Astrocytes play an important role in the pathogenesis of hepatic encephalopathy (HE) and ammonia toxicity, whereas little is known about microglia and neuroinflammation under these conditions. We therefore studied the effects of ammonia on rat microglia in vitro and in vivo and analyzed markers of neuroinflammation in post mortem brain tissue from patients with cirrhosis with and without HE and non-cirrhotic controls. In cultured rat microglia, ammonia stimulated cell migration and induced oxidative stress and an up-regulation of the microglial activation marker ionized calcium-binding adaptor molecule-1 (Iba-1). Up-regulation of Iba-1 was also found in the cerebral cortex from acutely ammonia-intoxicated rats and in the cerebral cortex from patients with cirrhosis who have HE, but not from patients with cirrhosis who do not have HE. However, ammonia had no effect on microglial glutamate release, prostaglandin synthesis, and messenger RNA (mRNA) levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and the proinflammatory cytokines interleukin (IL)-1?/?, tumor necrosis factor ?, or IL-6, whereas in cultured astrocytes ammonia induced the release of glutamate, prostaglandins, and increased IL-1? mRNA. mRNA and protein expression of iNOS and COX-2 or mRNA expression of proinflammatory cytokines and chemokine monocyte chemoattractive protein-1 in cerebral cortex from patients with liver cirrhosis and HE were not different from those found in patients with cirrhosis who did not have HE or control patients without cirrhosis.
Ca(+)-dependent signaling regulates the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. The activity of DCs is suppressed by glucocorticoids, potent immunosuppressive hormones. The present study explored whether the glucocorticoid dexamethasone influences the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in DCs. To this end, DCs were isolated from mouse bone marrow. According to fura-2 fluorescence, exposure of DCs to lipopolysaccharide (LPS, 100 ng/ml) increased [Ca(2+)](i), an effect significantly blunted by overnight incubation with 10 nM dexamethasone before LPS treatment. Dexamethasone did not affect the Ca(2+) content of intracellular stores, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2 and SERCA3 expression, ryanodine receptor (RyR)1 expression, or Ca(2+) entry through store-operated Ca(2+) channels. In contrast, dexamethasone increased the transcript level and the membrane protein abundance of the Na(+)/Ca(2+) exchanger NCX3. The activity of Na(+)/Ca(2+) exchangers was assessed by removal of extracellular Na(+) in the presence of external Ca(2+), a maneuver triggering the Ca(2+) influx mode. Indeed, Na(+) removal resulted in a rapid transient increase of [Ca(2+)](i) and induced an outwardly directed current as measured in whole cell patch-clamp experiments. Dexamethasone significantly augmented the increase of [Ca(2+)](i) and the outward current following removal of extracellular Na(+). The NCX blocker KB-R7943 reversed the inhibitory effect of dexamethasone on LPS-induced increase in [Ca(2+)](i). Dexamethasone blunted LPS-induced stimulation of CD86 expression and TNF-? production, an effect significantly less pronounced in the presence of NCX blocker KB-R7943. In conclusion, our results show that glucocorticoid treatment blunts LPS-induced increase in [Ca(2+)](i) in DCs by increasing expression and activity of Na(+)/Ca(2+) exchanger NCX3. The effect contributes to the inhibitory effect of the glucocorticoid on DC maturation.
Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 (sgk3(-/-)) and their wild-type littermates (sgk3(+/+)). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular Ca(2+) and entry of extracellular Ca(2+). Influx of extracellular Ca(2+) but not Ca(2+) release from intracellular stores was significantly blunted in sgk3(-/-) BMMCs compared with sgk3(+/+) BMMCs. Antigen stimulation further led to a rapid increase of a K(+)-selective conductance in sgk3(+/+) BMMCs, an effect again blunted in sgk3(-/-) BMMCs. In contrast, the Ca(2+) ionophore ionomycin activated K(+) currents to a similar extent in sgk3(-/-) and in sgk3(+/+) BMMCs. ?-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in sgk3(-/-) BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in sgk3(-/-) compared with sgk3(+/+) mice. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk3(-/-) than in sgk3(+/+) mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function.
TGR5 (Gpbar-1) is a membrane-bound bile acid receptor in the gastrointestinal tract and immune cells with pleiotropic actions. As shown in the present study, TGR5 is also expressed in astrocytes and neurons. Here, TGR5 may act as a neurosteroid receptor, which is activated by nanomolar concentrations of 5?-pregnan-3?-ol-20-one and micromolar concentrations of 5?-pregnan-3?-17?-21-triol-20-one and 5?-pregnan-3?-ol-20-one (allopregnanolone). TGR5 stimulation in astrocytes and neurons is coupled to adenylate cyclase activation, elevation of intracellular Ca(2+) and the generation of reactive oxygen species. In cultured rat astrocytes, TGR5 mRNA is downregulated in the presence of neurosteroids and ammonia already at concentrations of 0.5 mmol L(-1). Furthermore, TGR5 protein levels are significantly reduced in isolated rat astrocytes after incubation with ammonia. A marked downregulation of TGR5 mRNA is also found in cerebral cortex from cirrhotic patients dying with hepatic encephalopathy (HE) when compared with brains from noncirrhotic control subjects. It is concluded that TGR5 is a novel neurosteroid receptor in brain with implications for the pathogenesis of HE.
