Bronchipret® syrup containing thyme and ivy extracts suppresses bronchoalveolar inflammation and goblet cell hyperplasia in experimental bronchoalveolitis

Background/purposeAcute bronchitis (AB) is a common lung condition characterized by inflammation of the large bronchi in response to infection. Bronchipret^® syrup (BRO), a fixed combination of thyme and ivy extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to identify potential mechanisms of action.MethodsBronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o. once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24–72 h post LPS challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO).ResultsBRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular release of LTB₄ (IC₅₀ = 36 µg⋅ml⁻¹) and cysLT (IC₅₀ = 10 µg⋅ml⁻¹) and inhibited the activity of isolated 5-LO (IC₅₀ = 19 µg⋅ml⁻¹).ConclusionBRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may contribute to its observed clinical efficacy in AB.     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

Sepiapterin regulates cell proliferation and migration: its association with integrin α3β1 and p53 in human lung cancer cells

Tetrahydrobiopterin (BH₄) has been known to be an essential cofactor for nitric oxide synthase as well as the aromatic amino acid hydroxylases, which are involved in regulation of cellular fates including proliferation, migration and differentiation. In the present study, we report that sepiapterin, a stable form of BH₄ precursor, modulates proliferation and migration in human lung cancer cells. Sepiapterin induction of cell proliferation in p53 wild-type A549 cells, but not in p53-deficient H1299 cells, is accompanied by enhanced expression of cell cycle-related proteins such as cyclin-dependent kinase 4 (Cdk4), cyclin D and cyclin E, and reduced expression of Cdk inhibitor p21^WAF1/Cip1, demonstrating that sepiapterin-induced mitogenic responses might be associated with p53 expression status in lung cancer cells. In addition, sepiapterin enhances cell migration in A549 cells, but not H1299 cells. Finally, we show that sepiapterin induces A549 cell proliferation and migration through the activation of Akt and p70^S6K signaling pathways, as evidenced by using Akt and p70^S6K inhibitor. Collectively, these findings indicate that sepiapterin might play differential roles in regulation of cellular fates, depending on the status of p53 expression in lung cancer.     

Titanium dioxide induces different levels of IL-1β production dependent on its particle characteristics through caspase-1 activation mediated by reactive oxygen species and cathepsin B

Although titanium dioxide (TiO₂) is widely used, its inhalation can induce inflammatory diseases accompanied by interleukin-1β (IL-1β) production. The particle characteristics of TiO₂ are important factors in its biological effects. It is urgently necessary to investigate the relationship between the particle characteristics and biological responses for the development of safe forms of TiO₂. Here, we systematically compared the production of IL-1β in response to various forms of TiO₂ by macrophage-like human THP-1 cells using various sizes (nano to micro), crystal structures (anatase or rutile), and shapes (spherical or spicular) of TiO₂. The production of IL-1β depended dramatically on the characteristics of the TiO₂. Notably, smaller anatase and larger rutile particles provoked higher IL-1β production. In addition, IL-1β production depended on active cathepsin B and reactive oxygen species production independent of the characteristics of TiO₂. Our results provide basic information for the creation of safe and effective novel forms of TiO₂.     

γδ T cells cultured with artificial antigen-presenting cells and IL-2 show long-term proliferation and enhanced effector functions compared with γδ T cells cultured with only IL-2 after stimulation with zoledronic acid

