bluuebunny/crossref_metadata_2025 huggingface dataset:
Peritoneal Cavity B Cells Are Precursors of Splenic IgM Natural Antibody-Producing Cells
Authors: Kawahara Toshiyasu , Ohdan Hideki , Zhao Guiling , Yang Yong-Guang , Sykes Megan
Published: April 2014

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used α1,3-galactosyltransferase-deficient (GalT−/−) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galα1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT−/− and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT−/− and (for non-Gal specificities) wild-type mice. These cells are Mac-1− but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT−/− mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT−/−, Ig μ-chain mutant (μ−/−) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT−/− mice. When sorted GalT−/− Mac-1+ peritoneal cavity B cells were adoptively transferred to B6 GalT−/−, μ−/− mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1− B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1+ B-1 cells are precursors of Mac-1− splenic IgM Ab-secreting cells.
Immunodominance of an Antiviral Cytotoxic T Cell Response Is Shaped by the Kinetics of Viral Protein Expression
Authors: Probst Hans Christian , Tschannen Kathrin , Gallimore Awen , Martinic Marianne , Basler Michael , Dumrese Tilman , Jones Emma , van den Broek Maries F.
Published: April 2014

Lymphocytic choriomeningitis virus (LCMV) infection induces a protective CTL response consisting of gp- and nucleoprotein (NP)-specific CTL. We find that a small load of LCMV led to immunodominance of NP-CTL, whereas a large viral load resulted in dominance of gp-CTL. This is the first study describing that immunodominance is not fixed after infection with a given pathogen, but varies with the viral load instead. We assumed higher Ag sensitivity for NP-CTL, which would explain their preferential priming at low viral load, as well as their overstimulation resulting in selective exhaustion at high viral load. The higher Ag sensitivity of NP-CTL was due to faster kinetics of NP-epitope presentation. Thus, we uncover a novel factor that impinges upon immunodominance and is related to the kinetics of virus protein expression. We propose that CTL against early viral proteins swiftly interfere with virus replication, resulting in efficient protection. If these “early” CTL fail in immediate virus control, they are activated in the face of higher viral load compared with “late” CTL and are therefore prone to be exhausted. Thus, the observed absence of early CTL in persistent infections might not be the cause, but rather the consequence of viral persistence.
Autocrine/Paracrine IL-15 That Is Required for Type I IFN-Mediated Dendritic Cell Expression of MHC Class I-Related Chain A and B Is Impaired in Hepatitis C Virus Infection
Authors: Jinushi Masahisa , Takehara Tetsuo , Tatsumi Tomohide , Kanto Tatsuya , Groh Veronika , Spies Thomas , Suzuki Takahiro , Miyagi Takuya , Hayashi Norio
Published: April 2014

We previously reported that monocyte-derived dendritic cells activate resting NK cells by expressing MHC class I-related chain A and B (MICA/B), ligands for NKG2D, in response to IFN-α, but the MICA/B expression was severely impaired in patients with chronic hepatitis C virus (HCV) infection. In the present study, we examined induction of MICA/B on DCs by various innate cytokines and found that DCs from either healthy donors or HCV-infected individuals, upon IL-15 stimulation, express MICA/B and can activate NK cells, which is solely dependent on MICA/B-NKG2D interaction. Of interest is the finding that IL-15- and type I IFN-mediated induction of MICA/B in healthy donors is completely inhibited when DCs are incubated in the presence of anti-IFN-α/βR or anti-IL-15Rα, respectively, suggesting interdependent roles of these cytokines in MICA/B expression. Indeed, DCs produced IL-15 in response to type I IFN, whereas they directly produced IFN-β, in response to IL-15, which was followed by the production of IFN-α. In HCV-infected individuals, type I IFN-mediated production of IL-15 was virtually absent, but IL-15-mediated production of type I IFN was not compromised, which is consistent with the distinct ability of these cytokines to induce MICA/B in these patients. The present study demonstrates that IL-15 and type I IFN lead to DC expression of MICA/B and subsequent DC activation of NK cells, which is critically dependent on each other’s autocrine/paracrine effect, and suggests that impaired IL-15 production is one of the mechanisms of the aberrant response of DC to type I IFN in HCV-infected patients.
Differential Induction of TGF-β Regulates Proinflammatory Cytokine Production and Determines the Outcome of Lethal and NonlethalPlasmodium yoeliiInfections
Authors: Omer Fakhreldin M. , de Souza J. Brian , Riley Eleanor M.
Published: April 2014

