional activity of exogenous NF-κB p65 was also blocked by CIAS1. The nucleotide-binding and leucine-rich repeat regions, but not the pyrin domain of CIAS1, are responsible for this inhibition. These data suggest CIAS1/cryopyrin may act as a key regulator of inflammation, induced to dampen NF-κB-dependent proinflammatory signals.
Deletion of Naive CD8 T Cells Requires Persistent Antigen and Is Not Programmed by an Initial Signal from the Tolerogenic APC
Authors: Redmond William L. , Hernandez Javier , Sherman Linda A.
Published: April 2014

Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.
Human Dendritic Cells Express the IL-18R and Are Chemoattracted to IL-18
Authors: Gutzmer Ralf , Langer Katja , Mommert Susanne , Wittmann Miriam , Kapp Alexander , Werfel Thomas
Published: April 2014

IL-18 is secreted by a variety of cells such as epithelial cells, macrophages, and dendritic cells (DC), in particular, in areas of chronic inflammation. The effects of IL-18 are complex and not fully understood thus far.We sought to explore human DC as a new target for IL-18, since IL-18R expression has been described on myeloid cells such as macrophages and DC are likely to get in contact with IL-18 at sites of inflammatory reactions. We demonstrate the expression of the IL-18R on human DC in peripheral blood and epidermis, as well as monocyte-derived dendritic cells (MoDC). On MoDC, IL-18R expression is up-regulated by IFN-γ. IL-18 strongly up-regulated CD54 on MoDC, whereas the effect on MHC class II, CD83, and CD86 was only moderate and the expression of CD40 and CD80 was not affected. MoDC primed with IL-18 did not increase their capacity to stimulate the proliferation or IFN-γ production of autologous T cells. However, IL-18 had a direct migratory effect on MoDC as indicated by induction of filamentous actin polymerization and migration in Boyden chamber experiments. In epidermal DC, IL-18 was also able to induce filamentous actin polymerization. Therefore, IL-18 might represent a novel mechanism to recruit DC to areas of inflammation, in particular under Th1 cytokine conditions where IFN-γ is increased such as psoriasis or inflammatory bowel diseases.
IL-15 and Cognate Antigen Successfully Expand De Novo-Induced Human Antigen-Specific Regulatory CD4+ T Cells That Require Antigen-Specific Activation for Suppression
Authors: Koenen Hans J. P. M. , Fasse Esther , Joosten Irma
Published: April 2014

An important prerequisite in using regulatory T cells for immunotherapy is their ex vivo expansion without loss of suppressor function. Human anergic regulatory T cells are expandable by Ag-specific stimulation in the presence of IL-2. IL-15, like IL-2, is a T cell growth factor that, in contrast to IL-2, stimulates survival of T cells. In this study, we examined whether IL-15 could be exploited as a superior growth factor of human CD4+ anergic regulatory T cells that were generated by costimulation blockade. Next, IL-15, as compared with IL-2, was investigated with respect to expansion and function of these regulatory T cells. Optimal expansion required cognate allogeneic stimulation in the presence of exogenous IL-15. IL-15 resulted in enhanced survival that was paralleled by an increased number of Bcl-2-expressing cells. Moreover, IL-15 induced a distinct type of anergy characterized by hyperreactivity to IL-15, resulting in improved expansion. This is likely attributed to increased propensity of these cells to up-regulate both α- and γ-chains of the IL-2 and IL-15 receptor. Notably, IL-15-expanded regulatory CD4+ T cells suppressed both naive and memory T cells in a superior way. Immunosuppression required alloantigen-specific stimulation and appeared gamma-irradiation resistant and independent of IL-10, TGFβ, or CTLA-4 interactions. These regulatory T cells were stable suppressors, mediating bystander suppression upon TCR stimulation, but leaving recall responses unaffected in the absence of cognate Ag. Finally, human naturally occurring regulatory CD4+CD25+ T cells appeared important in generating regulatory T cells by costimulation blockade. In conclusion, IL-15-expanded, de novo-induced human anergic regulatory CD4+ T cells are of interest in Ag-specific immunotherapy.
Mouse Strain Differences in Plasmacytoid Dendritic Cell Frequency and Function Revealed by a Novel Monoclonal Antibody
Authors: Asselin-Paturel Carine , Brizard Géraldine , Pin Jean-Jacques , Brière Francine , Trinchieri Giorgio
Published: April 2014