Background aimsImmunotherapeutic approaches using γδ T cells have emerged as the function of γδ T cells in tumor surveillance and clearance has been discovered. In vitro expansion methods of γ9δ2 T cells have been based on phosphoantigens and cytokines, but expansion methods using feeder cells to generate larger numbers of γδ T cells have also been studied recently. However, there are no studies that directly compare γδ T cells cultured with phosphoantigens with those cultured with feeder cells. Therefore, this study aimed to compare the expansion, characteristics and effector functions of γδ T cells stimulated with K562-based artificial antigen-presenting cells (aAPCs) (aAPC-γδ T cells) and γδ T cells stimulated with only zoledronic acid (ZA) (ZA-γδ T cells).MethodsPeripheral blood mononuclear cells were stimulated with ZA for 7 days, and aAPC-γδ T cells were stimulated weekly with K562-based aAPCs expressing CD32, CD80, CD83, 4-1BBL, CD40L and CD70, whereas ZA-γδ T cells were stimulated with only IL-2. Cultured γδ T cells were analyzed by flow cytometry for the expression of co-stimulatory molecules, activating receptors and checkpoint inhibitors. Differentially expressed gene (DEG) analysis was also performed to determine the difference in gene expression between aAPC-γδ T cells and ZA-γδ T cells. In vitro cytotoxicity assay was performed with calcein AM release assay, and in vivo anti-tumor effect was compared using a U937 xenograft model.ResultsFold expansion on day 21 was 690.7 ± 413.1 for ZA-γδ T cells and 1415.2 ± 1016.8 for aAPC- γδ T cells. Moreover, aAPC-γδ T cells showed continuous growth, whereas ZA-γδ T cells showed a decline in growth after day 21. The T-cell receptor Vγ9⁺δ2⁺ percentages (mean ± standard deviation) on day 21 were 90.0 ± 2.7% and 87.0 ± 4.5% for ZA-γδ T cells and aAPC-γδ T cells, respectively. CD25 and CD86 expression was significantly higher in aAPC-γδ T cells. In DEG analysis, aAPC-γδ T cells and ZA-γδ T cells formed distinct clusters, and aAPC-γδ T cells showed upregulation of genes associated with metabolism and cytokine pathways. In vitro cytotoxicity revealed superior anti-tumor effects of aAPC-γδ T cells compared with ZA-γδ T cells on Daudi, Raji and U937 cell lines. In addition, in the U937 xenograft model, aAPC-γδ T-cell treatment increased survival, and a higher frequency of aAPC-γδ T cells was shown in bone marrow compared with ZA-γδ T cells.ConclusionsOverall, this study demonstrates that aAPC-γδ T cells show long-term proliferation, enhanced activation and anti-tumor effects compared with ZA-γδ T cells and provides a basis for using aAPC-γδ T cells in further studies, including clinical applications and genetic engineering of γδ T cells.     

Human mast cells decrease SLPI levels in type II – like alveolar cell model, in vitro

Background  

Mast cells are known to accumulate at sites of inflammation and upon activation to release their granule content, e.g. histamine, cytokines and proteases. The secretory leukocyte protease inhibitor (SLPI) is produced in the respiratory mucous and plays a role in regulating the activity of the proteases.     

Staphylococcal α-hemolysin does not induce cell damage in murine mast cells but it augments the degranulation induced by FcεRI cross-linking and ionomycin

Staphylococcal α-hemolysin (Hla) is a principal small β-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, β-hexosaminidase. However, Hla more than doubled the releases of β-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130?mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses.     

NK cell development, homeostasis and function: parallels with CD8⁺ T cells

Natural killer (NK) cells survey host tissues for signs of infection, transformation or stress and, true to their name, kill target cells that have become useless or are detrimental to the host. For decades, NK cells have been classified as a component of the innate immune system. However, accumulating evidence in mice and humans suggests that, like the B and T cells of the adaptive immune system, NK cells are educated during development, possess antigen-specific receptors, undergo clonal expansion during infection and generate long-lived memory cells. In this Review, we highlight the many stages that an NK cell progresses through during its remarkable lifetime, discussing similarities and differences with its close relative, the cytotoxic CD8(+) T cell.     

Lack of association or interactions between the IL-4, IL-4Rα and IL-13 genes, and rheumatoid arthritis

Introduction  

A feature of rheumatoid arthritis (RA) is an imbalance between proinflammatory and anti-inflammatory cytokines. Several recent studies have implicated polymorphism in the IL-4 signalling pathway in the development of erosive RA. The aim of the present study was to investigate the role of polymorphism in the IL-4, IL-4Rα and IL-13 genes in RA, including an examination of epistasis.     

Cinnamon extract reduces symptoms,inflammatory mediators and mast cell markers in murine IL-10−/− colitis

