bluuebunny/crossref_metadata_2025 huggingface dataset:
CD25+CD4+ Cells Contribute to Th2 Polarization during Helminth Infection by Suppressing Th1 Response Development
Authors: McKee Amy S. , Pearce Edward J.
Published: April 2014

Mice infected with Schistosoma mansoni develop polarized Th2 responses in which Th1 responses are prevented by IL-10-mediated suppression of IL-12 production. We show that dendritic cells from infected mice are primed to make IL-12 in response to CD40 ligation, and that IL-10 acts by inhibiting this process. In infected mice, two subpopulations of CD4+ cells, separable by their expression of CD25, make IL-10. CD25+CD4+ cells expressed forkhead box P3, inhibited proliferation of CD4+ T cells, and made IL-10, but little IL-5. In contrast, CD25−CD4+ cells failed to express forkhead box P3 or to inhibit proliferation and accounted for all the IL-5, IL-6, and IL-13 produced by unseparated splenic populations. Thus, CD25+ and CD25− subpopulations could be characterized as regulatory T cells (Treg cells) and Th2 cells, respectively. Consistent with their ability to make IL-10, both CD25+ and CD25−CD4+ T cells from infected mice were able, when stimulated with egg Ag, to suppress IL-12 production by CD40 agonist-stimulated dendritic cells. Additionally, in adoptive transfer experiments, both CD4+ subpopulations of cells were able to partially inhibit the development of Th1 responses in egg-immunized IL-10−/− mice. The relationship of Treg cells in infected mice to natural Treg cells was strongly suggested by the ability of CD25+CD4+ cells from naive mice to inhibit Th1 response development when transferred into egg-immunized or infected IL-10−/− mice. The data suggest that natural Treg cells and, to a lesser extent, Th2 cells play roles in suppressing Th1 responses and ensuring Th2 polarization during schistosomiasis.
CD154 Signaling Regulates the Th1 Response to Herpes Simplex Virus-1 and Inflammation in Infected Corneas
Authors: Xu Min , Lepisto Andrew J. , Hendricks Robert L.
Published: April 2014

Approximately 7 days after HSV-1 corneal infection, BALB/c mice develop tissue-destructive inflammation in the cornea termed herpes stromal keratitis (HSK), as well as periocular skin lesions that are characterized by vesicles, edema, and fur loss. CD4+ T cells and Th1 cytokines contribute to both the immunopathology in the cornea and the eradication of viral replication in the skin. We demonstrate that disruption of CD40/CD154 signaling does not impact the initial expansion of CD4+ T cells in the draining lymph nodes, but dramatically reduces the persistence and Th1 polarization of these cells. Despite the reduced Th1 response, CD154−/− mice developed HSK and periocular skin disease with similar kinetics and severity (as assessed by clinical examination) as wild-type (WT) mice. However, when the composition of the inflammatory infiltrate was examined by flow cytometric analysis, CD154−/− mice exhibited significantly fewer CD4+ and CD8+ T cells and neutrophils than WT mice at the peak of HSK. Moreover, CD4+ T cells from infected corneas of CD154−/− mice produced significantly less IFN-γ than those of WT mice when stimulated with viral Ags in vitro. The IFN-γ production of cells from infected corneas of WT mice was not affected by addition of anti-CD154 mAb to the stimulation cultures. This suggests that CD154 signaling is required at the inductive phase, but not at the effector phase, of the Th1 response within the infected cornea. We conclude that local disruption of CD40/CD154 signaling is not likely to be a useful therapy for HSK.
Schistosoma mansoni Worms Induce Anergy of T Cells via Selective Up-Regulation of Programmed Death Ligand 1 on Macrophages
Authors: Smith Philip , Walsh Caitriona M. , Mangan Niamh E. , Fallon Rosie E. , Sayers Jon R. , McKenzie Andrew N. J. , Fallon Padraic G.
Published: April 2014

Infectious pathogens can selectively stimulate activation or suppression of T cells to facilitate their survival within humans. In this study we demonstrate that the trematode parasite Schistosoma mansoni has evolved with two distinct mechanisms to suppress T cell activation. During the initial 4- to 12-wk acute stages of a worm infection both CD4+ and CD8+ T cells are anergized. In contrast, infection with male and female worms induced T cell anergy at 4 wk, which was replaced after egg laying by T cell suppression via a known NO-dependent mechanism, that was detected for up to 40 wk after infection. Worm-induced anergy was mediated by splenic F4/80+ macrophages (Mφ) via an IL-4-, IL-13-, IL-10-, TGF-β-, and NO-independent, but cell contact-dependent, mechanism. F4/80+ Mφ isolated from worm-infected mice were shown to induce anergy of naive T cells in vitro. Furthermore, naive Mφ exposed to live worms in vitro also induced anergy in naive T cells. Flow cytometry on in vivo and in vitro worm-modulated Mφ revealed that of the family of B7 costimulatory molecules, only programmed death ligand 1 (PD-L1) was selectively up-regulated. The addition of inhibitory mAb against PD-L1, but not PD-L2, to worm-modulated Mφ completely blocked the ability of these cells to anergize T cells. These data highlight a novel mechanism through which S. mansoni worms have usurped the natural function of PD-L1 to reduce T cell activation during early acute stages of infection before the subsequent emergence of egg-induced T cell suppression in the chronic stages of infection.
Human Dendritic Cells Respond to Helicobacter pylori, Promoting NK Cell and Th1-Effector Responses In Vitro
Authors: Hafsi Nadia , Voland Petra , Schwendy Susanne , Rad Roland , Reindl Wolfgang , Gerhard Markus , Prinz Christian
Published: April 2014

