Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein.

Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half-life and are resistant to genetic manipulation. Neutrophil-like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER-HoxB8 oncoprotein, but whether these cells can replace neutrophils in high-throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER-HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro-inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H2 O2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays.

The role of STIM proteins in neutrophil functions.

Stromal interaction molecule (STIM) proteins regulate store-operated Ca2+ entry (SOCE) in innate and adaptive immune cells and participate in the Ca2+ signals that control the functions of neutrophils, the first line of host defence against bacterial and fungal infections. Loss-of-function experiments in animal and cellular models indicate that both STIM1 and STIM2 regulate neutrophil functions, but the complexity of the SOCE machinery and the versatility of neutrophils complicate the evaluation of the results. This review aims to summarize the latest progress in the field, with special attention to the details of the experimental designs. Future study design should aim to improve the standardization of experimental procedures and to provide a more holistic understanding of the role of STIM proteins in neutrophils function.

A calcium-redox feedback loop controls human monocyte immune responses: The role of ORAI Ca2+ channels.

In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.

Influence of extracellular magnesium on phagocytosis and free cytosolic Mg levels in differentiated U937 and MH-S cells.

BACKGROUND: The influence of extracellular calcium on phagocytosis is limited, and the involvement of extracellular magnesium is unclear. AIMS: The role of extracellular calcium and magnesium on phagocytosis efficiency was investigated. RESULTS: Extracellular calcium had no influence on the internalization of 1 µm polystyrene particles by primary monocytes as has been shown before for the human lymphoma-derived, differentiated cell line U937 and murine alveolar macrophage-derived MH-S cells. In contrast, the phagocytosis by differentiated U937 cells was positively affected by the presence of extracellular magnesium whereas that of MH-S cells was not. An extracellular increase in the magnesium level did not cause an increase in the free cytosolic magnesium concentration in either cell line. In contrast to magnesium, extracellular calcium caused an increase in intracellular divalent cation levels in differentiated U937 cells. CONCLUSIONS: A phagocytosis-enhancing effect in the extracellular space was observed in relation to extracellular magnesium rather than with an intracellular increase in magnesium levels, indicating that murine and human immune cells might be regulated differently.

Inverse regulation of melanoma growth and migration by Orai1/STIM2-dependent calcium entry.

Spontaneous melanoma phenotype switching is controlled by unknown environmental factors and may determine melanoma outcome and responsiveness to anticancer therapy. We show that Orai1 and STIM2 are highly expressed and control store-operated Ca(2+) entry in human melanoma. Lower extracellular Ca(2+) or silencing of Orai1/STIM2 caused a decrease in intracellular Ca(2+) , which correlated with enhanced proliferation and increased expression of microphthalmia-associated transcription factor, a marker for proliferative melanoma phenotype. In contrast, the invasive and migratory potential of melanoma cells was reduced upon silencing of Orai1 and/or STIM2. Accordingly, markers for a non-proliferative, tumor-maintaining phenotype such as JARID1B and Brn2 decreased. Immunohistochemical staining of primary melanomas and lymph node metastases revealed a heterogeneous distribution of Orai1 and STIM2 with elevated expression in the invasive rim of the tumor. In summary, our results support a dynamic model in which Orai1 and STIM2 inversely control melanoma growth and invasion. Pharmacological tuning of Orai1 and particularly STIM2 might thus prevent metastatic spread and render melanomas more susceptible to conventional therapy.

How ORAI and TRP channels interfere with each other: interaction models and examples from the immune system and the skin.

Four types of Ca(2+) selective ion channels are known, ten voltage gated Ca(2+) (CaV) channels, four CatSper channels, three store operated CRAC channels (ORAI channels) and at least two members of the TRPV subfamily (TRPV5, TRPV6). Some of the other TRP channels also show some Ca(2+) selectivity like certain splice variants of TRPM3. In addition to Ca(2+) selective channels, various cation channels play an important role for Ca(2+) entry and furthermore, they may also regulate Ca(2+) entry through other channels by modulating the membrane potential or other means as outlined in this review. Of the different types of cation channels, TRP channels form one of the most prominent families of non-selective cation channels with functional relevance in electrically non-excitable and electrically excitable cell types. Among these, the seven channels of the TRPC subfamily are rather non-selective with very modest Ca(2+) selectivity, whereas in the other subfamilies, cation selectivity ranges from monovalent selectivity (i.e. TRPM4, TRPM5) to divalent selectivity (i.e. TRPM6, TRPM7) or Ca(2+) selectivity (i.e. TRPV5, TRPV6). Rather than discussing the heavily reviewed individual functions of ORAI or TRP channels, we summarize data and present models how TRP and ORAI may functionally interact to guide cellular functions. We focus on T lymphocytes representing a more ORAI-dominated tissue and skin as model system in which both ORAI and TRP channel have been reported to control relevant functions. We present several interaction models how ORAI and TRP may interfere with each other's function.

Hydroxylated derivatives of dimethoxy-1,4-benzoquinone as redox switchable earth-alkaline metal ligands and radical scavengers.

Benzoquinones (BQ) have important functions in many biological processes. In alkaline environments, BQs can be hydroxylated at quinoid ring proton positions. Very little is known about the chemical reaction leading to these structural transformations as well as about the properties of the obtained hydroxyl benzoquinones. We analyzed the behavior of the naturally occurring 2,6-dimethoxy-1,4-benzoquinone under alkaline conditions and show that upon substitution of methoxy-groups, poly-hydroxyl-derivatives (OHBQ) are formed. The emerging compounds with one or several hydroxyl-substituents on single or fused quinone-rings exist in oxidized or reduced states and are very stable under physiological conditions. In comparison with the parent BQs, OHBQs are stronger radical scavengers and redox switchable earth-alkaline metal ligands. Considering that hydroxylated quinones appear as biosynthetic intermediates or as products of enzymatic reactions, and that BQs present in food or administered as drugs can be hydroxylated by enzymatic pathways, highlights their potential importance in biological systems.