The non-genomic effects on Na+/H+-exchange 1 by progesterone and 20alpha-hydroxyprogesterone in human T cells

Progesterone is an endogenous immunomodulator and can suppress T-cell activation during pregnancy. We have previously shown that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)-induced T-cell proliferation. The presence of amiloride-sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20alpha-hydroxyprogesterone (20alpha-OHP). Furthermore, 20alpha-OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone-BSA demonstrated non-genomic action via plasma membrane sites. Finally, co-stimulation with PHA and progesterone or amiloride, (5-(N, N-dimethyl)-amiloride, DMA), inhibited PHA-induced T-cell proliferation, but this inhibition did not occur with 20alpha-OHP and PHA co-stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA-induced T-cell proliferation. This is the first study to show that progesterone causes a rapid non-genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20alpha-OHP. The inhibition of NHE1 leads to immunosuppressive T-cell proliferation and suggests that progesterone might exert a major rapid non-genomic suppressive effect on NHE1 activity at the maternal-fetal interface in vivo and that 20alpha-OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface.     

Non-genomic immunosuppressive actions of progesterone inhibits PHA-induced alkalinization and activation in T cells

Progesterone is an endogenous immunomodulator, and can suppress T-cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Therefore, the aim of this study was to investigate whether the non-genomic effects on ion fluxes by progesterone could immunosuppress phytohemagglutinin (PHA)-induced human peripheral T-cell activation. The new findings indicated that, first, only progesterone stimulated both [Ca2+]i elevation and pHi decrease; in contrast, estradiol or testosterone stimulated [Ca2+]i elevation and hydrocortisone or dexamethasone stimulated pHi decrease. Secondly, the [Ca2+]i increase by progesterone was dependent on Ca2+ influx, and the acidification was blocked by Na+/H+ exchange (NHE) inhibitor, 3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE-694) but not by 5-(N,N-dimethyl)-amiloride (DMA). Thirdly, progesterone blocked phorbol 12-myristate 13-acetate (PMA) or PHA-induced alkalinization, but PHA did not prevent progesterone-induced acidification. Fourthly, progesterone did not induce T-cell proliferation; however, co-stimulation progesterone with PHA was able to suppress PHA-induced IL-2 or IL-4 secretion and proliferation. When progesterone was applied 72 h after PHA stimulation, progesterone could suppress PHA-induced T-cell proliferation. Finally, immobilization of progesterone by conjugation to a large carrier molecule (BSA) also stimulated a rapid [Ca2+]i elevation, pHi decrease, and suppressed PHA-induced proliferation. These results suggested that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress the genomic responses to PHA. Progesterone might act directly through membrane specific nonclassical steroid receptors to cause immunomodulation and suppression of T-cell activation during pregnancy.     

Interferon‐γ suppresses Na+–H+ exchanger in cultured human endolymphatic sac epithelial cells

Adequate regulation of endolymphatic pH is essential for maintaining inner ear function. The Na⁺–H⁺ exchanger (NHE) is a major determinant of intracellular pH (pH_i), and facilitates Na⁺ and fluid absorption in various epithelia. We determined the functional and molecular expression of NHEs in cultured human endolymphatic sac (ES) epithelial cells and examined the effect of IFN‐γ on NHE function. Serial cultures of human ES epithelial cells were generated from tissue samples. The molecular expression of NHE1, ‐2, and ‐3 isoforms was determined by real‐time RT‐PCR. The functional activity of NHE isoforms was measured microfluorometrically using a pH‐sensitive fluorescent dye, 2′,7′‐bis(carbonylethyl)‐5(6)‐carboxyfluorescein (BCECF), and a NHE‐inhibitor, 3‐methylsulfonyl‐4‐piperidinobenzoyl guanidine methanesulfonate (HOE694). NHE1, ‐2, and ‐3 mRNAs were expressed in human ES epithelial cells. Functional activity of NHE1 and ‐2 was confirmed in the luminal membrane of ES epithelial cells by sequentially suppressing Na⁺‐dependent pH_i recovery from intracellular acidification using different concentrations of HOE694. Treatment with IFN‐γ (50 nM for 24 h) suppressed mRNA expression of NHE1 and ‐2. IFN‐γ also suppressed functional activity of both NHE1 and ‐2 in the luminal membrane of ES epithelial cells. This study shows that NHEs are expressed in cultured human ES epithelial cells and that treatment with IFN‐γ suppresses the expression and functional activity of NHE1 and ‐2. J. Cell. Biochem. 107: 965–972, 2009. © 2009 Wiley‐Liss, Inc.     