Helicobacter pylori infection leads to chronic gastric inflammation. The current study determined the response of human APCs, NK cells, and T cells toward the bacteria in vitro. Human monocyte-derived dendritic cells (DC) were incubated with bacteria for 48 h. Intact H. pylori at a multitude of infection 5 stimulated the expression of MHC class II (4- to 7-fold), CD80, and CD86 B7 molecules (10- to 12-fold) and the CD83 costimulatory molecule (>30-fold) as well as IL-12 secretion (>50-fold) in DCs, and thereby, strongly induced their maturation and activation. CD56+/CD4− NK cells, as well as CD4+/CD45RA+ naive T cells, were isolated and incubated with DCs pulsed with intact bacteria or different cellular fractions. Coculture of H. pylori-pulsed DCs with NK cells strongly potentiated the secretion of TNF-α and IFN-γ. Coculture of naive T cells with H. pylori-pulsed DCs significantly enhanced TNF-α, IFN-γ, and IL-2 secretion as well as T-bet mRNA levels, while GATA-3 mRNA was lowered. However, the effect appeared attenuated compared with coculture with Escherichia coli. A greater stimulation was seen with naive T cells and DCs pulsed with H. pylori membrane preparations. Intact H. pylori potently induced the maturation and activation of human monocyte-derived DC and thereby promote NK and Th1 effector responses. The strong activation of NK cells may be important for the innate immune response. Th1-polarized T cells were induced especially by incubation with membrane preparations of H. pylori, suggesting that membrane proteins may account for the specific adaptive immune response.
Fluid Shear Regulates the Kinetics and Receptor Specificity of Staphylococcus aureus Binding to Activated Platelets
Authors: Pawar Parag , Shin Pyong Kyun , Mousa Shaker A. , Ross Julia M. , Konstantopoulos Konstantinos
Published: April 2014

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1β. Peak adhesion efficiency occurs at low shear (100 s−1) and decreases with increasing shear. The molecular interaction of platelet αIIbβ3 with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (≤400 s−1), the involvement of platelet gpIb and staphylococcal protein A through von Willebrand factor bridging is essential for optimal recruitment of S. aureus cells by platelets in the high shear regime. IgG plays an inhibitory role in the adhesion process, presumably by interfering with the binding of von Willebrand factor to staphylococcal protein A. This study demonstrates that platelet activation and a fluid-mechanical environment representative of the vasculature affect platelet-S. aureus cell-adhesive interactions pertinent to the process of S. aureus-induced bloodstream infections.
Type I IFN Protects Permissive Macrophages fromLegionella pneumophilaInfection through an IFN-γ-Independent Pathway
Authors: Schiavoni Giovanna , Mauri Claudia , Carlei Davide , Belardelli Filippo , Castellani Pastoris Maddalena , Proietti Enrico
Published: April 2014

Legionella pneumophila is an intracellular pathogen whose replication in macrophages is mainly controlled by IFN-γ. Freshly isolated peritoneal macrophages elicited in vivo with thioglycolate (TG) from A/J mice are highly permissive to L. pneumophila growth in vitro, while TG-elicited macrophages from CD1 mice are resistant. In this study, we show that when CD1 TG-macrophages are cultured for 7 days, they become permissive to Legionella infection. We demonstrate that treatment with type I IFN (IFN-αβ) totally inhibits the growth of L. pneumophila in both freshly isolated A/J and in vitro-aged CD1 TG-macrophages. IFN-αβ protective effect on permissive macrophages was comparable to that induced by IFN-γ. Even low doses of either IFN-α or IFN-β alone were effective in inhibiting L. pneumophila multiplication in macrophage cultures. Notably, treatment of resistant, freshly isolated CD1 TG-macrophages with Ab to mouse IFN-αβ significantly enhanced their susceptibility to Legionella infection in vitro, thus implying a role of endogenous IFN-αβ in mediating the natural resistance of macrophages to L. pneumophila infection. Finally, addition of anti-IFN-γ-neutralizing Ab did not restore Legionella growth in IFN-α- or IFN-β-treated A/J or CD1 permissive macrophages, indicating that IFN-αβ effect was not mediated by IFN-γ. This observation was further confirmed by the finding that IFN-αβ was effective in inhibiting L. pneumophila replication in macrophages from IFN-γ receptor-deficient mice. Taken together, our results provide the first evidence for a role of IFN-αβ in the control of L. pneumophila infection in mouse models of susceptible macrophages and suggest the existence of different pathways for the control of intracellular bacteria in macrophages.
Inhibition of Farnesyltransferase Prevents Collagen-Induced Arthritis by Down-Regulation of Inflammatory Gene Expression through Suppression of p21ras-Dependent NF-κB Activation
Authors: Na Hee-Jun , Lee Seon-Jin , Kang Yun-Chul , Cho Young-Lai , Nam Woo-Dong , Kim Peter K. M. , Ha Kwon-Soo , Chung Hun-Taeg , Lee Hansoo , Kwon Young-Guen , Koh Jong Sung , Kim Young-Myeong
Published: April 2014

