EA 575^® is an ivy leaves dry extract (DER 5-7.5:1, 30% ethanol) used against diseases of the lower respiratory tract associated with productive cough. EA 575^® improves symptoms associated with chronic inflammatory bronchial conditions. Compared to its bronchospasmolytic and secretolytic properties, the anti-inflammatory effects of EA 575^® are mostly untried. Therefore, we addressed the question of whether the anti-inflammatory effect of EA 575^® is due to an impact on the NFκB pathway. NFκB nuclear translocation was visualized by immunofluorescence in J774.2 as well as HEK293 cells. In the latter, a luciferase-based reporter was used to monitor NFκB transcriptional activity. Phosphorylation of RelA and its inhibitor IκB was measured by Western blot analysis. Additionally, changes in the stability of NFκB:IκB complex were shown by protein fragment complementation. Decreased transcriptional activity of NFκB under treatment with EA 575^® was also shown for a human monocytic as well as a human lung epithelial cell line. EA 575^® is able to inhibit NFκB transcriptional activity by partially inhibiting its translocation to the nucleus after stimulation with TNFα. Furthermore, phosphorylation of IκBα is reduced while phosphorylation of RelA is enhanced after pre-incubation with EA 575^®, leading to an enhanced stability of NFκB:IκBα complex. EA 575^® has an regulatory impact on the NFκB pathway, possibly by switching specificity of IKK from IκBα to RelA, resulting in enhanced stability of NFκB:IκBα complex and reduced RelA translocation into the nucleus.

Antigen presentation by human lymphocyte antigen (HLA) class II peptide receptors alerts the immune system to infections. In antigen‐presenting cells (APCs), HLA class II, HLA‐DM, and associated invariant chain‐encoding genes are exclusively regulated by the interferon γ (IFNγ)‐inducible class II transactivator (CIITA). Control of CIITA expression could therefore govern expression of class II peptide receptors in the diverse group of APCs. We discovered that elevation of the HLA class III region encoded B‐associated transcript 3 (BAT3) increases and depletion of BAT3 decreases expression of HLA class II, HLA‐DM, and invariant chain. IFNγ strongly elevates BAT3 transcription in various tumor cell lines and in primary macrophages. BAT3 chaperones the simultaneously IFNγ‐induced CIITA. Following IFNγ‐treatment, both CIITA and BAT3 translocate from the cytosol to the nucleus. The nuclear import of CIITA mediated by IFNγ controls activation of HLA class II genes. BAT3 is a novel key regulator of components of the HLA class II processing pathway. We present a mechanism explaining how parallel IFNγ‐mediated regulation of CIITA and of its chaperone BAT3 controls the level of components of the HLA class II processing pathway.—Kämper, N., Franken, S., Temme, S., Koch, S., Bieber, T., Koch, K. γ‐Interferon‐regulated chaperone governs human lymphocyte antigen class II expression. FASEB J. 26, 104–116 (2012). www.fasebj.org

Chronic stress, an important factor in the development of depressive disorders, leads to an increased formation of cortisol, which causes a hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, cortisol mediates an adaptive effect on plasma membrane fluidity which may affect signal transduction of membrane-bound receptors and contribute to pathophysiological changes. Membrane fluidity was measured by fluorescence anisotropy using DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene). Changes in cellular content of phosphatidylcholine species was determined by pulse-chase experiments using deuterated choline and mass spectrometry. Single molecule tracking was used to examine the lateral mobility of β1-adrenoceptors and changes in cAMP formation were measured by ELISA. Chronic exposure (6 - 8 days) of C6 cells to cortisol dose-dependently decreased DPH and TMA-DPH fluorescence anisotropy, reflecting increased membrane fluidity. In contrast, cells pretreated with St. John's wort extract Ze117 showed increased DPH and TMA-DPH fluorescence anisotropy values, indicating a membrane rigidification effect which was mediated at least by the constituents hypericin, hyperforin, quercetin, amentoflavone and biapigenin. The observed membrane fluidizing effect of cortisol could be reversed by cotreatment with Ze117. The membrane rigidification of Ze117 was in line with the in parallel observed decrease in the phosphatidylcholine/phosphatidylethanolamine ratio determined in whole cell lipid extracts. Interestingly, pulse-chase experiments demonstrated, that Ze117 inhibited the incorporation of choline-D9 in phosphatidylcholine species with saturated or monounsaturated fatty acids compared to control cells, while the synthesis of phosphatidylcholine species with polyunsaturated fatty acids was not affected. C6 cells whose membranes have become more rigid by Ze117 showed altered lateral mobility of β1-adrenoceptors as well as reduced cAMP formation after stimulation with the β1-adrenoceptor agonist dobutamine. Obviously, the signaling of β1-adrenoceptors depends on the nature of the membrane environment. It can therefore be assumed that Ze117 has a normalizing effect not only on the membrane fluidity of "stressed" cells, but also on lateral mobility and subsequently on the signal transduction of membrane-associated receptors.