AFM‐ and NSOM‐based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4⁺ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4⁺ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4⁺ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4⁺ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.     

Ginsenosides promote proliferation of granulosa cells from chicken prehierarchical follicles through PKC activation and up‐regulated cyclin gene expression

The effect of GS (ginsenosides) on proliferation of chicken GCs (granulosa cells) from prehierarchical SYF (small yellow follicles) was evaluated, and involvement of the PKC (protein kinase C) signalling pathway as well as mRNA expression of cyclins and CDK (cyclin‐dependent kinase) were investigated. Whole SYF or GCs isolated from SYF were cultured in Medium 199 supplemented with 0.5% FCS (fetal calf serum). After 16 h, the cells were challenged with GS alone or in combination with PKC inhibitor H₇ or activator PMA (phorbol 12‐myristate 13‐acetate) for 24 h in serum‐free medium. Results showed that in both whole follicles and pure GCs monolayer culture system, GS (0.1–10 μg/ml) significantly increased the number of GCs in SYF in a dose‐dependent manner, and this stimulatory effect was inhibited by H₇, but enhanced by PMA. Meanwhile, the PCNA‐LI (proliferating cell nuclear antigen labelling index) of GCs displayed similar changes with the cell number. Mechanism of GS action was further evaluated in cultured GCs separated from SYF. Western blot analysis showed that 10 μg/ml GS increased PKC translocation from cytoplasm to the plasma membrane of the GCs to become the active state. This effect was blocked by H₇. Furthermore, GS up‐regulated the expression of cyclin D1/CDK6 and cyclin E/CDK2 mRNAs in GCs; however, inhibition of PKC with H₇ attenuated this stimulatory effect. These results indicated that GS could stimulate proliferation of chicken GCs through activated PKC‐involved up‐regulation of cyclin D1/CDK6 and cyclin E/CDK2 genes, subsequently promoting development of the chicken prehierarchical follicles.     

The signaling lipid phosphatidylinositol‐3,5‐bisphosphate targets plant CLC‐a anion/H+ exchange activity

Phosphatidylinositol‐3,5‐bisphosphate (PI(3,5)P₂) is a low‐abundance signaling lipid associated with endo‐lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on Arabidopsis indicated a critical role of PI(3,5)P₂ in vacuolar acidification and morphology during ABA‐induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch‐clamp recordings on Arabidopsis vacuoles, we show here that PI(3,5)P₂ does not affect the activity of vacuolar H⁺‐pyrophosphatase or vacuolar H⁺‐ATPase. Instead, PI(3,5)P₂ at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H⁺ pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC‐a), a member of the anion/H⁺ exchanger family formerly implicated in stomatal movements in Arabidopsis. H⁺‐dependent currents were absent in clc‐a knock‐out vacuoles, and canonical CLC‐a‐dependent nitrate/H⁺ antiport was inhibited by low concentrations of PI(3,5)P₂. Finally, using the pH indicator probe BCECF, we show that CLC‐a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P₂ and advance our knowledge about the regulation of vacuolar ion transport.     