Farnesylation of p21ras is an important step in the intracellular signaling pathway of growth factors, hormones, and immune stimulants. We synthesized a potent and selective farnesyltransferase inhibitor (LB42708) with IC50 values of 0.8 nM in vitro and 8 nM in cultured cells against p21ras farnesylation and examined the effects of this inhibitor in the settings of inflammation and arthritis. LB42708 suppressed NF-κB activation and iNOS promoter activity by suppressing the I-κB kinase activity and I-κBα degradation. The inhibitor suppressed the expression of inducible NO synthase, cyclooxygenase-2, TNF-α, and IL-1β and the production of NO and PGE2 in immune-activated macrophages and osteoblasts as well as LPS-administrated mice. Furthermore, in vivo administration of LB42708 significantly decreased the incidence and severity of arthritis as well as mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-α, and IL-1β in the paws of collagen-induced arthritic mice compared with controls. These observations indicate that the anti-inflammatory and antiarthritic effects of the farnesyltransferase inhibitor may be ascribed to the inhibition of I-κB kinase activity and subsequent suppression of NF-κB-dependent inflammatory gene expression through the suppression of p21ras farnesylation. Together, these findings reveal that the inhibitory effect of LB42708 on p21ras-dependent NF-κB activation may have potential therapeutic value for arthritis and other inflammatory diseases.
The Lectin-Like Domain of Complement Receptor 3 Protects Endothelial Barrier Function from Activated Neutrophils
Authors: Tsikitis Vassiliki L. , Morin Nicole A. , Harrington Elizabeth O. , Albina Jorge E. , Reichner Jonathan S.
Published: April 2014

The adhesion of neutrophils to endothelial cells is a central event leading to diapedesis and involves the binding of the I-domain of β2 integrins (CD11/CD18) to endothelial ICAMs. In addition to the I-domain, the β2 integrin complement receptor 3 (CR3) (CD11b/CD18) contains a lectin-like domain (LLD) that can alter leukocyte functions such as chemotaxis and cytotoxicity. The present study demonstrates that, in contrast to the CR3 I-domain, Ab blockade of the CR3 LLD has no role in mediating neutrophil-induced loss of endothelial barrier function. However, activation of CR3 with the LLD agonist β-glucan protects the barrier function of endothelial cells in the presence of activated neutrophils and reduces transendothelial migration without affecting adhesion of the neutrophils to the endothelium. The LLD site-specific mAb VIM12 obviates β-glucan protection while activation of the LLD by VIM12 cross-linking mimics the β-glucan response by both preserving endothelial barrier function and reducing neutrophil transendothelial migration. β-glucan has no direct effect on endothelial cell function in the absence of activated neutrophils. These findings demonstrate that signaling through the CR3 LLD prevents neutrophil-induced loss of endothelial barrier function and reduces diapedesis. This suggests that the LLD may be a suitable target for oligosaccharide-based anti-inflammatory therapeutics.
Differential Induction of IL-1β and TNF by CD40 Ligand or Cellular Contact with Stimulated T Cells Depends on the Maturation Stage of Human Monocytes
Authors: Burger Danielle , Molnarfi Nicolas , Gruaz Lyssia , Dayer Jean-Michel
Published: April 2014

Cellular contact with stimulated T cells potently induces cytokine production in monocytes, a mechanism that is likely to be relevant to chronic inflammation. Although the identity of surface molecules involved in this process remains elusive, CD40 and its ligand, CD40L, are thought to be implicated, considering that they are expressed at the inflammatory site. To ascertain the involvement of CD40L, we compared the activation of three different types of human monocytic cells, i.e., freshly isolated monocytes, monocytes primed with IFN-γ (IFN-γ-macrophages), and THP-1 cells. These cells were activated by either membranes isolated from stimulated T cells (HUT-78 or T lymphocytes) to mimic cellular contact, soluble extracts from isolated membranes, or CD40L trimer (CD40LT). The production of TNF and IL-1β was induced by membranes of stimulated T cells in the three types of target cells, whereas CD40LT induced TNF production in IFN-γ-macrophages only. Similar results were obtained with soluble extracts of T cell membranes, demonstrating that the difference between membranes and CD40LT was not due to the particulate form of membranes. CD40LT induced neither transcript nor protein of cytokines in monocytes, whereas in IFN-γ-macrophages, IL-1β and TNF mRNA were observed, but only TNF was measured in cell supernatants. Finally, anti-CD40L Abs failed to inhibit TNF and IL-1β production induced in IFN-γ-macrophages by solubilized membranes, whereas TNF production induced by CD40LT was inhibited. These results demonstrate that CD40L is not required in monocyte activation by direct cellular contact with stimulated T cells, although soluble CD40LT induces the production of TNF in IFN-γ-macrophages.
Induction and Maintenance of Airway Responsiveness to Allergen Challenge Are Determined at the Age of Initial Sensitization
Authors: Gelfand Erwin W. , Joetham Anthony , Cui Zhi-Hua , Balhorn Annette , Takeda Katsuyuki , Taube Christian , Dakhama Azzeddine
Published: April 2014

