Glucocorticoid-induced TNFR family-related protein (GITR)-mediated activation of JNK was shown to regulate the suppressive activity of CD4(+)CD25(+) naturally occurring T regulatory cells (nTregs) in wild-type (WT) hosts. In this study, CD4(+)CD25(+) T cells were shown to be capable of becoming pathogenic effector cells in sensitized and challenged CD8(-/-) recipient mice. Only GITR-expressing CD4(+)CD25(+) T cells, but neither GITR knocked-in CD4(+)CD25(-) T cells nor GITR-silenced CD4(+)CD25(+) T cells, enhanced development of lung allergic responses. Inhibition of JNK in WT nTregs or nTregs from GITR(-/-)and JNK2(-/-) mice failed to enhance lung allergic responses in sensitized and challenged CD8(-/-) recipient mice. The failure to enhance responses was associated with increased bronchoalveolar lavage fluid levels of IL-10 and TGF-? and decreased levels of IL-5, IL-6, and IL-13. In contrast, nTregs from JNK1(-/-) mice, similar to WT nTregs, were fully effective in enhancing responses. Thus, GITR stimulation of nTregs and signaling through JNK2, but not JNK1, triggered the loss of regulatory function while concomitantly gaining pathogenic CD4(+) T effector cell function responsible for exacerbating asthma-like immunopathology.
D-Amino acid oxidase (DAO) is structurally unstable and exhibits broad specificity to D-amino acids. In this work, we fabricated a stable liposomal DAO system with high apparent substrate specificity. Permeability of the membrane composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was highly selective between the d-forms of alanine (Ala) and serine (Ser). The permeability coefficient of d-Ala and d-Ser at 25 °C was 3.59 and 0.27 pm/s, respectively, as determined with the dialysis method. On the other hand, the chiral environment of POPC membrane showed no clear selectivity between the enantiomers of Ala or Ser. POPC liposomes encapsulating DAO from porcine kidney selectively catalyzed the oxidation of hydrophobic D-phenylalanine (D-Phe) over D-Ala and D-Ser because of their intrinsic membrane permeability. As a different type of liposomal DAO, the enzyme molecules were conjugated to the surface of activated lipids-bearing liposomes. The activity of liposome-conjugated DAO showed significantly higher stability at 50 °C than free DAO at low enzyme concentrations ranging from 2.5 to 10 mg/L. Then, the DAO-conjugated liposomes were coated with POPC bilayers to give the oligolamellar structure intercalated with the DAO molecules. The additional bilayers allowed to induce the permeability resistance-based substrate specificity and strengthened the stabilizing effect on the DAO activity. The oligolamellar liposomes fabricated can be a colloidal platform for integrating the functions of lipid membrane to stabilize DAO and to modulate its substrate specificity.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illnesses in infants worldwide. Both RSV-G and RSV-F glycoproteins play pathogenic roles during infection with RSV. The objective of this study was to compare the effects of anti-RSV-G and anti-RSV-F monoclonal antibodies (mAbs) on airway hyperresponsiveness (AHR) and inflammation after primary or secondary RSV infection in mice. In the primary infection model, mice were infected with RSV at 6 weeks of age. Anti-RSV-G or anti-RSV-F mAbs were administered 24 hours before infection or Day +2 postinfection. In a secondary infection model, mice were infected (primary) with RSV at 1 week (neonate) and reinfected (secondary) 5 weeks later. Anti-RSV-G and anti-RSV-F mAbs were administered 24 hours before the primary infection. Both mAbs had comparable effects in preventing airway responses after primary RSV infection. When given 2 days after infection, anti-RSV-G-treated mice showed significantly decreased AHR and airway inflammation, which persisted in anti-RSV-F-treated mice. In the reinfection model, anti-RSV-G but not anti-RSV-F administered during primary RSV infection in neonates resulted in decreased AHR, eosinophilia, and IL-13 but increased levels of IFN-? in bronchoalveolar lavage on reinfection. These results support the use of anti-RSV-G in the prevention and treatment of RSV-induced disease.