We report in this study the generation of a novel rat mAb that recognizes mouse plasmacytoid dendritic cells (pDC). This Ab, named 120G8, stains a small subset of CD11clow spleen cell with high specificity. This population produces high amounts of IFN-α upon in vitro viral stimulation. Both ex vivo- and in vitro-derived 120G8+ cells display a phenotype identical with that of the previously described mouse pDC (B220highLy6ChighGr1lowCD11b−CD11clow). Mice treated with 120G8 mAb are depleted of B220highLy6ChighCD11clow cells and have a much-reduced ability to produce IFN-α in response to in vivo CpG stimulation. The mAb 120G8 stains all and only B220highLy6ChighCD11clow pDC in all lymphoid organs. Immunohistochemical studies performed with this mAb indicate that pDC are located in the T cell area of spleen, lymph nodes, and Peyer’s patches. Although the Ag recognized by 120G8 is not yet known, we show that its expression is up-regulated by type I IFN on B cells and DC. Using this mAb in immunofluorescence studies demonstrates strain- and organ-specific differences in the frequency of pDC and other DC subsets. 129Sv mice have a much higher frequency of pDC, together with a lower frequency of conventional CD8α+CD11chigh DC, compared with C57BL/6 mice, both in spleen and blood. The higher ability of 129Sv mice to produce IFN-α in vivo is related to a higher number of pDC, but also to a higher ability of pDC from 129Sv mice to produce IFN-α in vitro in response to viral stimulation.
CD40-Induced Aggregation of MHC Class II and CD80 on the Cell Surface Leads to an Early Enhancement in Antigen Presentation
Authors: Clatza Abigail , Bonifaz Laura C. , Vignali Dario A. A. , Moreno José
Published: April 2014

Ligation of CD40 on B cells increases their ability to present Ag and to activate MHC class II (MHC-II)-restricted T cells. How this occurs is not entirely clear. In this study we demonstrate that CD40 ligation on Ag-presenting B cells (APC) for a short period between 30 min and 3 h has a rapid, augmenting effect on the ability of a B cell line and normal B cells to activate T cells. This is not due to alterations in Ag processing or to an increase in surface expression of CD80, CD86, ICAM-1, or MHC-II. This effect is particularly evident with naive, resting T lymphocytes and appears to be more pronounced under limiting Ag concentrations. Shortly after CD40 ligation on a B cell line, MHC-II and CD80 progressively accumulated in cholesterol-enriched microdomains on the cell surface, which correlated with an initial enhancement in their Ag presentation ability. Moreover, CD40 ligation induced a second, late, more sustained enhancement of Ag presentation, which correlates with a significant increase in CD80 expression by APC. Thus, CD40 signaling enhances the efficiency with which APC activate T cells by at least two related, but distinct, mechanisms: an early stage characterized by aggregation of MHC-II and CD80 clusters, and a late stage in which a significant increase in CD80 expression is observed. These results raise the possibility that one important role of CD40 is to contribute to the formation of the immunological synapse on the APC side.
The Selection of Marginal Zone B Cells Differs from That of B-1a Cells
Authors: Kretschmer Karsten , Jungebloud Anke , Stopkowicz Jana , Kleinke Tanja , Hoffmann Reinhard , Weiss Siegfried
Published: April 2014