Age is an important factor in determining the quantity and quality of immune responses when challenged with allergen. In a model of allergen-induced airway hyperresponsiveness and inflammation, where the sensitization phase and challenge phases can be dissociated in time, we examined the impact of age on these two phases. Sensitization of young mice (1–20 wk), but not older animals (30–40 wk), led to the development of airway hyperresponsiveness, airway eosinophilia, Th2 cytokine responses, and allergen-specific IgE, regardless of the age when the challenge phase was conducted. Thus, age at the time of initial sensitization was shown to be the critical factor dictating the nature of the response to later allergen challenge, as older mice remained responsive to allergen challenge if sensitized at a young age. These effects were shown to be mediated by lung T cells from sensitized young mice. Moreover, the failure of old sensitized mice to mediate these effects was shown not to be the result of active suppression of the responses. These data define the importance of age at initial allergen exposure in dictating subsequent responses in the lung when exposed to allergen and may help to define why asthma, even in adults, is most often initiated in early childhood.
Pneumolysin-Induced Lung Injury Is Independent of Leukocyte Trafficking into the Alveolar Space
Authors: Maus Ulrich A. , Srivastava Mrigank , Paton James C. , Mack Matthias , Everhart M. Brett , Blackwell Timothy S. , Christman John W. , Schlöndorff Detlef , Seeger Werner , Lohmeyer Jürgen
Published: April 2014

Pneumolysin (PLY) is a major virulence factor released by Streptococcus pneumoniae and has been implicated in the pathogenesis of pneumococcal pneumonia. In this study, we evaluated the contribution of newly recruited neutrophils and monocytes and resident alveolar macrophages to the pathogenesis of PLY-induced lung injury. Mice received either adhesion-blocking Abs to inhibit alveolar leukocyte trafficking or liposomal clodronate to deplete alveolar macrophages before intratracheal application of native PLY or its noncytotoxic derivative PdB. We found that treatment with PLY but not PdB resulted in increased lung vascular permeability. In addition, PLY also induced a decrease in the resident alveolar macrophage population, and the recruitment of peripheral blood neutrophils and monocytes into the alveolar space. Blockade of PLY-induced alveolar leukocyte trafficking by pretreatment of mice with anti-CD18 plus anti-CD49d Abs or depletion of circulating neutrophils did not attenuate the increase in lung permeability observed in response to intratracheal PLY. In addition, depletion of resident alveolar macrophages with clodronated liposomes did not reduce alveolar injury developing in response to PLY. PLY-induced lung injury was associated with only a small increase in bronchoalveolar lavage concentrations of cytokines. These data indicate that PLY-induced lung injury results from direct pneumotoxic effects on the alveolar-capillary barrier and is independent of both resident and recruited phagocytic cells.
TNF-Induced β2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK
Authors: Bouaouina Mohamed , Blouin Eric , Halbwachs-Mecarelli Lise , Lesavre Philippe , Rieu Philippe
Published: April 2014

We previously demonstrated that the TNF-α-induced inside-out signaling leading to β2 integrin activation is redox regulated. To identify kinases involved in this pathway, the effects of kinase inhibitors on the expression of β2 integrin activation neoepitope (clone 24) were investigated. We show that both p38 MAPK (inhibited by SB203580) and Src kinases (inhibited by PP2) are involved in β2 integrin activation by TNF and oxidants in human neutrophils. Src kinases appeared constitutively active in resting neutrophils and not further activated by TNF or oxidants in nonadherent conditions. However, PP2 blocked both TNF-induced expression of the 24 epitope and cell adhesion promoted by the integrin activating anti-CD18 KIM185 mAb, showing that both the inside-out and the outside-in signaling involve Src kinases. p38 MAPK was activated by TNF and oxidants in nonadherent conditions i.e., with 10 mM EDTA. This activation in EDTA resulted in CD11b, CD35 and CD66 up-regulation and in an oxidative response, all blocked by SB203580 and PP2. p38 MAPK was not activated upon direct integrin activation by KIM185 mAb. Thus, p38 activation allows the study to distinguish the initial transduction pathway leading to β2 integrin activation from the signaling resulting from integrin engagement. Finally, p38 MAPK activation by TNF was blocked by diphenylene iodonium, an inhibitor of flavoprotein oxidoreductase, and by the free radical scavenger N-acetylcystein. Taken together, these results demonstrate, for the first time, that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to β2 integrin activation.
E-Prostanoid-3 Receptors Mediate the Proinflammatory Actions of Prostaglandin E2 in Acute Cutaneous Inflammation
Authors: Goulet Jennifer L. , Pace Amy J. , Key Mikelle L. , Byrum Robert S. , Nguyen MyTrang , Tilley Stephen L. , Morham Scott G. , Langenbach Robert , Stock Jeffrey L. , McNeish John D. , Smithies Oliver , Coffman Thomas M. , Koller Beverly H.
Published: April 2014

PGs are derived from arachidonic acid by PG-endoperoxide synthase (PTGS)-1 and PTGS2. Although enhanced levels of PGs are present during acute and chronic inflammation, a functional role for prostanoids in inflammation has not been clearly defined. Using a series of genetically engineered mice, we find that PTGS1 has the capacity to induce acute inflammation, but PTGS2 has negligible effects on the initiation of this response. Furthermore, we show that the contribution of PTGS1 is mediated by PGE2 acting through the E-prostanoid (EP)3 receptor. Moreover, in the absence of EP3 receptors, inflammation is markedly attenuated, and the addition of nonsteroidal anti-inflammatory agents does not further impair the response. These studies demonstrate that PGE2 promotes acute inflammation by activating EP3 receptors and suggest that EP3 receptors may be useful targets for anti-inflammatory therapy.
Immunoregulatory Role of Nitric Oxide in Kilham Rat Virus-Induced Autoimmune Diabetes in DR-BB Rats
Authors: Mendez Israel I. , Chung Young-Hwa , Jun Hee-Sook , Yoon Ji-Won
Published: April 2014