Activation of the alternative pathway of complement plays a critical role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in mice. Endogenous factor H, a potent inhibitor of the alternative pathway, is increased in the airways of sensitized and challenged mice, but its role in regulating inflammation or AHR has been unknown. We found that blocking the tissue-binding function of factor H with a competitive antagonist increased complement activation and tissue inflammation after allergen challenge of sensitized mice. Conversely, administration of a fusion protein that contains the iC3b/C3d binding region of complement receptor 2 linked to the inhibitory region of factor H, a molecule directly targeting complement-activating surfaces, protected mice in both primary and secondary challenge models of AHR and lung inflammation. Thus, although endogenous factor H does play a role in limiting the development of AHR, strategies to deliver the complement-regulatory region of factor H specifically to the site of inflammation provide greater protection than that afforded by endogenous regulators. Such an agent may be an effective therapy for the treatment of asthma.
Notch signaling pathways govern immune function and the regulation of Th1 and Th2 differentiation. We previously demonstrated essential interactions between Notch on CD4+ T cells and Jagged1 on antigen-presenting cells in Th2 differentiation for the full development of allergen-induced airway hyperresponsiveness (AHR) and allergic airway inflammation.
Naturally occurring CD4(+)CD25(+)Foxp3(+) T regulatory cells (nTregs) regulate lung allergic responses through production of IL-10 and TGF-?. nTregs from CD8(-/-) mice failed to suppress lung allergic responses and were characterized by reduced levels of Foxp3, IL-10, and TGF-?, and high levels of IL-6. Administration of anti-IL-6 or anti-IL-6R to wild-type recipients prior to transfer of CD8(-/-) nTregs restored suppression. nTregs from IL-6(-/-) mice were suppressive, but lost this capability if incubated with IL-6 prior to transfer. The importance of CD8 in regulating the production of IL-6 in nTregs was demonstrated by the loss of suppression and increases in IL-6 following transfer of nTregs from wild-type donors depleted of CD8(+) cells. Transfer of nTregs from CD8(-/-) donors reconstituted with CD8(+) T cells was suppressive, and accordingly, IL-6 levels were reduced. These data identify the critical role of CD8-T regulatory cell interactions in regulating the suppressive phenotype of nTregs through control of IL-6 production.
The aim of this study was to evaluate the role of home medical care support system to relieve the symptom and regional alliances for elderly cancer patients. We investigated clinical parameters to study the features of this system. The home medical care support system is designed for patients who are B75-year-old with decrease in activities of daily living and severe dementia. The support system plays a significant role in patients with impaired oral ingestion, dyspnea, delirium, and a poor general status.
Invariant NKT cells (iNKT cells) play a pivotal role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation. However, it is unclear what role they play in the initiation (sensitization) phase as opposed to the effector (challenge) phase. The role of iNKT cells during sensitization was examined by determining the response of mice to intratracheal transfer of OVA-pulsed or OVA-alpha-galactosylceramide (OVA/alphaGalCer)-pulsed bone marrow-derived dendritic cells (BMDCs) prior to allergen challenge. Wild-type (WT) recipients of OVA-BMDCs developed AHR, increased airway eosinophilia, and increased levels of Th2 cytokines in bronchoalveolar lavage fluid, whereas recipients of OVA/alphaGalCer BMDCs failed to do so. In contrast, transfer of these same OVA/alphaGalCer BMDCs into IFN-gamma-deficient (IFN-gamma(-/-)) mice enhanced the development of these lung allergic responses, which was reversed by exogenous IFN-gamma treatment following OVA-BMDC transfer. Further, Jalpha18-deficient recipients, which lack iNKT cells, developed the full spectrum of lung allergic responses following reconstitution with highly purified WT liver or spleen iNKT cells and transfer of OVA-BMDCs, whereas reconstituted recipients of OVA/alphaGalCer BMDCs failed to do so. Transfer of iNKT cells from IFN-gamma(-/-) mice restored the development of these responses in Jalpha18-deficient recipients following OVA-BMDC transfer; the responses were enhanced following OVA/alphaGalCer BMDC transfer. iNKT cells from these IFN-gamma(-/-) mice produced higher levels of IL-13 in vitro compared with WT iNKT cells. These data identify IFN-gamma as playing a critical role in dictating the consequences of iNKT cell activation in the initiation phase of the development of AHR and airway inflammation.