Transforming growth factor-β is an essential moderator of malaria-induced inflammation in mice. In this study, we show that the virulence of malaria infections is dependent upon the cellular source of TGF-β and the timing of its production. C57BL/6 mice infected with a nonlethal (Py17X) strain of Plasmodium yoelii produce TGF-β from 5 days postinfection; this correlates with resolution of parasitemia, down-regulation of TNF-α, and full recovery. In contrast, infection with the lethal strain Py17XL induces high levels of circulating TGF-β within 24 h; this is associated with delayed and blunted IFN-γ and TNF-α responses, failure to clear parasites, and 100% mortality. Neutralization of early TGF-β in Py17XL infection leads to a compensatory increase in IL-10 production, while simultaneous neutralization of TGF-β and IL-10R signaling leads to up-regulation of TNF-α and IFN-γ, prolonged survival in all, and ultimate resolution of infection in 40% of Py17XL-infected animals. TGF-β production can be induced in an Ag-specific manner from splenocytes of infected mice, and by cross-linking surface CTLA-4. CD25+ and CD8+ cells are the primary source of TGF-β following Py17X stimulation of splenocytes, whereas Py17XL induces significant production of TGF-β from adherent cells. In mice immunized against Py17XL, the early TGF-β response is inhibited and is accompanied by significant up-regulation of IFN-γ and TNF-α and rapid resolution of challenge infections.
TRANCE-RANK Costimulation is Required for IL-12 Production and the Initiation of a Th1-Type Response toLeishmania majorInfection in CD40L-Deficient Mice
Authors: Padigel Udaikumar M. , Kim Nacksung , Choi Yongwon , Farrell Jay P.
Published: April 2014

Blockade of TNF-related activation-induced cytokine (TRANCE)-receptor activator of NF-κB (RANK) interaction reverses healing in CD40L−/− mice infected with Leishmania major. Although previous studies demonstrated a requirement for CD40-CD40L interaction in production of IL-12 and the development of resistance to Leishmania infection, we recently showed that CD40L−/− mice control infection when inoculated with low numbers of parasites and that cells from these mice produce IL-12. Here, we show that in vivo treatment with a TRANCE receptor fusion protein results in a decrease in numbers of IL-12 producing cells as well as a shift from a dominant Th1 to Th2 type response in infected mice. These results demonstrate that CD40L−/− mice use the TRANCE-RANK costimulatory pathway to promote IL-12 production and the activation of a protective Th1 type response.
A Loss-of-Function Polymorphism in the Human P2X7 Receptor Abolishes ATP-Mediated Killing of Mycobacteria
Authors: Saunders Bernadette M. , Fernando Suran L. , Sluyter Ronald , Britton Warwick J. , Wiley James S.
Published: April 2014

Protective immunity to mycobacterial infections requires activation of the antibacterial mechanisms of infected macrophages. It has previously been reported that ATP treatment of mycobacteria-infected macrophages induces apoptosis mediated via the P2X7 pathway and that this leads to the death of both the host cell and the internalized bacilli. We have recently identified a single nucleotide polymorphism in the P2X7 gene (1513A→C), with 1–2% prevalence in the homozygous state, which codes for a nonfunctional receptor. IFN-γ-primed, mycobacteria-infected macrophages from wild-type individuals were incubated with ATP and this induced apoptosis and reduced mycobacterial viability by 90%. Similar treatment of macrophages from individuals homozygous for the 1513C polymorphism failed to induce apoptosis and did not lead to mycobacterial killing via the P2X7-mediated pathway. These data demonstrate that a single nucleotide polymorphism in the P2X7 gene can allow survival of mycobacteria within infected host cells.
Localized Reactive Oxygen and Nitrogen Intermediates Inhibit Escape ofListeria monocytogenesfrom Vacuoles in Activated Macrophages
Authors: Myers Jesse T. , Tsang Albert W. , Swanson Joel A.
Published: April 2014

Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of nonactivated macrophages within 30 min of infection. Macrophages activated with IFN-γ, LPS, IL-6, and a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91phox−/− and NO synthase 2−/− mice showed that vacuolar retention required ROI and was augmented by RNI. Live cell imaging with the fluorogenic probe dihydro-2′,4,5,6,7,7′-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and RNI to vacuoles, and by their combined actions in a small space
Increased CD8+ T Cell Memory to Concurrent Infection at the Expense of Increased Erosion of Pre-existing Memory: The Paradoxical Role of IL-15
Authors: Chapdelaine Yvan , Smith Dean K. , Pedras-Vasconcelos Joao A. , Krishnan Lakshmi , Sad Subash
Published: April 2014

The use of cytokines during vaccination, particularly IL-15, is being considered due to the unique ability of IL-15 to enhance the proliferation of memory CD8+ T cells. However, as homeostatic mechanisms limit excessive lymphocyte expansion, we addressed the consequences of this enhancement of T cell memory by IL-15. Infection of mice with either recombinant Mycobacterium bovis (BCG) expressing IL-15 (BCG-IL-15) or BCG and purified IL-15 resulted in an increased CD44, IL-2Rβ expression and increased frequency of IFN-γ-secreting CD8+ T cells. Surprisingly, the enhancement of memory to concurrent infection by IL-15 exacerbated the attrition of pre-existing memory. Infection of mice with Listeria monocytogenes expressing OVA resulted in potent OVA257–264-specific CD8+ T cell memory, and a challenge of these mice with either BCG-IL-15 or BCG and purified IL-15 resulted in an increased erosion of OVA257–264-specific CD8+ T cell memory, relative to BCG. Enhancement in the erosion of OVA-specific CD8+ T cell memory by BCG-IL-15 resulted in a consequently greater impairment in protection against a challenge with OVA-expressing tumor cells. We thus raise important questions regarding vaccinations that are aimed at maximizing T cell memory without considering the impact on pre-existing T cell memory.
The Role of the 19-kDa Region of Merozoite Surface Protein 1 and Whole-Parasite-Specific Maternal Antibodies in Directing Neonatal Pups’ Responses to Rodent Malaria Infection
Authors: Stanisic Danielle I. , Martin Laura B. , Good Michael F.
Published: April 2014