Inflammatory bowel disease (IBD) shows an increasing prevalence and harm in western countries. Conventional therapies are associated with bad compliance and adverse side effects. Natural substances like cinnamon extract (CE) could be an additional therapy. We found recently that CE acts anti-inflammatory on mast cells — discussed of being relevant in IBD. Here, we analysed the effects of CE on murine IL-10^−/− colitis as model for IBD. Mice were treated 12 weeks with or without CE in drinking water. Clinical scores and disease activity index were assessed. Colonic tissue samples were analysed for infiltration, tissue damage, bowel wall thickness, expression of pro-inflammatory mediators, mast cell proteases, tight junction proteins, and NF-κB signaling. Following treatment with CE, symptoms of murine colitis as well as increased infiltration of immune cells, tissue damage and bowel wall thickness in colon tissue of IL-10^−/− mice were diminished significantly. MIP-2, TNF, IFNγ, CCL2, CCL3, CCL4 and IL-1β as well as MC-CPA, MCP-1 and MCP-4 were strongly upregulated in IL-10^−/− mice compared to WT, but noteworthy not in CE group. Expression of tight junction proteins was not influenced by CE. Phosphorylation of IκB was slightly down-regulated in CE treated IL-10^−/− mice compared to IL-10^−/− controls. In summary, CE decreases inflammatory symptoms and expression of inflammatory markers in murine IL-10^−/− colitis. CE has no influence on tight junction proteins, but seems acting via reducing pro-inflammatory mediators and recruitment of neutrophil granulocytes probably by inhibiting NF-κB signaling.     

Increase in CD30 ligand/CD153 and TNF-α expressing mast cells in basal cell carcinoma

Mast cells are a significant source of tumor necrosis factor (TNF) superfamily members, such as TNF-α, CD30 ligand/CD153 (CD30L) and CD40L/CD154. Furthermore, the expression of some of these proteins in mast cells has been associated with tumorigenesis, and mast cells have been found to be increased in number in the basal cell carcinoma (BCC) lesion. In this study, we have examined the expression of TNF-α, CD30L and CD40L immunoreactivity in mast cells in the healthy-looking skin and lesional skin of ten patients with superficial spreading BCC. Also, the counterparts of these molecules, TNF receptor (TNFR) I and II as well as CD30 and CD40, were analysed immunohistochemically. We found that numbers of mast cells and Kit-positive cells were significantly increased in the dermal BCC lesion. The percentage of CD30L-positive mast cells and the number of CD30-positive cells were significantly increased in the upper dermis of the BCC lesion as well. In addition, the numbers of TNF-α-positive mast cells and cells with TNFRI and TNFRII were markedly increased in the upper lesional dermis. In contrast, no mast cells positive for CD40L could be detected, even though the lesional dermis contained increased numbers of CD40 positive cells. The BCC epithelium was positive for TNFRI, TNFRII and CD40, but not for CD30, though the larger basal buds appeared to be less intensely stained for TNFRI and CD40. In conclusion, mast cells positive for CD30L and TNF-α, but not CD40L, are increased in number in the lesional dermis in BCC. These data suggest plausible pathways whereby mast cells can be activated and to interact with other cells and thereby contribute to the tumorigenesis in BCC.     

IL-21 promotes skin recruitment of CD4(+) cells and drives IFN-γ-dependent epidermal hyperplasia

Psoriasis is a chronic inflammatory disorder of the skin characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. T cell-derived cytokines, such as IFN-γ and IL-17A, play a major role in the psoriasis-associated epidermal hyperplasia, even though factors/mechanisms that regulate the production of these cytokines are not fully understood. We have recently shown that IL-21 is synthesized in excess in psoriatic skin lesions and causes epidermal hyperplasia when injected intradermally in mice. Moreover, in the human psoriasis SCID mouse model, neutralization of IL-21 reduces both skin thickening and expression of inflammatory molecules, thus supporting the pathogenic role of IL-21 in psoriasis. However, the basic mechanism by which IL-21 promotes skin pathology remains unknown. In this study, we show that CD4(+) cells accumulate early in the dermis of IL-21-treated mice and mediate the development of epidermal hyperplasia. Indeed, IL-21 fails to induce skin damage in RAG1-deficient mice and CD4(+) cell-depleted wild-type mice. The majority of CD4(+) cells infiltrating the dermis of IL-21-treated mice express IFN-γ and, to a lesser extent, IL-17A. Studies in cytokine knockout mice show that IFN-γ, but not IL-17A, is necessary for IL-21-induced epidermal hyperplasia. Finally, we demonstrate that IFN-γ-producing CD4(+) cells infiltrating the human psoriatic plaque express IL-21R, and abrogation of IL-21 signals reduces IFN-γ expression in cultures of psoriatic CD4(+) cells. Data indicate that IL-21 induces an IFN-γ-dependent pathogenic response in vivo, thus contributing to elucidate a mechanism by which IL-21 sustains skin-damaging inflammation.     

EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.