The β₂-adrenergic receptor (β₂AR) is relevant for surfactant formation in alveolar type 2 cells and reduction of intracellular calcium concentration in bronchial muscle cells and thus for secretolytic and bronchospasmolytic effects. Herbal medicinal products that affect the β₂AR system are used to treat common cold and bronchitis accompanied with mucus covered and narrowed airways. The present work compares the influence of an ivy preparation and an ivy/thyme combination on the β₂-adrenergic signal transduction. For receptor binding studies and characterization of the lateral mobility of β₂AR we have used single molecule detection by fluorescence correlation spectroscopy and single particle tracking. For the determination of both the second messenger cAMP and the internalization of β₂AR we have generated luciferase based reporter cell lines, which produce a cAMP-dependent luciferase in the cytosol and express β₂AR with extracellular luciferase moiety in the plasma membrane. While both preparations increased the β₂AR binding, a significant increase of the cAMP level was observed only for the ivy preparation, which can be explained by the inhibited internalization of HiBiT-tagged β₂AR under stimulating conditions. In contrast, isoprenaline-mediated internalization of HiBiT-tagged β₂AR of ivy/thyme combination pre-treated cells was not inhibited. Cells comparatively pre-treated with a thyme preparation did not show inhibition of ß₂AR internalization either. Furthermore, SNAP-tagged β₂AR of ivy preparation pre-treated cells, which were not internalized after isoprenaline stimulation, showed a redistribution from fast-to-slowly diffusing β₂AR. A corresponding redistribution of these receptors was not observed after pre-treatment with both the ivy/thyme combination and the thyme preparation. Comparable to the ivy/thyme combination, no decrease in the intratrack transitioning probability ratio (p23/p32) for fast and slow diffusing β₂AR was found for the thyme preparation, which, however, significantly decreased for control cells and for pre-treatment with the ivy preparation under stimulating conditions. It can therefore be concluded that the thyme fluid extract fraction in the ivy/thyme combination may have in part a negative effect on the β₂-adrenergic signal transduction.

β₂-adrenergic receptor (β₂-AR) stimulation activates the G protein/cAMP pathway, which is opposed by the GRK2/β-arrestin 2 pathway. The latter is undesirable in the treatment of respiratory diseases. EA 575® is capable of mediating a biased β₂-adrenergic signaling pathway. The impact of the ivy leaves dry extract EA 575® on β₂-adrenergic signaling was tested in a dynamic mass redistribution assay in HEK wild-type and in HEK β-arrestin knock-out cells. cAMP formation and recruitment of β-arrestin 2 were investigated using GloSensor™ and PathHunter® assays, respectively. NFκB transcriptional activity was determined in both HEK wild-type as well as HEK β-arrestin knock-out cells. EA 575® inhibits the recruitment of β-arrestin 2 and thereby enhances G protein/cAMP signaling under β₂-stimulating conditions, as evidenced by a corresponding increase in cAMP formation. While β₂-AR-mediated inhibition of NFκB transcriptional activity is β-arrestin-dependent, EA 575® leads to significant inhibition of NFκB transcriptional activity in β-arrestin knock-out cells and thus via a β-arrestin-independent signaling pathway. EA 575® is the first active phytopharmaceutical ingredient for which biased β₂-adrenergic activation has been described. This shift towards G protein/cAMP signaling provides the molecular basis for the clinically proven efficacy of EA 575® in the treatment of lower respiratory tract diseases. In this light, EA 575® could potentially reduce β-arrestin-mediated adverse effects in new combinatorial therapeutic approaches.