Macrophages play a critical role in the pathogenesis of Kilham rat virus (KRV)-induced autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats. This investigation was initiated to determine the role of macrophage-derived soluble mediators, particularly NO, in the pathogenesis of KRV-induced diabetes in DR-BB rats. We found that the expression of inducible NO synthase (iNOS), an enzyme responsible for NO production, was significantly increased during the early phase of KRV infection. Inhibition of iNOS by aminoguanidine (AG) treatment resulted in the prevention of diabetes in KRV-infected animals. The expression of IL-1β, TNF-α, and IL-12 was significantly decreased in the spleen of AG-treated, KRV-infected DR-BB rats compared with PBS-treated, KRV-infected control rats. Subsequent experiments revealed that AG treatment exerted its preventive effect in KRV-infected rats by maintaining the finely tuned immune balance normally disrupted by KRV, evidenced by a significant decrease in the expression of IFN-γ, but not IL-4, and a decrease in Th1-type chemokine receptors CCR5, CXCR3, and CXCR4. We also found that iNOS inhibition by AG decreased the KRV-induced expression of MHC class II molecules and IL-2R α-chain, resulting in the suppression of T cell activation, evidenced by the decreased cytolytic activity of CD8+ T cells. We conclude that NO plays a critical immunoregulatory role by up-regulating macrophage-derived proinflammatory cytokines, up-regulating the Th1 immune response, and activating T cells, leading to type 1 diabetes after KRV infection, whereas suppression of NO production by AG treatment prevents KRV-induced autoimmune diabetes in DR-BB rats.
Regulation of Endotoxin-Induced Proinflammatory Activation in Human Coronary Artery Cells: Expression of Functional Membrane-Bound CD14 by Human Coronary Artery Smooth Muscle Cells
Authors: Stoll Lynn L. , Denning Gerene M. , Li Wei-Gen , Rice James B. , Harrelson Allan L. , Romig Sara A. , Gunnlaugsson Skuli T. , Miller Francis J. , Weintraub Neal L.
Published: April 2014

Low-level endotoxemia has been identified as a powerful risk factor for atherosclerosis. However, little is known about the mechanisms that regulate endotoxin responsiveness in vascular cells. We conducted experiments to compare the relative responses of human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) to very low levels of endotoxin, and to elucidate the mechanisms that regulate endotoxin responsiveness in vascular cells. Endotoxin (≤1 ng/ml) caused production of chemotactic cytokines in HCAEC. Endotoxin-induced cytokine production was maximal at LPS-binding protein:soluble CD14 ratios <1, typically observed in individuals with subclinical infection; higher LPS-binding protein:soluble CD14 ratios were inhibitory. Endotoxin potently activated HCASMC, with cytokine release >10-fold higher in magnitude at >10-fold lower threshold concentrations (10–30 pg/ml) compared with HCAEC. This remarkable sensitivity of HCASMC to very low endotoxin concentrations, comparable to that found in circulating monocytes, was not due to differential expression of TLR4, which was detected in HCAEC, HCASMC, and intact coronary arteries. Surprisingly, membrane-bound CD14 was detected in seven different lines of HCASMC, conferring responsiveness to endotoxin and to lipoteichoic acid, a product of Gram-positive bacteria, in these cells. These results suggest that the low levels of endotoxin associated with increased risk for atherosclerosis are sufficient to produce inflammatory responses in coronary artery cells. Because CD14 recognizes a diverse array of inflammatory mediators and functions as a pattern recognition molecule in inflammatory cells, expression of membrane-bound CD14 in HCASMC implies a potentially broader role for these cells in transducing innate immune responses in the vasculature.
Antineuroinflammatory Effect of NF-κB Essential Modifier-Binding Domain Peptides in the Adoptive Transfer Model of Experimental Allergic Encephalomyelitis
Authors: Dasgupta Subhajit , Jana Malabendu , Zhou You , Fung Yiu K. , Ghosh Sankar , Pahan Kalipada
Published: April 2014

It has been shown that peptides corresponding to the NF-κB essential modifier-binding domain (NBD) of IκB kinase α or IκB kinase β specifically inhibit the induction of NF-κB activation without inhibiting the basal NF-κB activity. The present study demonstrates the effectiveness of NBD peptides in inhibiting the disease process in adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. Clinical symptoms of EAE were much lower in mice receiving wild-type (wt)NBD peptides compared with those receiving mutated (m)NBD peptides. Histological and immunocytochemical analysis showed that wtNBD peptides inhibited EAE-induced spinal cord mononuclear cell invasion and normalized p65 (the RelA subunit of NF-κB) expression within the spinal cord. Analysis of lymph node cells isolated from donor and recipient mice showed that wtNBD peptides but not mNBD peptides were able to shift the immune response from a Th1 to a Th2 profile. Consistently, wtNBD peptides but not mNBD peptides inhibited the encephalitogenicity of myelin basic protein-specific T cells. Furthermore, i.p. injection of wtNBD peptides but not mNBD peptides was also able to reduce LPS- and IFN-γ-induced expression of inducible NO synthase, IL-1β, and TNF-α in vivo in the cerebellum. Taken together, our results support the conclusion that NBD peptides are antineuroinflammatory, and that NBD peptides may have therapeutic effect in neuroinflammatory disorders such as multiple sclerosis.
Regulatory Role of C5a on Macrophage Migration Inhibitory Factor Release from Neutrophils
Authors: Riedemann Niels C. , Guo Ren-Feng , Gao Hongwei , Sun Lei , Hoesel Marco , Hollmann Travis J. , Wetsel Rick A. , Zetoune Firas S. , Ward Peter A.
Published: April 2014