Respiratory syncytial virus (RSV) bronchiolitis in infants may be followed by the development of asthma-like symptoms. Age at first infection dictates consequences upon reinfection. Reinfection of mice initially exposed as neonates to RSV enhanced development of airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus hyperproduction. RSV lower respiratory tract disease is associated with activation of the leukotriene pathway.
Poor functional fitness of the lower extremities is a potentially modifiable risk factor for falls. This study compared the Aotake stepping exercise, a unique indoor program, to walking and examined improvements in lower-extremity functional fitness.
Jagged1, a Notch ligand, and Notch have been implicated in Th2 differentiation, but their role in initiating IL-4 production and Th2 differentiation in vivo and the development of allergic airway responses has not been defined. In this study, we show that Jagged1 is up-regulated on bone marrow-derived dendritic cells (BMDCs) pulsed with allergen and that the transfer of these BMDCs before allergen challenge induces airway hyperresponsiveness (AHR) and eosinophilic airway inflammation. Treatment of CD4(+) T cells with a gamma-secretase inhibitor (GSI), which inhibits Notch signaling, resulted in decreased cytokine production when the cells were cocultured with allergen-pulsed, Jagged1-expressing BMDCs and, after the transfer of allergen-pulsed BMDCs, IL-4-deficient (IL-4(-/-)) recipients of GSI-treated naive CD4(+) T cells developed lower levels of AHR, reduced numbers of eosinophils, and lower Th2 cytokine levels when challenged with allergen. In vivo treatment of wild-type mice with Jagged1-Fc enhanced AHR and airway inflammation, whereas the transfer of BMDC transfected with Jagged1 small interfering RNA (siRNA) cells into WT or IL-4(-/-) mice before transfer of CD4(+) T cells resulted in decreased AHR, inflammation, and Th2 cytokines, indicating the critical role for Jagged1 expression on APCs. These data identify the essential role of the interactions between Notch on CD4(+) T cells and Jagged1 on APCs in the initiation of IL-4 production and Th2 differentiation for the development of AHR and allergic airway inflammation.
Naturally occurring Foxp3(+)CD4(+)CD25(+) T cells isolated from lungs of naive mice regulate lung allergic airway hyperresponsiveness, inflammation, levels of Th2 cytokines, and mucus production. OVA-specific (alphabetaTCR(+)) CD4(+)CD25(+) T cells suppressed ragweed-induced airway hyperresponsiveness and inflammation as did anti-TCR-treated OVA-specific CD4(+)CD25(+) T cells, suggesting that Ag-specificity was not required for expression of regulatory activities. Suppression was associated with increased levels of IL-10 and TGF-beta; decreased levels of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid; and reduced recruitment and activation of CD8(+) T cells in the airways. Following intratracheal administration, OVA-specific CD4(+)CD25(+) T cells were identified in both the airway lumens and lung parenchyma, and in some instances in close proximity to host CD8(+) T cells. These results demonstrate that the regulatory activities of naturally occurring Foxp3(+)CD4(+)CD25(+) T cells on lung allergic responses are Ag-nonspecific and thus, independent of Ag-specific recognition.
Previous studies have shown that leukotriene B4 (LTB4), a proinflammatory lipid mediator, is linked to the development of airway hyperresponsiveness through the accumulation of IL-13-producing CD8+ T cells, which express a high affinity receptor for LTB4, BLT1 (Miyahara et al., Am J Respir Crit Care Med 2005;172:161-167; J Immunol 2005;174:4979-4984). By using leukotriene A4 hydrolase-deficient (LTA4H-/-) mice, which fail to synthesize LTB4, we determined the role of this lipid mediator in allergen-induced airway responses. Two approaches were used. In the first, LTA4H-/- mice and wild-type (LTA4H+/+) mice were systemically sensitized and challenged via the airways to ovalbumin. In the second, mice were passively sensitized with anti-ovalbumin IgE and exposed to ovalbumin via the airways. Mast cells were generated from bone marrow of LTA4H+/+ mice or LTA4H-/- mice. After active sensitization and challenge, LTA4H-/- mice showed significantly lower airway hyperresponsiveness compared with LTA4H+/+ mice, and eosinophil numbers and IL-13 levels in the bronchoalveoloar lavage of LTA4H-/- mice were also significantly lower. LTA4H-/- mice also showed decreased airway reactivity after passive sensitization and challenge. After LTA4H+/+ mast cell transfer, LTA4H-/- mice showed increased airway reactivity after passive sensitization and challenge, but not after systemic sensitization and challenge. These data confirm the important role for LTB4 in the development of altered airway responses and suggest that LTB4 secretion from mast cells is critical to eliciting increased airway reactivity after passive sensitization with allergen-specific IgE.