Aberrant dynamin 2‐dependent Na+/H+ exchanger‐1 trafficking contributes to cardiomyocyte apoptosis

Sarcolemmal Na⁺/H⁺ exchanger 1 (NHE1) activity is essential for the intracellular pH (pH_i) homeostasis in cardiac myocytes. Emerging evidence indicates that sarcolemmal NHE1 dysfunction was closely related to cardiomyocyte death, but it remains unclear whether defective trafficking of NHE1 plays a role in the vital cellular signalling processes. Dynamin (DNM), a large guanosine triphosphatase (GTPase), is best known for its roles in membrane trafficking events. Herein, using co‐immunoprecipitation, cell surface biotinylation and confocal microscopy techniques, we investigated the potential regulation on cardiac NHE1 activity by DNM. We identified that DNM2, a cardiac isoform of DNM, directly binds to NHE1. Overexpression of a wild‐type DNM2 or a dominant‐negative DNM2 mutant with defective GTPase activity in adult rat ventricular myocytes (ARVMs) facilitated or retarded the internalization of sarcolemmal NHE1, whereby reducing or increasing its activity respectively. Importantly, the increased NHE1 activity associated with DNM2 deficiency led to ARVMs apoptosis, as demonstrated by cell viability, terminal deoxynucleotidyl transferase–mediated dUTP nick‐end labelling assay, Bcl‐1/Bax expression and caspase‐3 activity, which were effectively rescued by pharmacological inhibition of NHE1 with zoniporide. Thus, our results demonstrate that disruption of the DNM2‐dependent retrograde trafficking of NHE1 contributes to cardiomyocyte apoptosis.     

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

Precise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pH_i, pH_e) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na⁺/H⁺ exchanger NHE1 and Na⁺, HCO₃^? transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pH_e to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pH_i changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pH_i regulation by the two transporters.     

Recovery of poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) from Ralstonia eutropha cultures with non‐halogenated solvents

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA‐based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate‐co‐hydroxyhexanoate) (P(HB‐co‐HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non‐halogenated solvents. Several non‐halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17–30 mol%) remained completely in solution, while polymer with a lower HHx content (11–16 mol%) formed a gel‐like phase. All PHA in solution could be precipitated by addition of threefold volumes of n‐hexane or n‐heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non‐halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA. Biotechnol. Bioeng. 2013; 110: 461–470. © 2012 Wiley Periodicals, Inc.     

Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

We tested whether NHE3 and NHE2 Na⁺/H⁺ exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na⁺/H⁺ exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na⁺/H⁺ exchange function (NH₄-prepulse acid load sustained in Na⁺-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na⁺ removal revealed that only NHE3-CFP translocated when medium Na⁺ was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na⁺-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Membrane‐bound HSP70‐engineered myeloma cell‐derived exosomes stimulate more efficient CD8+ CTL‐ and NK‐mediated antitumour immunity than exosomes released from heat‐shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour‐derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T‐cell immune responses. To enhance EXO‐based antitumour immunity, we generated an engineered myeloma cell line J558_HSP expressing endogenous P1A tumour antigen and transgenic form of membrane‐bound HSP70 and heat‐shocked J558_HS expressing cytoplasmic HSP70, and purified EXO_HSP and EXO_HS from J558_HSP and J558_HS tumour cell culture supernatants by ultracentrifugation. We found that EXO_HSP were able to more efficiently stimulate maturation of DCs with up‐regulation of Ia^b, CD40, CD80 and inflammatory cytokines than EXO_HS after overnight incubation of immature bone‐marrow‐derived DCs (5 × 10⁶ cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A‐specific T‐cell responses after immunization. We demonstrate that EXO_HSP are able to stimulate type 1 CD4⁺ helper T (Th1) cell responses, and more efficient P1A‐specific CD8⁺ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO_HS. In addition, we further elucidate that EXO_HSP‐stimulated antitumour immunity is mediated by both P1A‐specific CD8⁺ CTL and non‐P1A‐specific natural killer (NK) responses. Therefore, membrane‐bound HSP70‐expressing tumour cell‐released EXO may represent a more effective EXO‐based vaccine in induction of antitumour immunity.     

Synthesis and Na+/H+ Exchanger‐1 Inhibitory Activity of Substituted (Quinolinecarbonyl)guanidine Derivatives

The Na⁺/H⁺ exchanger (NHE) is a protein expressed in many mammalian cell types. It is involved in intracellular pH (pH_i) homeostasis by exchanging extracellular Na⁺ for intracellular H⁺. To date, nine NHE isoforms (NHE1–NHE9) have been identified. NHE1 is the most predominant isoform expressed in mammalian cardiac muscle. A novel series of substituted (quinolinecarbonyl)guanidine derivatives were designed and synthesized as NHE inhibitors. Most compounds can inhibit NHE1‐mediated platelet swelling in a concentration‐dependent manner, among which compound 7f was the most active and more potent than cariporide. Furthermore, compound 7f has also been demonstrated to exhibit the in vivo cardioprotective effects against SD rat myocardial ischemic‐reperfusion injury superior to those of cariporide.     