There is evidence that C5a and macrophage migration inhibitory factor (MIF) both play important roles in experimental sepsis. Humans with sepsis also show elevated levels of both mediators in the blood. Regulation of MIF during sepsis is poorly understood. We now demonstrate that neutrophil depletion greatly reduced serum MIF levels in rats and mice during the onset of sepsis after cecal ligation and puncture. In vitro, C5a induced MIF release from rat and mouse neutrophils. In vivo blockade of C5aR or absence of C5aR led to significantly reduced MIF generation during the onset of sepsis. C5a-induced release in vitro of MIF from neutrophils appeared to be due to up-regulation of MIF in cytoplasmic granules of neutrophils via activation of the protein kinase B signaling pathway together with involvement of PI3K. Our data suggest that C5a plays a role in enhancing MIF release from neutrophils in vitro and during sepsis. These findings represent a previously unrecognized function of C5a and neutrophils in the appearance of MIF in sepsis.
Blockade of Allergic Airway Inflammation Following Systemic Treatment with a B7-Dendritic Cell (PD-L2) Cross-Linking Human Antibody
Authors: Radhakrishnan Suresh , Iijima Koji , Kobayashi Takao , Rodriguez Moses , Kita Hirohito , Pease Larry R.
Published: April 2014

We present a novel immunotherapeutic strategy using a human B7-DC cross-linking Ab that prevents lung inflammation, airway obstruction, and hyperreactivity to allergen in a mouse model of allergic inflammatory airway disease. Dendritic cells (DC) have the ability to skew the immune response toward a Th1 or Th2 polarity. The sHIgM12 Ab functions in vitro by cross-linking the costimulatory family molecule B7-DC (PD-L2) on DC up-regulating IL-12 production, homing to lymph nodes, and T cell-activating potential of these APCs. Using chicken OVA as a model Ag, the administration of sHIgM12 Ab to BALB/c mice blocked lung inflammation, airway pathology, and responsiveness to methacholine, even after animals were presensitized and a Th2-polarized immune response was established. This therapeutic strategy was ineffective in STAT4-deficient animals, indicating that IL-12 production is critical in this system. Moreover, the polarity of the immune response upon in vitro restimulation with Ag is changed in wild-type mice, with a resulting decrease in Th2 cytokines IL-4 and IL-5 and an increase in the immunoregulatory cytokine IL-10. These studies demonstrate that the immune response of hypersensitized responders can be modulated using B7-DC cross-linking Abs, preventing allergic airway disease upon re-exposure to allergen.
An Advanced Intercross Line ResolvesEae18into Two Narrow Quantitative Trait Loci Syntenic to Multiple Sclerosis Candidate Loci
Authors: Jagodic Maja , Becanovic Kristina , Sheng Jian Rong , Wu Xingchen , Bäckdahl Liselotte , Lorentzen Johnny C. , Wallström Erik , Olsson Tomas
Published: April 2014

Identification of polymorphic genes regulating inflammatory diseases may unravel crucial pathogenic mechanisms. Initial steps to map such genes using linkage analysis in F2 intercross or backcross populations, however, result in broad quantitative trait loci (QTLs) containing hundreds of genes. In this study, an advanced intercross line in combination with congenic strains, was used to fine-map Eae18 on rat chromosome 10 in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE). Myelin oligodendrocyte glycoprotein-induced EAE is a chronic relapsing disease that closely mimics key features of multiple sclerosis. Congenic DA.ACI rat strains localized Eae18 to an ∼30-Mb large region. Fine-mapping was then performed in an advanced intercross line consisting of a (DA × PVG.1AV1)F7 intercross, resulting in two adjacent EAE-regulating QTLs designated Eae18a and Eae18b. The two QTLs span 5.5 and 3 Mb, respectively, and the 3-Mb Eae18b contains as few as 10 genes, including a cluster of chemokine genes (CCL1, CCL2, CCL7, and CCL11). Eae18a and Eae18b are syntenic to human chromosome 17p13 and 17q11, respectively, which both display linkage to multiple sclerosis. Thus, Eae18 consists of at least two EAE-regulating genes, providing additional evidence that clustering of disease-regulating genes in QTLs is an important phenomenon. The overlap between Eae18a and Eae18b with previously identified QTLs in humans and mice further supports the notion that susceptibility alleles in inflammatory disease are evolutionary conserved between species.
Activated Ets2 Is Required for Persistent Inflammatory Responses in the Motheaten Viable Model
Authors: Wei Guo , Guo Jianping , Doseff Andrea I. , Kusewitt Donna F. , Man Albert K. , Oshima Robert G. , Ostrowski Michael C.
Published: April 2014