Transgenic (Tg) L2 mice expressing high levels of the λ2 (315) L chain contain only B cell populations involved in the first line of defense, i.e., B-1 and marginal zone (MZ) B cells. The strongly oligoclonal IgH chain repertoire of Tg B-1a cells in such mice was attributed to strong positive selection by autoantigens. In this study, we show that the MZ B cells of L2 mice correspond very closely to MZ B cells of normal mice, as revealed by surface marker expression and gene expression profiling. We demonstrate that the IgH chain repertoire of these Tg MZ B cells is extremely heterogeneous. This is in sharp contrast to the oligoclonality found in B-1a cells of the same mice, which was attributed to strong positive selection mediated by autoantigens. Therefore, the strong positive selection of the IgH chain repertoire in L2 mice is B-1a specific. Thus, our data demonstrate that despite common functional properties, MZ B and B-1a cells exhibit striking differences in their selection and/or maintenance requirements.
Alloreactive CD4 T Cell Activation In Vivo: An Autonomous Function of the Indirect Pathway of Alloantigen Presentation
Authors: Reed Amy J. , Noorchashm Hooman , Rostami Susan Y. , Zarrabi Yasaman , Perate Alison R. , Jeganathan Arjun N. , Caton Andrew J. , Naji Ali
Published: April 2014

Activation of alloreactive CD4 T cells occurs via the direct and indirect pathways of alloantigen presentation. A novel TCR/alloantigen transgenic system was designed that permitted in vivo visualization of CD4 T cell priming through these pathways. When both pathways of alloantigen presentation were intact, CD4 T cell activation in response to cardiac allografts was rapid and systemic by day 4 after transplantation, in contrast to that seen in response to skin allografts, which was delayed until 10–12 days after transplantation. Despite this systemic CD4 T cell activation in response to cardiac allografts, there was a paucity of activated graft-infiltrating CD4 T cells at 4 days posttransplantation. This finding suggests that the initial priming of alloimmune CD4 T cell responses occurs within draining lymphoid organs. Furthermore, alloantigens derived from cardiac allografts failed to promote thymic negative selection of developing thymocytes expressing the alloreactive TCR clonotype. In the absence of a functional direct pathway, the kinetics of activation, anatomic localization, and effector function of alloreactive CD4 T cells remained unchanged. Overall, the present study defines the anatomic and temporal characteristics of CD4 T cell alloimmune responses and demonstrates that CD4 T cell priming via the indirect pathway proceeds optimally in the absence of the direct pathway of alloantigen presentation.
Hypoxia-Inducible Factor Regulates Survival of Antigen Receptor-Driven T Cells
Authors: Makino Yuichi , Nakamura Hiroshi , Ikeda Eiji , Ohnuma Kei , Yamauchi Kenji , Yabe Yutaka , Poellinger Lorenz , Okada Yasunori , Morimoto Chikao , Tanaka Hirotoshi
Published: April 2014

Peripheral T lymphocytes undergo activation by antigenic stimulation and function in hypoxic areas of inflammation. We demonstrated in CD3-positive human T cells accumulating in inflammatory tissue expression of the hypoxia-inducible factor-1α (HIF-1α), indicating a role of hypoxia-mediated signals in regulation of T cell function. Surprisingly, accumulation of HIF-1α in human T cells required not only hypoxia but also TCR/CD3-mediated activation. Moreover, hypoxia repressed activation-induced cell death (AICD) by TCR/CD3 stimulation, resulting in an increased survival of the cells. Microarray analysis suggested the involvement of HIF-1 target gene product adrenomedullin (AM) in this process. Indeed, AM receptor antagonist abrogated hypoxia-mediated repression of AICD. Moreover, synthetic AM peptides repressed AICD even in normoxia. Taken together, we propose that hypoxia is a critical determinant of survival of the activated T cells via the HIF-1α-AM cascade, defining a previously unknown mode of regulation of peripheral immunity.
CMRF-35-Like Molecule-1, a Novel Mouse Myeloid Receptor, Can Inhibit Osteoclast Formation
Authors: Chung Dong-Hui , Humphrey Mary Beth , Nakamura Mary C. , Ginzinger David G. , Seaman William E. , Daws Michael R.
Published: April 2014

