Eicosanoids and their role in immune modulation in fish—a brief overview

Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E₂, leukotriene B₄ and lipoxin A₄. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B₄ and lipoxin A₄ have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.     

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.     

On the move,lysosomal CAX drives Ca2+ transport and motility

Acidic Ca²⁺ stores are important sources of Ca²⁺ during cell signaling but little is known about how Ca²⁺ enters these stores. In this issue, Melchionda et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510019) identify a Ca²⁺/H⁺ exchanger (CAX) that is required for Ca²⁺ uptake and cell migration in vertebrates.Intracellular Ca²⁺ signaling is of fundamental importance in processes such as cell migration but we do not fully understand the contribution made by different intracellular Ca²⁺ stores to this particular function. Elevation of cytosolic Ca²⁺ by 10- to 100-fold the normal resting levels can occur by entry of external Ca²⁺ across the plasma membrane and release of Ca²⁺ from intracellular organelles such as the ER. Ca²⁺ ions are transported across membranes by ligand-gated ion channels, energy-dependent pumps, and transporters (Berridge et al., 2003; Lloyd-Evans et al., 2010). Intracellular Ca²⁺ levels are regulated in this manner from simple organisms, such as yeast, through to complex multicellular organisms, suggesting a degree of conservation across the taxonomic kingdoms (Patel and Cai, 2015). Recent evidence has indicated that “acidic Ca²⁺ stores” such as lysosomes in mammalian cells are a key intracellular Ca²⁺ signaling store, like the ER (Lloyd-Evans and Platt, 2011; Patel and Muallem, 2011). The Ca²⁺ concentration of the lysosome (500 µM) is similar to the ER (Christensen et al., 2002; Lloyd-Evans et al., 2008) but lysosomes are smaller in volume and their impact on cellular Ca²⁺ signaling seems localized to events that regulate endocytosis, vesicular fusion, and recycling (Ruas et al., 2010; López-Sanjurjo et al., 2013). However, there is a significant amount of evidence emerging that lysosomes are capable of triggering much larger changes in cytosolic Ca²⁺ during signaling via the induction of Ca²⁺ release from the ER. This effect appears to be mediated by the most potent intracellular Ca²⁺–releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which triggers Ca²⁺ release from lysosomes via two-pore channels (Brailoiu et al., 2009; Calcraft et al., 2009). In addition to two-pore channels, acidic stores also express other Ca²⁺-permeable channels (summarized in Fig. 1).Open in a separate windowFigure 1.Lysosomal Ca²⁺ transporters and channels. Our current understanding of lysosomal Ca²⁺ transport and the proteins that regulate the transport of Ca²⁺ into and out of the lysosome is heavily stacked in favor of Ca²⁺ release channels. To date, voltage-gated (CaV2.1/CACNA1A), ligand-gated (TRPML1 and TRPM2), and nucleotide-gated (TPC1, TPC2, and P2X4) channels have all been identified or implicated in lysosomal Ca²⁺ release (Patel and Cai, 2015). Much less is known about the mechanisms of Ca²⁺ entry into lysosomes. In lower order organisms, CAX mediates lysosomal Ca²⁺ entry against the proton gradient. In this issue, Melchionda et al. (2016) provide the first evidence for a mammalian lysosomal Ca²⁺ uptake mechanism in nonplacental mammals. These findings provide further support for the key role of the lysosome as an intracellular Ca²⁺ store.Despite recent advances in our knowledge of lysosomal Ca²⁺ release channels, we have so far failed to identify the transport proteins that fill the lysosome with Ca²⁺. Ca²⁺ entering the cell by endocytosis is removed by the early endosome after the initiation of endosomal acidification by the vATPase; therefore, it is likely that lysosomes have their own transporters or pumps to take up Ca²⁺ (Gerasimenko et al., 1998; Christensen et al., 2002). Although there have been studies suggesting the presence of ATPases and putative ion exchangers on mammalian cells (Styrt et al., 1988), the identity of the proteins that mediate lysosomal Ca²⁺ uptake remains elusive. In this issue, Melchionda et al. describe the first lysosomal CAX in nonplacental mammals and link lysosomal Ca²⁺ import via CAX to the maintenance of normal cellular migration during development.To identify novel regulators of Ca²⁺ transport in vertebrates, Melchionda et al. (2016) searched gene databases for homologues of the CAX proteins, which are known to use the proton gradient across the vacuole to drive Ca²⁺ uptake in plant and yeast cells (Dunn et al., 1994). They identified putative CAX genes in many species, from sea urchins and frogs to reptiles and birds. The CAX homologues discovered in the genomes of the platypus and Tasmanian devil are the first lysosomal Ca²⁺ exchangers to be identified in any mammalian species. This new work is a significant finding as it suggests that these mechanisms do clearly exist in some mammalian cells and are required for lysosomal Ca²⁺ store filling. To examine