The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKC?. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKC? inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 ?M) increased the K(+) channel conductance to similar values in both genotypes. ?-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKC? inhibitor rottlerin increased ?-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKC? and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells.
Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immunity. Ca(2+)-dependent signaling plays a central regulatory role in DC responses to diverse antigens. DCs are a primary target of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a secosteroid hormone, that, in addition to its well-established action on Ca(2+) homeostasis, possesses immunomodulatory properties. Surprisingly, nothing is known about its effects on DC cytosolic Ca(2+) activity. The present study explored whether 1,25(OH)(2)D(3) modifies the intracellular Ca(2+) concentration ([Ca(2+)](i)) in DCs. Here we show that mouse DCs expressed K(+)-independent (NCX1-3) and K(+)-dependent (NCKX1, 3, 4, and 5) Na(+)/Ca(2+) exchangers. Acute application of LPS (100 ng/ml) to DCs increased [Ca(2+)](i), an effect significantly blunted by prior incubation with 1,25(OH)(2)D(3). 1,25(OH)(2)D(3) increased the membrane abundance of the NCKX1 protein, up-regulated the K(+)- and Na(+)-dependent Ca(2+) entry and enhanced the K(+)-dependent Na(+)/Ca(2+) exchanger currents. The NCKX blocker 3,4-dichlorobenzamyl (DBZ) reversed the inhibitory effect of 1,25(OH)(2)D(3) on the LPS-induced increase of [Ca(2+)](i). Expression of the costimulatory molecule CD86 was down-regulated by 1,25(OH)(2)D(3), an effect reversed by DBZ. In summary, 1,25(OH)(2)D(3) blunts the LPS-induced increase in [Ca(2+)](i) by stimulation of Na(+)/Ca(2+) exchanger-dependent Ca(2+) extrusion, an effect that contributes to 1,25(OH)(2)D(3)-mediated immunosuppression. The results disclose completely novel mechanisms in the regulation of DC maturation and function.
The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). The BMMC number as well as CD117, CD34, and FcepsilonRI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the FcepsilonRI receptor, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in sgk1(-/-) BMMCs. The currents through Ca(2+)-activated K+ channels induced by Ag were significantly higher in sgk1(+/+) BMMCs than in sgk1(-/-) BMMCs. Treatment with the Ca(2+) ionophore ionomycin (1 microM) led to activation of the K+ channels in both genotypes, indicating that the Ca(2+)-activated K+ channels are similarly expressed and sensitive to activation by Ca(2+) in sgk1(+/+) and sgk1(-/-) BMMCs, and that blunted stimulation of Ca(2+)-activated K+ channels was secondary to decreased Ca(2+) entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1(-/-) BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substantially reduced in sgk1(-/-) mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1(-/-) than in sgk1(+/+)mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction.
Peptidoglycans (PGN) from bacterial cell walls may modify the course of an infection with bacterial pathogens. The present study explored the effect of PGN on cytosolic Ca2+ activity, cytokine production and phagocytosis of mouse dendritic cells (DCs), essential cells in the initiation and direction of antigen-specific T cell responses. Exposure of DCs to PGN was followed by a rapid increase in cytosolic Ca2+ activity ([Ca2+]i), which was due to Ca2+ release from intracellular stores and influx of extracellular Ca2+ across the cell membrane. In DCs isolated from Toll-like receptor 2 (TLR2) deficient mice the effect of PGN on [Ca2+]i was dramatically impaired. The PGN-induced increase of [Ca2+]i was dependent on voltage-gated K+ (Kv) channel activity. PGN-induced increase of [Ca2+]i was significantly blunted by margatoxin (MgTx) and perhexiline maleate (PM), inhibitors of Kv1.3 and Kv1.5, respectively. PGN further stimulated the release of tumour necrosis factor alpha (TNFalpha), interleukin-12 (IL-12) and interleukin-10 (IL-10), an effect significantly blunted by PM and the specific blocker of store-operated Ca2+ channels SKF-96365. Moreover, phagocytic capacity was dramatically increased in PGN-stimulated DCs in the presence of either Kv channel inhibitors or SKF-96365. The observations disclose Ca2+ and Kv channel-dependent cytokine production and phagocytosis in PGN-stimulated DCs.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.