By homology to triggering receptor expressed by myeloid cells-2, we screened the mouse expressed sequence tag database and isolated a new single Ig domain receptor, which we have expressed and characterized. The receptor is most similar in sequence to the human CMRF-35 receptor, and thus we have named it CMRF-35-like molecule (CLM)-1. By screening the mouse genome, we determined that CLM-1 was part of a multigene family located on a small segment of mouse chromosome 11. Each contains a single Ig domain, and they are expressed mainly in cells of the myeloid lineage. CLM-1 contains multiple cytoplasmic tyrosine residues, including two that lie in consensus immunoreceptor tyrosine-based inhibitory motifs, and we demonstrate that CLM-1 can associate with Src-homology 2 containing phosphatase-1. Expression of CLM-1 mRNA is down-regulated by treatment with receptor activator of NF-κB ligand (RANKL), a cytokine that drives osteoclast formation. Furthermore, expression of CLM-1 in the osteoclastogenic cell line RAW (RAW.CLM-1) prevents osteoclastogenesis induced by RANKL and TGF-β. RAW.CLM-1 cells fail to multinucleate and do not up-regulate calcitonin receptor, but they express tartrate-resistant acid phosphatase, cathepsin K, and β3 integrin, suggesting that osteoclastogenesis is blocked at a late-intermediate stage. Thus, we define a new family of myeloid receptors, and demonstrate that the first member of this family, CLM-1, is an inhibitory receptor, able to block osteoclastogenesis.
Expression and Characterization of the C345C/NTR Domains of Complement Components C3 and C5
Authors: Thai Chuong-Thu , Ogata Ronald T.
Published: April 2014

Complement components C3, C4, and C5 are members of the thioester-containing α-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their α-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high β-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.
Regulation of Inducible Nitric Oxide Synthase Expression by p300 and p50 Acetylation
Authors: Deng Wu-Guo , Wu Kenneth K.
Published: April 2014

To determine whether p300 is involved in inducible NO synthase (iNOS) transcriptional regulation, we evaluated the effect of p300 overexpression on iNOS expression and characterized p300 binding to iNOS promoter in RAW 264.7 cells. p300 overexpression increased iNOS expression which was abrogated by deletion of the histone acetyltransferase (HAT) domain (Δ1472–1522). DNA-binding and chromatin immunoprecipitation assays revealed binding of p300 to several DNA-bound transactivators at basal state. Following stimulation with LPS plus IFN-γ, binding of p300, p50/p65 NF-κB, and IFN-regulatory factor-1 was increased by ∼2-fold. Nuclear p50 was complexed with and acetylated by p300 at the basal binding state which was increased by LPS and IFN-γ stimulation. p300 overexpression resulted in increased p50 acetylation which was reduced by HAT mutation. p50 acetylation correlated with increased NF-κB binding and enhanced p300 recruitment. Co-overexpression of E1A abolished the augmentation of p50 acetylation and p50 binding induced by p300 overexpression, and a correlative suppression of p300 recruitment to the complex. We conclude that p300 is essential for iNOS transcription. Our results suggest that p300 HAT acetylates the p50 subunit of NF-κB, thereby increasing NF-κB binding and NF-κB mediated transactivation.
Identification, Isolation, and Culture of HLA-A2-Specific B Lymphocytes Using MHC Class I Tetramers
Authors: Mulder Arend , Eijsink Chantal , Kardol Marrie J. , Franke-van Dijk Marry E. I. , van der Burg Sjoerd H. , Kester Michel , Doxiadis Ilias I. N. , Claas Frans H. J.
Published: April 2014