Maternal Abs generated as a result of prior exposure to infectious agents such as the malaria parasite are transferred from the mother through the placenta to the fetus. Numerous studies have attributed the resistance to malaria infection observed in neonates and infants up to 6 mo of age to the presence of maternally derived Abs. However, recent studies have produced conflicting results suggesting that alternative protective mechanisms may be responsible. Although the presence of maternally derived Abs in the infant is not disputed, their exact role in the infant is unknown. Even less clear is the effect that maternally derived Abs, if generated in response to vaccination, may have on the infant’s ability to respond to malaria infection. Studies on mouse pups were performed to determine the role of the 19-kDa region of merozoite surface protein 1 (MSP119) and Plasmodium yoelii-specific Abs in neonatal malaria infection and to examine their effect on the development of a specific immune response in the pup. It was shown that P. yoelii- and MSP119-specific Abs transferred to the pup from the mother act to suppress the growth of the parasite in the pup. However, the maternally derived Abs interfered with the development of the pups’ own Ab response to the parasite by altering the fine specificity of the response. These results suggest that immunizing women of child-bearing age with a malaria vaccine candidate such as MSP119 would not prevent the infant from producing Abs in response to malaria infection, but it may affect the region of the Ag to which it responds.
Eosinophils and T Lymphocytes Possess Distinct Roles in Bleomycin-Induced Lung Injury and Fibrosis
Authors: Huaux Francois , Liu Tianju , McGarry Bridget , Ullenbruch Matt , Xing Zhou , Phan Sem H.
Published: April 2014

Leukocyte infiltration is characteristic of lung injury and fibrosis, and its role during tissue repair and fibrosis is incompletely understood. We found that overexpression of IL-5 in transgenic mice (IL-5TG) or by adenoviral gene transfer increased bleomycin (blm)-induced lung injury, fibrosis, and eosinophilia. Surprisingly, blm-treated IL-5-deficient (IL-5−/−) mice also developed pronounced pulmonary fibrosis but characterized by marked T lymphocyte infiltration and absence of eosinophilia. In both murine strains however, induction of lung TGF-β expression was evident. Purified lung eosinophils from blm-treated IL-5TG mice stimulated α-smooth muscle actin and collagen expression in mouse lung fibroblasts, without affecting proliferation. Furthermore instillation of purified eosinophils into murine lungs resulted in extension of blm-induced lung fibrosis, thus confirming a role for eosinophils. However, lung T lymphocytes from blm-treated IL-5−/− mice were able to stimulate fibroblast proliferation but not α-smooth muscle actin or collagen expression. Blocking T cell influx by anti-CD3 Abs abrogated lung fibrosis, thus also implicating T lymphocytes as a key participant in fibrosis. Pulmonary fibrosis in IL-5TG mice was preferentially associated with type 2 cytokines (IL-4 and IL-13), whereas fibrotic lesions in IL-5−/− animals were accompanied by proinflammatory cytokine (TNF-α, IL-1β, and IFN-γ) expression. We suggest that eosinophils and T cells contribute distinctly to the development of blm-induced lung fibrosis potentially via their production of different cytokine components, which ultimately induce TGF-β expression that is intimately involved with the fibrosis.
IL-4 Down-Regulates Lipopolysaccharide-Induced Formyl Peptide Receptor 2 in Murine Microglial Cells by Inhibiting the Activation of Mitogen-Activated Protein Kinases
Authors: Iribarren Pablo , Cui You-Hong , Le Yingying , Ying GuoGuang , Zhang Xia , Gong Wanghua , Wang Ji Ming
Published: April 2014

Microglial cells actively participate in proinflammatory responses in the CNS. Upon stimulation with the bacterial LPS, microglial cells express a functional formyl peptide receptor 2 which mediates the chemotactic and activating effects of a variety of polypeptide agonists including amyloid β (Aβ1–42), a critical pathogenic agent in Alzheimer’s disease. In the present study, we found that LPS-induced expression and function of formyl peptide receptor 2 in microglial cells was markedly inhibited by IL-4, a Th2-type cytokine. Our effort to elucidate the mechanistic basis revealed that IL-4 attenuated LPS-stimulated activation of NF-κB, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase, and the effect of IL-4 was associated with a phosphoinositide 3-kinase pathway-dependent increase in serine/threonine phosphatase activity. These results suggest that IL-4 may play an important role in the maintenance of homeostasis of CNS and in the regulation of the disease process characterized by microglial activation in response to proinflammatory stimulants.
Platelet-Activating Factor Mediates CD40-Dependent Angiogenesis and Endothelial-Smooth Muscle Cell Interaction
Authors: Russo Simona , Bussolati Benedetta , Deambrosis Ilaria , Mariano Filippo , Camussi Giovanni
Published: April 2014