NHE1 mediates migration and invasion of HeLa cells via regulating the expression and localization of MT1‐MMP

Na⁺/H⁺ exchanger 1 (NHE1), acting as an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been known to play a key role in the metastasis of many solid tumours. However, the exact mechanism underlying these processes, especially in cervical cancer, is still poorly understood. In the current study, we first showed that the inhibition of NHE1 activity by the specific inhibitor cariporide could suppress migration and invasion of HeLa cells in vitro. Moreover, cariporide also reversed the enhanced migration and invasion in HeLa cells by overexpressed membrane‐type 1 matrix metalloproteinase (MT1‐MMP). Subsequently, our results showed that NHE1 regulated the expression of MT1‐MMP at both messenger RNA and protein levels as well as its localization. Meanwhile, we observed slight modification in the morphology of HeLa cell after treating with cariporide. The present work indicates that NHE1 mediates HeLa cell metastasis via regulating the expression and localization of MT1‐MMP and provides a theoretical basis for the development of novel therapeutic strategies targeting cervical cancer. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of the simultaneous administration of glucocorticoids and IL-15 on human NK cell phenotype,proliferation and function

We have previously reported a synergistic effect between hydrocortisone (HC) and IL-15 on promoting natural killer (NK) cell expansion and function. In the present study, we extend our findings to methylprednisolone (MeP) and dexamethasone (Dex), thus ascribing to glucocorticoids (GCs) a general feature as positive regulators of IL-15-mediated effects on NK cells. We demonstrate that each GC when combined with IL-15 in cultures of peripheral blood (PB)-derived CD56⁺ cells induces increased expansion of CD56⁺CD3⁻ cells displaying high cytolytic activity, IFN-γ production potential and activating receptor expression, including NKp30, NKp44, NKp46, 2B4, NKG2D and DNAM-1. Furthermore, GCs protected NK cells from IL-15-induced cell death. The combination of IL-15 with GCs favored the expansion of a relatively more immature CD16^low/neg NK cell population, with high expression of NKG2A and CD94, and significantly lower expression of KIR (CD158a and CD158b) and CD57, compared to IL-15 alone. IL-15-expanded NK cells, in the presence or absence of GCs, did not express CD62L, CXCR1 or CCR7. However, the presence of GCs significantly increased the density of CXCR3 and induced strong CXCR4 expression on the surface of NK cells. Our data indicate that IL-15/GC-expanded NK cells, apart from their increased proliferation rate, retain their functional integrity and exhibit a migratory potential rendering them useful for adoptive transfer in NK cell-based cancer immunotherapy.     

A Model of Na/H Exchanger and Its Central Role in Regulation of pH and Na in Cardiac Myocytes

A new kinetic model of the Na⁺/H⁺ exchanger (NHE) was developed by fitting a variety of major experimental findings, such as ion-dependencies, forward/reverse mode, and the turnover rate. The role of NHE in ion homeostasis was examined by implementing the NHE model in a minimum cell model including intracellular pH buffer, Na⁺/K⁺ pump, background H⁺, and Na⁺ fluxes. This minimum cell model was validated by reconstructing recovery of pH_i from acidification, accompanying transient increase in [Na⁺]_i due to NHE activity. Based on this cell model, steady-state relationships among pH_i, [Na⁺]_I, and [Ca²⁺]_i were quantitatively determined, and thereby the critical level of acidosis for cell survival was predicted. The acidification reported during partial blockade of the Na⁺/K⁺ pump was not attributed to a dissipation of the Na⁺ gradient across the membrane, but to an increase in indirect H⁺ production. This NHE model, though not adapted to the dimeric behavioral aspects of NHE, can provide a strong clue to quantitative prediction of degree of acidification and accompanying disturbance of ion homeostasis under various pathophysiological conditions.     