The Ets2 transcription factor is constitutively phosphorylated on residue Thr72 in macrophages derived from mice homozygous for the motheaten viable (me-v) allele of the hemopoietic cell phosphatase (Hcph) gene. To genetically test the importance of signaling through residue Thr72 of Ets2 during inflammation, the Ets2A72 mutant allele, which cannot be phosphorylated on Thr72, was combined with the Hcphme-v allele in mice. Ets2A72/A72 moderated the inflammation-related pathology of Hcphme-v/me-v mice, as demonstrated by the increased life span and the decreased macrophage infiltration in skin and lungs of these mice. Macrophage apoptosis induced by cytokine withdrawal was also increased in the double-mutant mice. Importantly, the Ets2A72/A72 allele resulted in decreased expression of inflammatory response genes, including TNF-α, CCL3, matrix metalloprotease 9, integrin αM, and Bcl-X in alveolar macrophage. Ets2 phosphorylation was required for persistent activation of TNF-α following LPS stimulation of bone marrow-derived macrophages. The phosphorylation of Ets2 functions in the severe inflammatory phenotype of the me-v model by mediating both macrophage survival and inflammatory gene expression.
A3 Adenosine Receptor Signaling Contributes to Airway Inflammation and Mucus Production in Adenosine Deaminase-Deficient Mice
Authors: Young Hays W. J. , Molina Jose G. , Dimina Dawn , Zhong Hongyan , Jacobson Marlene , Chan Lee-Nien L. , Chan Teh-Sheng , Lee James J. , Blackburn Michael R.
Published: April 2014

Adenosine signaling has been implicated in chronic lung diseases such as asthma and chronic obstructive pulmonary disease; however, the specific roles of the various adenosine receptors in processes central to these disorders are not well understood. In this study, we have investigated the role(s) of the A3 adenosine receptor in adenosine-dependent pulmonary inflammation observed in adenosine deaminase (ADA)-deficient mice. The A3 receptor (A3R) was found to be expressed in eosinophils and mucus-producing cells in the airways of ADA-deficient mice. Treatment of ADA-deficient mice with MRS 1523, a selective A3R antagonist, prevented airway eosinophilia and mucus production. Similar findings were seen in the lungs of ADA/A3 double knockout mice. Although eosinophils were decreased in the airways of ADA-deficient mice following antagonism or removal of the A3R, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3R is needed for the migration of eosinophils into the airways. These findings identify an important role for the A3R in regulating lung eosinophilia and mucus production in an environment of elevated adenosine.
Mucosal T Cells Bearing TCRγδ Play a Protective Role in Intestinal Inflammation
Authors: Inagaki-Ohara Kyoko , Chinen Takatoshi , Matsuzaki Goro , Sasaki Atsuo , Sakamoto Yukiko , Hiromatsu Kenji , Nakamura-Uchiyama Fukumi , Nawa Yukifumi , Yoshimura Akihiko
Published: April 2014

Intestinal intraepithelial lymphocytes (IEL) bearing TCRγδ represent a major T cell population in the murine intestine. However, the role of γδ IEL in inflammatory bowel diseases (IBD) remains controversial. In this study, we show that γδ IEL is an important protective T cell population against IBD. γδ T cell-deficient (Cδ−/−) mice developed spontaneous colitis with age and showed high susceptibility to Th1-type 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis at a young age. Transfer of γδ IEL to Cδ−/− mice ameliorated TNBS-induced colitis, which correlated with decrease of IFN-γ and TNF-α production and an increase of TGF-β production by IEL. Furthermore, a high level of IL-15, which inhibits activation-induced cell death to terminate inflammation, was expressed more in intestinal epithelial cells (EC) from TNBS-treated Cδ−/− mice than in those from wild-type mice. EC from wild-type mice significantly suppressed the IFN-γ production of IEL from TNBS-treated Cδ−/− mice, whereas EC from TNBS-treated Cδ−/− mice did not. These data indicate that γδ IEL play important roles in controlling IBD by regulating mucosal T cell activation cooperated with EC function. Our study suggests that enhancement of regulatory γδ T cell activity is a possible new cell therapy for colitis.
Blockade of Late Stages of Autoimmune Diabetes by Inhibition of the Receptor for Advanced Glycation End Products
Authors: Chen Yali , Yan Shirley ShiDu , Colgan John , Zhang Hui-Ping , Luban Jeremy , Schmidt Ann Marie , Stern David , Herold Kevan C.
Published: April 2014

Ligation of the receptor for advanced glycation end products (RAGE) occurs during inflammation. Engagement of RAGE results in enhanced expression of addressins and it is therefore, not surprising that previous studies have shown a role of RAGE/ligand interactions in immune responses including cell/cell contact but the role of RAGE in spontaneous autoimmunity has not been clearly defined. To study the role of RAGE/ligand interactions in autoimmune diabetes, we tested the ability of soluble RAGE, a scavenger of RAGE ligands, in late stages of diabetes development in the NOD mouse-disease transferred with diabetogenic T cells and recurrent disease in NOD/scid recipients of syngeneic islet transplants. RAGE expression was detected on CD4+, CD8+, and B cells from diabetic mice and transferred to NOD/scid recipients. RAGE and its ligand, S100B, were found in the islets of NOD/scid mice that developed diabetes. Treatment of recipient NOD/scid mice with soluble RAGE prevented transfer of diabetes and delayed recurrent disease in syngeneic islet transplants. RAGE blockade was associated with increased expression of IL-10 and TGF-β in the islets from protected mice. RAGE blockade reduced the transfer of disease with enriched T cells, but had no effect when diabetes was transferred with the activated CD4+ T cell clone, BDC2.5. We conclude that RAGE/ligand interactions are involved in the differentiation of T cells to a mature pathogenic phenotype during the late stages of the development of diabetes.
Essential Role of MD-2 in TLR4-Dependent Signaling during Helicobacter pylori-Associated Gastritis
Authors: Ishihara Shunji , Rumi Mohammad Azharul Karim , Kadowaki Yasunori , Ortega-Cava Cesar Francisco , Yuki Takafumi , Yoshino Nagisa , Miyaoka Youichi , Kazumori Hideaki , Ishimura Norihisa , Amano Yuji , Kinoshita Yoshikazu
Published: April 2014