The aim of the present study was to investigate whether stimulation of CD40 expressed by endothelial or smooth muscle cells triggers the synthesis of platelet-activating factor (PAF), an inflammatory mediator with angiogenic properties, and whether PAF contributes to CD40-induced neoangiogenesis. The results obtained indicate that the interaction of CD40 with soluble CD154 or with CD154 expressed on the membrane of leukocytes (CD154-transfected J558 cells) or of activated platelets, stimulated the synthesis of PAF by endothelial cells but not by smooth cells. The synthesis of PAF triggered by activated platelets was inhibited by a soluble CD40-murine Ig fusion protein that prevents the interaction between membrane CD40 and CD154. Studies with specific inhibitors and evaluation of protein phosphorylation indicated the involvement in PAF synthesis of two intracellular signaling pathways leading to cytosolic phospholipase A2 activation: a phospholipase Cγ-protein kinase C-Raf-p42/p44-mitogen-activated protein kinase (MAPK) and a MAPK kinase-3/6-dependent activation of p38 MAPK. PAF synthesized by endothelial cells after CD40 stimulation was instrumental in the in vitro migration and vessel-like organization of endothelial cells, and in the interaction between endothelial cells and smooth muscle cells, as inferred by the inhibitory effect of two different PAF receptor antagonists, WEB2170 and CV3988. In vivo, blockade of PAF receptors prevented the angiogenic effect triggered by CD40 stimulation in a murine model of s.c. Matrigel implantation. In conclusion, these observations indicate that PAF synthesis induced by stimulation of endothelial CD40 contributes to the formation and organization of new vessels. This may be relevant in the vascular remodeling associated with tumor and inflammatory neoangiogenesis.
Inhibition of Airway Inflammation by Amino-Terminally Modified RANTES/CC Chemokine Ligand 5 Analogues Is Not Mediated through CCR3
Authors: Chvatchko Yolande , Proudfoot Amanda E. I. , Buser Raphaële , Juillard Pierre , Alouani Sami , Kosco-Vilbois Marie , Coyle Anthony J. , Nibbs Robert J. , Graham Gerry , Offord Robin E. , Wells Timothy N. C.
Published: April 2014

Chemokines play a key role in the recruitment of activated CD4+ T cells and eosinophils into the lungs in animal models of airway inflammation. Inhibition of inflammation by N-terminally modified chemokines is well-documented in several models but is often reported with limited dose regimens. We have evaluated the effects of doses ranging from 10 ng to 100 μg of two CC chemokine receptor antagonists, Met-RANTES/CC chemokine ligand 5 (CCL5) and aminooxypentane-RANTES/CCL5, in preventing inflammation in the OVA-sensitized murine model of human asthma. In the human system, aminooxypentane-RANTES/CCL5 is a full agonist of CCR5, but in the murine system neither variant is able to induce cellular recruitment. Both antagonists showed an inverse bell-shaped inhibition of cellular infiltration into the airways and mucus production in the lungs following allergen provocation. The loss of inhibition at higher doses did not appear to be due to partial agonist activity because neither variant showed activity in recruiting cells into the peritoneal cavity at these doses. Surprisingly, neither was able to bind to the major CCR expressed on eosinophils, CCR3. However, significant inhibition of eosinophil recruitment was observed. Both analogues retained high affinity binding for murine CCR1 and murine CCR5. Their ability to antagonize CCR1 and CCR5 but not CCR3 was confirmed by their ability to prevent RANTES/CCL5 and macrophage inflammatory protein-1β/CCL4 recruitment in vitro and in vivo, while they had no effect on that induced by eotaxin/CCL11. These results suggest that CCR1 and/or CCR5 may be potential targets for asthma therapy.
Role of γδT Cells in the Inflammatory Response of Experimental Colitis Mice
Authors: Tsuchiya Takahiro , Fukuda Sumiaki , Hamada Hiromasa , Nakamura Akihiro , Kohama Yasuhiro , Ishikawa Hiromichi , Tsujikawa Kazutake , Yamamoto Hiroshi
Published: April 2014

We examined the severity of experimental colitis induced by dextran sulfate sodium (DSS) using immunologically manipulated mice. C57BL/6 mice showed more severe colitis than BALB/c mice, but mice of both strains recovered fully from the disease after the removal of DSS from their drinking water. The infiltrated cells at the lesions were mainly granulocytes in normal littermates. However, C.B-17 scid, IL-7Rα deficient, and TCR-Cβδ double-deficient mice showed severe colitis and did not recover from the disease even after the removal of DSS. It was found that the infiltrated cells at the lesions in the lethal strains were monocytes. Although both TCR-Cδ−/− and TCR-Cβ−/− mice showed severe colitis phenotypes, infiltration in the former is monocyte-dominant while that in the latter is granulocyte-dominant. Thus the type of cells that infiltrate at the lesions of DSS-induced experimental colitis may be controlled by functional T cell subsets. Immunohistological and RT-PCR analyses of the inflamed colon revealed that the murine homologue of human GROα released by some cells under the control of γδT cells is a possible candidate determining the severity of DSS-induced experimental colitis.
A Potent Human C5a Receptor Antagonist Protects against Disease Pathology in a Rat Model of Inflammatory Bowel Disease
Authors: Woodruff Trent M. , Arumugam Thiruma V. , Shiels Ian A. , Reid Robert C. , Fairlie David P. , Taylor Stephen M.
Published: April 2014