Although Pneumocystis infection might be one of the causes of secondary pulmonary alveolar proteinosis (PAP), the mechanism of its pathogenesis is uncertain. We analyzed a mouse model of secondary PAP resulting from Pneumocystis infection using mice deficient in CD40 (CD40KO), and evaluated the mechanism of the pathogenesis of secondary PAP from the viewpoint of surfactant-associated protein (SP) homeostasis, the overproduction of SP by type II alveolar epithelial cells, and the phagocytic function of alveolar macrophages (AMs). The effect of CD40 on SP production was also investigated in vitro using the H441 cell line, which has a phenotype similar to type II alveolar epithelial cells and primary alveolar epithelial cells. After long-term exposure to ovalbumin, CD40KO mice showed Pneumocystis infection and accumulation of surfactants in the alveoli (ApCD40KO). The amounts of SP production were up-regulated in ApCD40KO mice compared with wild-type mice treated using the same procedure. On the other hand, AMs from ApCD40KO mice did not show either phagocytic dysfunction or down-regulation of PU.1 expression. Furthermore, the stimulation of CD40-CD40 ligand (CD154) pathway regulated the production of SPs in H441 cells or primary alveolar epithelial cells. These results suggested that CD40KO mice could be one of the models useful for developing secondary PAP resulting from Pneumocystis infection. Surfactant accumulation was due to the overproduction in our model of secondary PAP. The CD40-CD154 interaction plays an important role in the regulation of surfactant-associated protein production.
gammadelta T cells regulate airway reactivity, but their role in ozone (O3)-induced airway hyperresponsiveness (AHR) is not known. Our objective was to determine the role of gammadelta T cells in O3-induced AHR. Different strains of mice, including those that were genetically manipulated or antibody-depleted to render them deficient in total gammadelta T cells or specific subsets of gammadelta T cells, were exposed to 2.0 ppm of O3 for 3 hours. Airway reactivity to inhaled methacholine, airway inflammation, and epithelial cell damage were monitored. Exposure of C57BL/6 mice to O3 resulted in a transient increase in airway reactivity, neutrophilia, and increased numbers of epithelial cells in the lavage fluid. TCR-delta(-/-) mice did not develop AHR, although they exhibited an increase in neutrophils and epithelial cells in the lavage fluid. Similarly, depletion of gammadelta T cells in wild-type mice suppressed O3-induced AHR without influencing airway inflammation or epithelial damage. Depletion of Vgamma1+, but not of Vgamma4+ T cells, reduced O3-induced AHR, and transfer of total gammadelta T cells or Vgamma1+ T cells to TCR-delta(-/-) mice restored AHR. After transfer of Vgamma1+ cells to TCR-delta(-/-) mice, restoration of AHR after O3 exposure was blocked by anti-TNF-alpha. However, AHR could be restored in TCR-delta(-/-)mice by transfer of gammadelta T cells from TNF-alpha-deficient mice, indicating that another cell type was the source of TNF-alpha. These results demonstrate that TNF-alpha and activation of Vgamma1+ gammadelta T cells are required for the development of AHR after O3 exposure.