Characterizing the individual B cells that participate in the production of anti-HLA Abs requires isolation and culture of these cells and a suitable assay for detection of Abs produced in these B cell cultures. We previously showed that B cell precursors, programmed for anti-HLA Ab secretion, are present at measurable frequencies in peripheral blood of women immunized by pregnancy. In this study, we show that tetrameric HLA-A2, although designed for characterization of CTLs, provides a suitable affinity ligand for isolation of allospecific B cells, which subsequently can be induced to produce HLA-A2 Ab in a CD40-driven culture system. The validity of this concept was established by assaying human hybridomas, producing anti-HLA Abs, for specific tetrameric HLA-A2 binding. The availability of anti-HLA Ab-producing B cell cultures that are established without immortalization will be of value when T-B cell interaction is studied at an alloantigen-specific level.
Neutrophil Gelatinase-Associated Lipocalin Is Up-Regulated in Human Epithelial Cells by IL-1β, but Not by TNF-α
Authors: Cowland Jack B. , Sørensen Ole E. , Sehested Maxwell , Borregaard Niels
Published: April 2014

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1β induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-α, IL-6, and LPS had no effect. Similar IL-1β selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-κB consensus site was found to control the IL-1β-specific up-regulation. Involvement of the NF-κB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-κB pathway, and by EMSA. TNF-α activation of NF-κB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-κB to the NGAL promoter was observed in vitro. IL-1β specificity was not contained within the NF-κB site of the NGAL promoter, as determined by exchanging the NGAL promoter′s NF-κB-binding sequence with that of the IL-8 promoter or with the NF-κB consensus sequence and by testing the NF-κB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.
Anti-CD8 Antibodies Can Inhibit or Enhance Peptide-MHC Class I (pMHCI) Multimer Binding: This Is Paralleled by Their Effects on CTL Activation and Occurs in the Absence of an Interaction between pMHCI and CD8 on the Cell Surface
Authors: Wooldridge Linda , Hutchinson Sarah L. , Choi Ed M. , Lissina Anna , Jones Emma , Mirza Fareed , Dunbar P. Rod , Price David A. , Cerundolo Vincenzo , Sewell Andy K.
Published: April 2014

Cytotoxic T lymphocytes recognize short peptides presented in association with MHC class I (MHCI) molecules on the surface of target cells. The Ag specificity of T lymphocytes is conferred by the TCR, but invariable regions of the peptide-MHCI (pMHCI) molecule also interact with the cell surface glycoprotein CD8. The distinct binding sites for CD8 and the TCR allow pMHCI to be bound simultaneously by both molecules. Even before it was established that the TCR recognized pMHCI, it was shown that CTL exhibit clonal heterogeneity in their ability to activate in the presence of anti-CD8 Abs. These Ab-based studies have since been interpreted in the context of the interaction between pMHCI and CD8 and have recently been extended to show that anti-CD8 Ab can affect the cell surface binding of multimerized pMHCI Ags. In this study, we examine the role of CD8 further using point-mutated pMHCI Ag and show that anti-CD8 Abs can either enhance or inhibit the activation of CTL and the stable cell surface binding of multimerized pMHCI, regardless of whether there is a pMHCI/CD8 interaction. We further demonstrate that multimerized pMHCI Ag can recruit CD8 in the absence of a pMHCI/CD8 interaction and that anti-CD8 Abs can generate an intracellular activation signal resulting in CTL effector function. These results question many previous assumptions as to how anti-CD8 Abs must function and indicate that CD8 has multiple roles in CTL activation that are not necessarily dependent on an interaction with pMHCI.
The Antimicrobial Peptide LL-37 Activates Innate Immunity at the Airway Epithelial Surface by Transactivation of the Epidermal Growth Factor Receptor
Authors: Tjabringa G. Sandra , Aarbiou Jamil , Ninaber Dennis K. , Drijfhout Jan Wouter , Sørensen Ole E. , Borregaard Niels , Rabe Klaus F. , Hiemstra Pieter S.
Published: April 2014

Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.