The complement system is implicated in the pathogenesis of human inflammatory bowel disease, but the specific role of C5a has never been examined. We have compared the efficacy of an orally active human C5a receptor antagonist (AcPhe[Orn-Pro-d-cyclohexylalanine-Trp-Arg]), prednisolone, and infliximab against trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. The drugs were administered either 2 days before or 24 h after TNBS instillation, and rats were then examined after 8 days. Drug-free colitis control rats showed severe disease pathology with significant mortality (39%). Rats pre or posttreated with the C5a antagonist (10 mg/kg/day peroral, 0.3 mg/kg/day s.c.) had reduced mortality and significantly improved macroscopic scores, colon edema, colon myeloperoxidase levels, reduced concentrations of TNF-α levels in the colon and serum, and had greater food intake resulting in greater weight gains than colitis-only rats. Rats pretreated with prednisolone (1 mg/kg/day s.c.) displayed significant improvement in parameters measured, but posttreatment was ineffective. Single dose pretreatment with the TNF-α inhibitor infliximab (3 mg/kg i.v.) also had significant improvements in the parameters measured. Rats pretreated with a combination of the C5a antagonist and prednisolone showed no greater improvements than either drug alone. These findings suggest a central role for complement, particularly C5a, in the pathology of TNBS-induced colitis in rats, indicating a possible therapeutic role for C5a antagonists in inflammatory bowel disease.
CiC3-1a-Mediated Chemotaxis in the Deuterostome InvertebrateCiona intestinalis(Urochordata)
Authors: Pinto Maria Rosaria , Chinnici Cinzia M. , Kimura Yuko , Melillo Daniela , Marino Rita , Spruce Lynn A. , De Santis Rosaria , Parrinello Nicolò , Lambris John D.
Published: April 2014

Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms. However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown. To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C. intestinalis hemocytes. We found that the migration of C. intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab. As is true for mammalian C3a, the chemotactic activity of C. intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a59–76) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a59–75) retained most of the directional hemocyte migration activity. The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is Gi protein coupled. Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3′-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells. Our findings indicate that CiC3-1a mediates chemotaxis of C. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes.
Chemokines Accumulate in the Lungs of Rats with Severe Pulmonary Embolism Induced by Polystyrene Microspheres
Authors: Zagorski John , Debelak Jacob , Gellar Michael , Watts John A. , Kline Jeffrey A.
Published: April 2014

Pulmonary thromboembolism (PEm) is a serious and life threatening disease and the most common cause of acute pulmonary vascular occlusion. Even following successful treatment of PEm, many patients experience long-term disability due to diminished heart and lung function. Considerable damage to the lungs presumably occurs due to reperfusion injury following anti-occlusive treatments for PEm and the resulting chronic inflammatory state in the lung vasculature. We have used a rat model of irreversible PEm to ask whether pulmonary vascular occlusion in the absence of reperfusion is itself sufficient to induce an inflammatory response in lungs. By adjusting the severity of the vascular occlusion, we were able to generate hypertensive and nonhypertensive PEm, and then examine lung tissue for expression of CXC and C-C chemokine genes and bronchoalveolar lavage (BAL) fluid for the presence of chemokine proteins. Hypertensive and nonhypertensive PEm resulted in increased expression of both CXC and C-C chemokines genes in lung tissues. Hypertensive PEm was also associated with a 50–100-fold increase in protein content in lung BAL fluid, which included the CXC chemokines cytokine-induced neutrophil chemoattractant and macrophage-inflammatory protein 2. The presence of chemokines in BALs was reflected by a potent neutrophil chemotactic activity in in vitro chemotaxis assays. Abs to cytokine-induced neutrophil chemoattractant blocked the in vitro neutrophil chemotactic activity of BAL by 44%. Our results indicate that the ischemia and hypertension associated with PEm are sufficient to induce expression of proinflammatory mediators such as chemokines, and establish a proinflammatory environment in the ischemic lung even before reperfusion.
Inhibition of Fibrocyte Differentiation by Serum Amyloid P
Authors: Pilling Darrell , Buckley Christopher D. , Salmon Mike , Gomer Richard H.
Published: April 2014