TLR4, a member of pattern recognition receptors, is the main receptor of LPS. MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. Helicobacter pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated in this study the role of MD-2 in TLR4-dependent signaling in H. pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H. pylori infection and four gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-κB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H. pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-κB and IL-8 promoter upon stimulation with H. pylori LPS. Live H. pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H. pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H. pylori LPS in the stomach.
Production of Profibrotic Cytokines by Invariant NKT Cells Characterizes Cirrhosis Progression in Chronic Viral Hepatitis
Authors: de Lalla Claudia , Galli Grazia , Aldrighetti Luca , Romeo Raffaella , Mariani Margherita , Monno Antonella , Nuti Sandra , Colombo Massimo , Callea Francesco , Porcelli Steven A. , Panina-Bordignon Paola , Abrignani Sergio , Casorati Giulia , Dellabona Paolo
Published: April 2014

Invariant (inv)NKT cells are a subset of autoreactive lymphocytes that recognize endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines. We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls (healthy subjects or patients with nonviral benign or malignant focal liver damage and minimal inflammatory response) or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and undergo a substantial modification in their effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13. Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection, and suggest that these cells, possibly triggered by the recognition of CD1d associated with viral- or stress-induced lipid ligands, contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.
The Myelin-Associated Oligodendrocytic Basic Protein Region MOBP15–36 Encompasses the Immunodominant Major Encephalitogenic Epitope(s) for SJL/J Mice and Predicted Epitope(s) for Multiple Sclerosis-Associated HLA-DRB1*1501
Authors: de Rosbo Nicole Kerlero , Kaye Joel F. , Eisenstein Miriam , Mendel Itzhack , Hoeftberger Romana , Lassmann Hans , Milo Roni , Ben-Nun Avraham
Published: April 2014

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15–36 as their major immunodominant epitope. Accordingly, MOBP15–36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-As with MOBP15–36, together with analysis of the MOBP15–36-specific T cell response to truncated peptides, suggests MOBP20–28 as the core sequence for I-As-restricted recognition of the encephalitogenic region MOBP15–36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vβ genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15–36 sequence suggests the potential relevance of T cell reactivity against MOBP15–36 to MS. The reactivity to MOBP15–36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.
Crisscross CTL Induction by SYT-SSX Junction Peptide and Its HLA-A*2402 Anchor Substitute
Authors: Ida Kazunori , Kawaguchi Satoshi , Sato Yuriko , Tsukahara Tomohide , Nabeta Yuki , Sahara Hiroeki , Ikeda Hideyuki , Torigoe Toshihiko , Ichimiya Shingo , Kamiguchi Kenjiro , Wada Takuro , Nagoya Satoshi , Hiraga Hiroaki , Kawai Akira , Ishii Takeshi , Araki Nobuhito , Myoui Akira , Matsumoto Seiichi , Ozaki Toshifumi , Yoshikawa Hideki , Yamashita Toshihiko , Sato Noriyuki
Published: April 2014

To investigate the effects of anchor substitutions in SYT-SSX junction peptide, an HLA-A24 anchor residue (position 9) of the SYT-SSX B peptide (GYDQIMPKK) was substituted to more favorable residues according to the HLA-A24-binding motif. Among four substitutes constructed, a substitute with isoleucine (termed K9I peptide) most apparently enhanced the affinity for HLA-A24 molecule. Subsequent in vitro CTL induction analysis using PBMCs of 15 HLA-A24+ synovial sarcoma patients revealed that the original B peptide allowed to induce synovial sarcoma-specific CTLs from 7 patients (47%), whereas such CTLs were inducible from 12 patients (80%) with K9I peptide. Moreover, the extent of cytotoxicity against HLA-A24+ synovial sarcoma cell lines was higher in K9I peptide-induced CTLs than B peptide-induced CTLs. Influence of anchor substitution on peptide/TCR interaction was evaluated by cytotoxicity assays against autologous cells and tetramer analysis. CTLs induced from a synovial sarcoma patient using K9I peptide did not lyse autologous PHA blasts or EBV-infected B cells. In vitro stimulations of PBMCs from 5 HLA-A24+ synovial sarcoma patients with K9I peptide increased the frequency of T cells reacting with both HLA-A24/K9I peptide tetramer and HLA-A24/B peptide tetramer. In contrast, the frequency of T cells reacting with HLA/HIV-derived peptide tetramer remained low. These findings support the validity in design of anchor residue substitution in SYT-SSX fusion gene-derived peptide, and provide a potential clue to the current stagnation in vaccination trials of fusion gene-derived natural junction peptides.