the regulation of lysosomal Ca²⁺ uptake by vertebrate CAX transporters, the authors cloned full-length CAX from the frog and found that expression of frog CAX could rescue Ca²⁺ transport in yeast lacking their own CAX. Furthermore, the authors show that the frog CAX channels correctly localize to lysosomes when expressed in human cell lines and that these CAX are capable of manipulating lysosomal and cytosolic Ca²⁺ levels (in a manner perhaps comparable to plasma membrane Ca²⁺ ATPases). The findings reported in Melchionda et al. (2016) also have significance for researchers who are using simpler model organisms to characterize mechanisms regulating acidic store Ca²⁺. A study by Churchill et al. (2002) that used acidic stores purified from sea urchin egg homogenate to monitor acidic store Ca²⁺ entry concluded that vanadate-sensitive Ca²⁺ pumps were absent and suggested instead the presence of a CAX. This now appears to be the case through the reported cloning of sea urchin CAX. The findings of Melchionda et al. (2016) are a step forward in unraveling the molecular mechanisms of Ca²⁺ handling in model animals.Ca²⁺ signaling plays an important role in development, particularly for cellular migration, where localized elevations in intracellular Ca²⁺ drive rearrangement of the cytoskeleton, cellular contraction, and adhesion (Wei et al., 2009; Sumoza-Toledo et al., 2011; Praitis et al., 2013). A concentration gradient of Ca²⁺ exists across the migrating cell, with higher levels at the rear that contribute to cellular detachment and contraction (Praitis et al., 2013). Recent evidence has highlighted the presence of Ca²⁺ flickers at the leading edge of the migrating cell that have been shown to underlie changes in direction (Wei et al., 2009). Despite the clear importance of Ca²⁺ in mediating cellular migration events and the emergent role of lysosomes in maintaining intracellular Ca²⁺ signaling, very little is known about the roles of lysosomal Ca²⁺ stores in cellular migration. ER Ca²⁺ channels including the inositol 1,4,5-trisphosphate receptors and ryanodine receptors as well as the secretory pathway Ca²⁺ ATPase and lysosomal TRPM2 have all been implicated in regulating changes in intracellular Ca²⁺ to mediate cellular migration, but to date no lysosomal transporters have been implicated in this process (Wei et al., 2009; Sumoza-Toledo et al., 2011; Praitis et al., 2013). Melchionda et al. (2016) investigated the migration of neural crest cells during frog development to find out whether or not CAX transporters control cell motility. CAX proteins are expressed in the neural crest of developing frogs and morpholino-mediated knockdown of CAX expression increased cytosolic Ca²⁺ levels and impeded neural crest cell migration. Confocal imaging of neural crest tissue in vitro revealed the dynamic recruitment of CAX-containing vesicles to the protrusions that contain focal adhesion complexes at the leading edge of the migrating neural cells. Loss of CAX protein expression reduced the ability of neural crest cells to form stable focal adhesions and undergo the initial cell spreading required for migration. The work presented by Melchionda et al. (2016) is a significant discovery providing evidence that lysosomal Ca²⁺ uptake is involved in cell migration and that lower organisms are useful model systems to investigate the role of acidic store Ca²⁺ in this critical cellular function during embryo development.Melchionda et al. (2016) have made a significant step forward in our understanding of the mechanisms that regulate lysosomal/vacuolar Ca²⁺ entry. However, we remain in the dark about the identity of the transporters that pump Ca²⁺ into the lysosomes of placental mammals. What led to the loss of CAX genes in these organisms is as much a mystery as the identity of the transporters that have replaced CAX. Evidence from a study using purified mammalian lysosomes to observe Ca²⁺ uptake indicates that the process is ATP-dependent (Styrt et al., 1988). Placental mammals may have completely different ATP-dependent mechanisms governing lysosomal Ca²⁺ uptake compared with lower order organisms and nonplacental mammals. Interestingly, defects in lysosomal Ca²⁺ uptake are associated with two human diseases, Niemann-Pick type C and Chediak-Higashi syndrome (CHS; Styrt et al., 1988; Lloyd-Evans et al., 2008). The lysosomal accumulation of sphingosine, a Ca²⁺ ATPase inhibitor (Lloyd-Evans and Platt, 2011), leads to reduced lysosomal Ca²⁺ levels in Niemann-Pick type C disease cells and defects in NAADP-mediated lysosomal Ca²⁺ release (Lloyd-Evans et al., 2008). In CHS, there have been reports of enhanced lysosomal Ca²⁺ ATPase transporter activity in neutrophils (Styrt et al., 1988). Interestingly, CHS leukocytes show alterations in chemotaxis with a reduced response to chemotactic factors (Clark and Kimball, 1971), which is supportive of the findings of Melchionda et al. (2016). Much remains to be elucidated about the enigma of mammalian lysosomal Ca²⁺ uptake, but the work of Melchionda et al. (2016) begins to pick this mystery apart.     

Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium

Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the acidic compartment (which we define as the late endosome and the lysosome) protein, NPC1. The function of NPC1 is unknown, but when it is dysfunctional, sphingosine, glycosphingolipids, sphingomyelin and cholesterol accumulate. We have found that NPC1-mutant cells have a large reduction in the acidic compartment calcium store compared to wild-type cells. Chelating luminal endocytic calcium in normal cells with high-affinity Rhod-dextran induced an NPC disease cellular phenotype. In a drug-induced NPC disease cellular model, sphingosine storage in the acidic compartment led to calcium depletion in these organelles, which then resulted in cholesterol, sphingomyelin and glycosphingolipid storage in these compartments. Sphingosine storage is therefore an initiating factor in NPC1 disease pathogenesis that causes altered calcium homeostasis, leading to the secondary storage of sphingolipids and cholesterol. This unique calcium phenotype represents a new target for therapeutic intervention, as elevation of cytosolic calcium with curcumin normalized NPC1 disease cellular phenotypes and prolonged survival of the NPC1 mouse.     

Glucosylceramide and glucosylsphingosine modulate calcium mobilization from brain microsomes via different mechanisms

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673-21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were approximately 10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.     

The Relationship between Therapeutic Alliance and Service User Satisfaction in Mental Health Inpatient Wards and Crisis House Alternatives: A Cross-Sectional Study

Background

Poor service user experiences are often reported on mental health inpatient wards. Crisis houses are an alternative, but evidence is limited. This paper investigates therapeutic alliances in acute wards and crisis houses, exploring how far stronger therapeutic alliance may underlie greater client satisfaction in crisis houses.

Methods and Findings

Mixed methods were used. In the quantitative component, 108 crisis house and 247 acute ward service users responded to measures of satisfaction, therapeutic relationships, informal peer support, recovery and negative events experienced during the admission. Linear regressions were conducted to estimate the association between service setting and measures, and to model the factors associated with satisfaction. Qualitative interviews exploring therapeutic alliances were conducted with service users and staff in each setting and analysed thematically.

Results

We found that therapeutic alliances, service user satisfaction and informal peer support were greater in crisis houses than on acute wards, whilst self-rated recovery and numbers of negative events were lower. Adjusted multivariable analyses suggest that therapeutic relationships, informal peer support and negative experiences related to staff may be important factors in accounting for greater satisfaction in crisis houses. Qualitative results suggest factors that influence therapeutic alliances include service user perceptions of basic human qualities such as kindness and empathy in staff and, at service level, the extent of loss of liberty and autonomy.

Conclusions and Implications

We found that service users experience better therapeutic relationships and higher satisfaction in crisis houses compared to acute wards, although we cannot exclude the possibility that differences in service user characteristics contribute to this. This finding provides some support for the expansion of crisis house provision. Further research is needed to investigate why acute ward service users experience a lack of compassion and humanity from ward staff and how this could be changed.