Wound healing and the dysregulated events leading to fibrosis both involve the proliferation and differentiation of fibroblasts and the deposition of extracellular matrix. Whether these fibroblasts are locally derived or from a circulating precursor population is unclear. Fibrocytes are a distinct population of fibroblast-like cells derived from peripheral blood monocytes that enter sites of tissue injury to promote angiogenesis and wound healing. We have found that CD14+ peripheral blood monocytes cultured in the absence of serum or plasma differentiate into fibrocytes within 72 h. We purified the factor in serum and plasma that prevents the rapid appearance of fibrocytes, and identified it as serum amyloid P (SAP). Purified SAP inhibits fibrocyte differentiation at levels similar to those found in plasma, while depleting SAP reduces the ability of plasma to inhibit fibrocyte differentiation. Compared with sera from healthy individuals and patients with rheumatoid arthritis, sera from patients with scleroderma and mixed connective tissue disease, two systemic fibrotic diseases, were less able to inhibit fibrocyte differentiation in vitro and had correspondingly lower serum levels of SAP. These results suggest that low levels of SAP may thus augment pathological processes leading to fibrosis. These data also suggest mechanisms to inhibit fibrosis in chronic inflammatory conditions, or conversely to promote wound healing.
Direct Inhibition of NF-κB Blocks Bone Erosion Associated with Inflammatory Arthritis
Authors: Clohisy John C. , Roy Bhabesh C. , Biondo Christine , Frazier Elfaridah , Willis David , Teitelbaum Steven L. , Abu-Amer Yousef
Published: April 2014

Inflammatory arthritis is associated with devastating joint tissue destruction and periarticular bone erosion. Although secreted products of infiltrating immune cells perpetuate the inflammatory response, the osteolytic component of this disease is a direct result of localized recruitment and activation of osteoclasts. Given that NF-κB plays a central role in both processes, the function of this transcription factor was examined. Using a mouse model of autoreactive Ig transfer that engenders inflammatory arthritis, we show numerous osteoclasts in the articular joint tissue associated with progressive periarticular osteolytic lesions. Moreover, cells retrieved from these joints exhibit heightened NF-κB activity. Importantly, direct administration of dominant negative∗I-κB or tyrosine 42-mutated I-κB (Y42F∗I-κB) proteins into mice before induction of the disease attenuates in vivo activation of the transcription factor. More importantly, these I-κB mutant forms significantly inhibit in vivo production of TNF and receptor activator of NF-κB ligand, and block joint swelling, osteoclast recruitment, and osteolysis. Thus, NF-κB appears to be the centerpiece of inflammatory-osteolytic arthritis and direct inhibition of this transcription factor by unique and novel I-κB mutant proteins blocks manifestation of the disease.
Tolerance to Noninherited Maternal MHC Antigens in Mice
Authors: Andrassy Joachim , Kusaka Satoshi , Jankowska-Gan Ewa , Torrealba Jose R. , Haynes Lynn D. , Marthaler Brodie R. , Tam Robert C. , Illigens Ben M.-W. , Anosova Natalie , Benichou Gilles , Burlingham William J.
Published: April 2014

The phenomenon of tolerance to noninherited maternal Ags (NIMA) is poorly understood. To analyze the NIMA effect C57BL/6 (H-2b/b) males were mated with B6D2F1 (H-2b/d) females, whereby 50% of the offspring are H-2b/b mice that have been exposed to maternal H-2d alloantigens. Controls were H-2b/b offspring of C57BL/6 mothers, either inbred C57BL/6 mice or F1 backcross mice from breedings with H-2b/d fathers. We found that 57% of the H-2b/b offspring of semiallogeneic (H-2b/d) mothers accepted fully allogeneic DBA/2 (H-2d/d) heart grafts for >180 days, while similar transplants were all rejected by day 11 in controls (p < 0.0004). Foster nursing studies showed that both oral and in utero exposure to NIMA are required for this tolerogenic effect. An effect of NIMA was also found to extend the survival of skin grafts from a semiallogeneic donor (p < 0.02). Pretransplant analysis of splenocytes showed a 40–90% reduction of IL-2-, IL-5-, and IFN-γ-producing T cells responding to H-2d-expressing APC in NIMAd-exposed vs control mice. Injection of pregnant BALB/c-dm2 (H-2Ld-negative) female mice i.v. with H-2Ld61–80 peptide profoundly suppressed the offspring’s indirect pathway alloreactive CD4+ T cell response to H-2Ld. These results suggest that the natural exposure of the fetus and newborn to maternal cells and/or soluble MHC Ags suppresses NIMA-allospecific T cells of the offspring, predisposing to organ transplant tolerance in adult mice.
Impaired Germinal Center Maturation in Adenosine Deaminase Deficiency
Authors: Aldrich Melissa B. , Chen Wilma , Blackburn Michael R. , Martinez-Valdez Hector , Datta Surjit K. , Kellems Rodney E.
Published: April 2014

Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2′-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.
A Blunted Blood Plasmacytoid Dendritic Cell Response to an Acute Systemic Viral Infection Is Associated with Increased Disease Severity
Authors: Pichyangkul Sathit , Endy Timothy P. , Kalayanarooj Siripen , Nisalak Ananda , Yongvanitchit Kosol , Green Sharone , Rothman Alan L. , Ennis Francis A. , Libraty Daniel H.
Published: April 2014