Oligolamellar phospholipid vesicles incorporated with d-amino acid oxidase from porcine kidney (OV-DAO) were prepared by encapsulating pre-formed enzyme-bound unilamellar vesicles (UV-DAO) with bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The bilayer of UV-DAO was composed of POPC, 30 mol% of cholesterol and 15 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (NGPE) that was responsible for covalent linking to D-amino acid oxidase (DAO). OV-DAO and UV-DAO showed the activity to catalyze the oxidation of D-alanine as measured based on the hydrogen peroxide produced. The oligolamellar and unilamellar structure of OV-DAO and UV-DAO, respectively was elucidated based on the quenching characteristics of bilayers-incorporated fluorescent lipid 7-nitro-2,1,3-benzoxadiazol-4-yl-phosphoethanolamine (NBD-PE) and the size distribution of the vesicles measured with the dynamic light scattering method. The enzyme activity of OV-DAO and UV-DAO was significantly stabilized at 50°C compared to that of free DAO at the fixed enzyme concentration of 3.29 ?g/mL. At the temperature, OV-DAO and UV-DAO showed the remaining activity of 52.7 and 29.6%, respectively at the incubation time of 20 min while free DAO was completely deactivated. Thus the dimeric form of DAO could be stabilized by its coupling to the surface of UV-DAO membrane being the inner bilayer of OV-DAO. Furthermore, the thermal denaturation of DAO and dissociation of flavin adenine dinucleotide (FAD) from the subunits of enzyme were prevented in the aqueous phase formed between the bilayers of OV-DAO.
Asthma is regarded as a multifactorial inflammatory disorder arising as a result of inappropriate immune responses in genetically susceptible individuals to common environmental antigens. However, the precise molecular basis is unknown. To identify genes for susceptibility to three asthma-related traits, airway hyperresponsiveness (AHR), eosinophil infiltration, and allergen-specific serum IgE levels, we conducted a genetic analysis using SMXA recombinant inbred (RI) strains of mice. Quantitative trait locus analysis detected a significant locus for AHR on chromosome 17. For eosinophil infiltration, significant loci were detected on chromosomes 9 and 16. Although we could not detect any significant loci for allergen-specific serum IgE, analysis of consomic strains showed that chromosomes 17 and 19 carried genes that affected this trait. We detected genetic susceptibility loci that separately regulated the three asthma-related phenotypes. Our results suggested that different genetic mechanisms regulate these asthma-related phenotypes. Genetic analyses using murine RI and consomic strains enhance understanding of the molecular mechanisms of asthma in human.
Recent studies revealed a critical role for thymic stromal lymphopoietin (TSLP) released from epithelial cells and OX40 ligand (OX40L) expressed on dendritic cells (DCs) in T(H)2 priming and polarization.
The provirus integration site for Moloney murine leukemia virus (Pim) 1 kinase is an oncogenic serine/threonine kinase implicated in cytokine-induced cell signaling, whereas Runt-related transcription factor (Runx) has been implicated in the regulation of T-cell differentiation. The interaction of Pim1 kinase and Runx3 in the pathogenesis of peanut allergy has not been defined.
Naturally occurring Foxp3(+)CD4(+)CD25(+) T regulatory cell (nTreg)-mediated suppression of lung allergic responses is abrogated following ligation of glucocorticoid-induced tumor necrosis receptor (GITR) family-related protein. In vitro stimulation of nTregs with GITR ligand increased phosphorylation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated protein kinase (ERK) or p38 MAPK. SP600125, a known JNK inhibitor, prevented GITR-mediated phosphorylation of JNK. Activation of JNK was associated with increases in the upstream mitogen-activated protein kinase kinase 7 (MKK7) and the downstream transcription factor NF-??. Phosphorylated c-Jun (p-c-Jun), indicative of the activation of JNK, was detected in the immunoprecipitates of nTregs from wild-type but not JNK- or GITR-deficient mice. Treatment with an inhibitor of JNK phosphorylation resulted in complete reversal of all GITR-induced changes in nTreg phenotype and function, with full restoration of suppression of in vivo lung allergic responses and in vitro proliferation of activated CD4(+)CD25(-) T cells. Thus, regulation of JNK phosphorylation plays a central role in T regulatory cell function with therapeutic implications for the treatment of asthma and autoimmune diseases.
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