Recycling and LFA‐1‐dependent trafficking of ICAM‐1 to the immunological synapse

Little is known about how adhesion molecules on APCs accumulate at immunological synapses. We show here that ICAM‐1 on APCs is continuously internalized and rapidly recycled back to the interface after antigen‐priming T‐cell contact. The internalization rate is high in APCs, including Raji B cells and dendritic cells, but low in endothelial cells. Internalization is significantly reduced by inhibitors of Na⁺/H⁺ exchangers (NHEs), suggesting that members of the NHE‐family regulate this process. Once internalized, ICAM‐1 is co‐localized with MHC class II in the polarized recycling compartment. Surprisingly, not only ICAM‐1, but also MHC class II, is targeted to the immunological synapse through LFA‐1‐dependent adhesion. Cytosolic ICAM‐1 is highly mobile and forms a tubular structure. Inhibitors of microtubule or actin polymerization can reduce ICAM‐1 mobility, and thereby block accumulation at immunological synapses. Membrane ICAM‐1 also moves to the T‐cell contact zone, presumably through an active, cytoskeleton‐dependent mechanism. Collectively, these results demonstrate that ICAM‐1 can be transported to the immunological synapse through the recycling compartment. Furthermore, the high‐affinity state of LFA‐1 on T cells is critical to induce targeted movements of both ICAM‐1 and MHC class II to the immunological synapse on APCs. J. Cell. Biochem. 111: 1125–1137, 2010. © 2010 Wiley‐Liss, Inc.     

IL‐35Ig–expressing dendritic cells induce tolerance via Arginase 1

The cytokine interleukin IL‐35 is known to exert strong immunosuppressive functions. Indoleamine 2,3‐dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL‐35 and the activity of those enzymes. We transfected a single chain IL‐35Ig gene construct in murine splenic DCs (DC₃₅) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL‐35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC₃₅, and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed‐type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC₃₅ was associated with the detection of CD25⁺CD39⁺, rather than Foxp3⁺, regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC₃₅ appears to be an early event in IL‐35Ig–mediated immunosuppression.     

Development of Bifidobacterium lactis Bb 12 on β‐(2,6)‐Linked Fructan‐Containing Substrate

β‐(2,1)‐linked fructan of plant origin (inulin) and the related oligosaccharides (FOS) as non‐digestible carbohydrates, i.e., potent prebiotics, can stimulate the growth of various probiotic lactic acid bacteria, including a number of bifidobacteria strains. The related β‐(2,6)‐linked fructans of microbial origin (levan and FOS), however, have scarcely been investigated in this respect. Therefore, the bifidogenic properties of various fructans, i.e., inulin, levan, fructooligosaccharides (FOS) and fructan syrup (FS), were tested as glucose substitutes in MRS media and were compared concerning their effect on the commercial strain Bifidobacterium lactis Bb 12. Although glucose was the preferred substrate for growth and biomass formation, FS exhibited a comparable cell growth (8.4 × 10⁷ counts/mL and 1.0 × 10⁷ counts/mL, respectively) and acidification power (84 °T and 74 °T, respectively) during 48 h of fermentation, as well as an increase in lactic acid and decrease in acetic acid formation. Bifidobacterium lactis Bb 12 did not utilize inulin as a sole carbon source as judged from the 60 % decrease in cell count and the insignificant (0.1 pH unit) acidification of the growth medium, whereas levan provided a noticeable increase in cell count and acidification (0.4 pH units) during 48 h of fermentation. FOS preparation appeared to be a satisfactory carbon source for this strain, but lower acidification power (56 °T) and cell counts were observed as compared to glucose‐ or FOS‐containing media (2.6 % and 22 %, respectively). The products obtained under conditions of mild lactic acid hydrolysis of levan (37 °C, pH 3.3, 24 h) enhanced the cell count (7–10 %) and acidification power (by a factor of 2.7) of Bifidobacterium lactis Bb 12.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.