At least two distinct human dendritic cell (DC) subsets are produced in the bone marrow and circulate in the peripheral blood-precursor myeloid DCs (pre-mDCs) and plasmacytoid DCs (PDCs). Both lineages of DCs are instrumental in antiviral innate immunity and shaping Th1 adaptive immune responses. PDCs are the most potent IFN-α-producing cells to viral pathogens. Dengue, an acute flavivirus disease, provides a model to study DC responses to a self-limited human viral infection. We analyzed circulating DC subsets in a prospective study of children with dengue across a broad range of illness severities: healthy controls; mild, nondengue, presumed viral infections; moderately ill dengue fever; and, the most severe form of illness, dengue hemorrhagic fever. We also examined PDC responses in monkeys with asymptomatic dengue viremia and to dengue virus exposure in vitro. The absolute number and frequency of circulating pre-mDCs early in acute viral illness decreased as illness severity increased. Depressed pre-mDC blood levels appeared to be part of the typical innate immune response to acute viral infection. The frequency of circulating PDCs trended upward and the absolute number of circulating PDCs remained stable early in moderately ill children with dengue fever, mild other, nondengue, febrile illness, and monkeys with asymptomatic dengue viremia. However, there was an early decrease in circulating PDC levels in children who subsequently developed dengue hemorrhagic fever. A blunted blood PDC response to dengue virus infection was associated with higher viremia levels, and was part of an altered innate immune response and pathogenetic cascade leading to severe disease.
Unopposed Matrix Metalloproteinase-9 Expression in Human Tuberculous Granuloma and the Role of TNF-α-Dependent Monocyte Networks
Authors: Price Nicholas M. , Gilman Robert H. , Uddin Jasim , Recavarren Sixto , Friedland Jon S.
Published: April 2014

Tuberculosis is characterized by granuloma formation and caseous necrosis, but the factors causing tissue destruction are poorly understood. Matrix metalloproteinase (MMP)-9 (92-kDa gelatinase) secretion from monocytes is stimulated by Mycobacterium tuberculosis (M. tb) and associated with local tissue injury in tuberculosis patients. We demonstrate strong immunohistochemical MMP-9 staining in monocytic cells at the center of granuloma and adjacent to caseous necrosis in M. tb-infected patient lymph nodes. Minimal tissue inhibitor of MMPs-1 staining indicated that MMP-9 activity is unopposed. Because granulomas characteristically contain few mycobacteria, we investigated whether monocyte-monocyte cytokine networks amplify MMP-9 secretion. Conditioned medium from M. tb-infected primary human monocytes or THP-1 cells (CoMTB) stimulated MMP-9 gene expression and a >10-fold increase in MMP-9 secretion by monocytes at 3–4 days (p < 0.009, vs controls). Although CoMTB stimulated dose-dependent MMP-9 secretion, MMP-1 (52-kDa collagenase) was not induced. Anti-TNF-α Ab but not IL-1R antagonist pretreatment decreased CoMTB-induced MMP-9 secretion by 50% (p = 0.0001). Anti-TNF-α Ab also inhibited MMP-9 secretion from monocytic cells by 50%, 24 h after direct M. tb infection (p = 0.0002). Conversely, TNF-α directly stimulated dose-dependent MMP-9 secretion. Pertussis toxin inhibited CoMTB-induced MMP-9 secretion and enhanced the inhibitory effect of anti-TNF-α Ab (p = 0.05). Although chemokines bind to G protein-linked receptors, CXCL8, CXCL10, CCL2, and CCL5 did not stimulate monocyte MMP-9 secretion. However, the response to cholera toxin confirmed that G protein signaling pathways were intact. In summary, MMP-9 within tuberculous granuloma is associated with tissue destruction, and TNF-α, critical for antimycobacterial granuloma formation, is a key autocrine and paracrine regulator of MMP-9 secretion.
Peptide-MHC Class II Dimers as Therapeutics to Modulate Antigen-Specific T Cell Responses in Autoimmune Diabetes
Authors: Masteller Emma L. , Warner Matthew R. , Ferlin Walter , Judkowski Valeria , Wilson Darcy , Glaichenhaus Nicolas , Bluestone Jeffrey A.
Published: April 2014

Type 1 diabetes is an autoimmune disorder caused by autoreactive T cells that mediate destruction of insulin-producing β cells of the pancreas. Studies have shown that T cell tolerance can be restored by inducing a partial or altered signal through the TCR. To investigate the potential of bivalent peptide-MHC class II/Ig fusion proteins as therapeutics to restore Ag-specific tolerance, we have developed soluble peptide I-Ag7 dimers for use in the nonobese diabetic mouse model of diabetes. I-Ag7 dimers with a linked peptide specific for islet-reactive BDC2.5 TCR transgenic CD4+ T cells were shown to specifically bind BDC2.5 T cells as well as a small population of Ag-specific T cells in nonobese diabetic mice. In vivo treatment with BDC2.5 peptide I-Ag7 dimers protected mice from diabetes mediated by the adoptive transfer of diabetogenic BDC2.5 CD4+ T cells. The dimer therapy resulted in the activation and increased cell death of transferred BDC2.5 CD4+ T cells. Surviving cells were hypoproliferative to challenge by Ag and produced increased levels of IL-10 and decreased levels of IFN-γ compared with cells from control I-Ag7 dimer-treated mice. Anti-IL-10R therapy reversed the tolerogenic effects of the dimer. Thus, peptide I-Ag7 dimers induce tolerance of BDC2.5 TCR T cells through a combination of the induction of clonal anergy and anti-inflammatory cytokines.
IFN-γ Polymorphisms (IFN-γ +2109 and IFN-γ +3810) Are Associated with Severe Hepatic Fibrosis in Human Hepatic Schistosomiasis (Schistosoma mansoni)
Authors: Chevillard Christophe , Moukoko Carole Eboumbou , Elwali Nasr-Eldin M. A. , Bream Jay H. , Kouriba Bourema , Argiro Laurent , Rahoud Siddig , Mergani Adil , Henri Sandrine , Gaudart Jean , Mohamed-Ali Qurashi , Young Howard A. , Dessein Alain J.
Published: April 2014

Schistosome infection is a major public health concern affecting millions of people living in tropical regions of Africa, Asia, and South America. Schistosomes cause mild clinical symptoms in most subjects, whereas a small proportion of individuals presents severe clinical disease (as periportal fibrosis (PPF)) that may lead to death. Severe PPF results from an abnormal deposition of extracellular matrix proteins in the periportal spaces due to a chronic inflammation triggered by eggs and schistosome Ags. Extracellular matrix protein production is regulated by a number of cytokines, including IFN-γ. We have now screened putative polymorphic sites within this gene in a population living in an endemic area for Schistosoma mansoni. Two polymorphisms located in the third intron of the IFN-γ gene are associated with PPF. The IFN-γ +2109 A/G polymorphism is associated with a higher risk for developing PPF, whereas the IFN-γ +3810 G/A polymorphism is associated with less PPF. The polymorphisms result in changes in nuclear protein interactions with the intronic regions of the gene, suggesting that they may modify IFN-γ mRNA expression. These results are consistent with the results of previous studies. Indeed, PPF is controlled by a major locus located on chromosome 6q22-q23, closely linked to the gene encoding the α-chain of the IFN-γ receptor, and low IFN-γ producers have been shown to have an increased risk of severe PPF. Together, these observations support the view that IFN-γ expression and subsequent signal transduction play a critical role in the control of PPF in human hepatic schistosome infection (S. mansoni).
Local Somatic Hypermutation and Class Switch Recombination in the Nasal Mucosa of Allergic Rhinitis Patients
Authors: Coker Heather A. , Durham Stephen R. , Gould Hannah J.
Published: April 2014

Immunoglobulin E is produced by nasal B cells in response to allergen. We have analyzed IgE VH region sequences expressed in the nasal mucosa of patients suffering from allergic rhinitis. VH region sequences were amplified by RT-PCR from IgE+ B cells from nasal biopsies. In two of six patients, sequence analysis clearly demonstrated the presence of closely related IgE+ B cell clones: cells displaying identical signature regions across CDR3/FWR4, indicating a common clonal ancestry, but a mixture of shared and diverse somatic mutations across the VH region. Furthermore, in one of the two patients exhibiting related IgE+ B cell clones, five IgA+ B cell clones, related to the IgE+ B cell family, were also isolated from the patient’s nasal mucosa. This evidence, combined with the local expression of mRNA transcripts encoding activation-induced cytidine deaminase, suggests that local somatic hypermutation, clonal expansion, and class switch recombination occur within the nasal mucosa of allergic rhinitics. The presence of related B cells in the nasal mucosa does not appear to result from the random migration of IgE+ cells from the systemic pool, as analysis of a nonatopic subject with highly elevated serum IgE did not exhibit any detectable VH-Cε transcripts in the nasal mucosa. We have provided evidence that suggests for the first time that the nasal mucosa of allergic rhinitics is an active site for local somatic hypermutation, clonal expansion, and class switch recombination, making it of major significance for the targeting of future therapies.
Development of a DNA Vaccine Designed to Induce Cytotoxic T Lymphocyte Responses to Multiple Conserved Epitopes in HIV-1
Authors: Wilson Cara C. , McKinney Denise , Anders Michelle , MaWhinney Samantha , Forster Jeri , Crimi Claire , Southwood Scott , Sette Alessandro , Chesnut Robert , Newman Mark J. , Livingston Brian D.
Published: April 2014

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-γ ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.
Conversion of Monophasic to Recurrent Autoimmune Disease by Autoreactive T Cell Subsets
Authors: Shao Hui , Lei Song , Sun Sheher L. , Kaplan Henry J. , Sun Deming
Published: April 2014

Autoimmune uveitis has been elicited in susceptible rodents by several ocular-specific Ags. In most of these animal models the induced uveitis is acute and monophasic. Because recurrent uveitis poses the highest risk for blinding ocular complications in human disease, a spontaneous relapsing animal model would be most helpful in understanding the disease pathogenesis. In our current study we have observed that the adoptive transfer of interphotoreceptor retinoid-binding protein residues 1177–1191-specific T cells to naive Lewis rats induced a chronic relapsing disease, in contrast to the monophasic disease induced by immunization with interphotoreceptor retinoid-binding protein residues 1177–1191 emulsified in CFA. The chronic relapsing uveitis induced by autoreactive T cell subsets is dependent on the number of autoreactive T cells generated as well as their activation status. Our study documented a spontaneous model of recurrent uveitis in the rat, which should assist us in the study of disease pathogenesis and the design of specific therapy.