The effect of extracellular nucleotides on cytokine production and phagocytosis of macrophages

Bacterial sepsis is a severe clinical condition, leading to severe sepsis, septic shock, and death. The complex pathophysiology of sepsis is not yet fully understood. Cytokines, released by immune cells such as macrophages, play an important role in the pathophysiology of sepsis. Kupffer cells are the largest population of macrophages in the body. Purinergic signaling, mediated by different nucleosides and nucleotides, and purinergic receptors, has been shown to have various effects on cytokine release, inflammatory processes and the immune system. In our work with in vitro experiments we studied the effect of extracellular nucleotides on the release of TNFα by primary murine Kupffer cells, and the effect of extracellular nucleotides on the phagocytosis of murine RAW 264.7 and human U-937 cell culture macrophages. Secretion of TNFα was measured using ELISA, phagocytosis of bio particles was measured using a plate reader phagocytosis assay and flow cytometry. Our experiments show, that extracellular LPS stimulate release of TNFα in murine Kupffer cells and that extracellular nucleotides inhibit this effect in a dose dependent matter. Our other experiments show phagocytosis of fluorescence labeled bio particles by both macrophage cell lines RAW 264.7 and U-937 in a dose dependent manner. The experiments could not show an effect of extracellular nucleotides on phagocytosis of cell culture macrophages.

Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.

Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation.

Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.

Active eosinophilic esophagitis is characterized by epithelial barrier defects and eosinophil extracellular trap formation.

BACKGROUND Eosinophilic esophagitis (EoE) exhibits esophageal dysfunction owing to an eosinophil-predominant inflammation. Activated eosinophils generate eosinophil extracellular traps (EETs) able to kill bacteria. There is evidence of an impaired barrier function in EoE that might allow pathogens to invade the esophagus. This study aimed to investigate the presence and distribution of EETs in esophageal tissues from EoE patients and their association with possible epithelial barrier defects. METHODS Anonymized tissue samples from 18 patients with active EoE were analyzed. The presence of DNA nets associated with eosinophil granule proteins forming EETs and the expression of filaggrin, the protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI), antimicrobial peptides, and cytokines were evaluated by confocal microscopy following immune fluorescence staining techniques. RESULTS Eosinophil extracellular trap formation occurred frequently and was detected in all EoE samples correlating with the numbers of infiltrating eosinophils. While the expression of both filaggrin and LEKTI was reduced, epithelial antimicrobial peptides (human beta-defensin-2, human beta-defensin-3, cathelicidin LL-37, psoriasin) and cytokines (TSLP, IL-25, IL-32, IL-33) were elevated in EoE as compared to normal esophageal tissues. There was a significant correlation between EET formation and TSLP expression (P = 0.02) as well as psoriasin expression (P = 0.016). On the other hand, a significant negative correlation was found between EET formation and LEKTI expression (P = 0.016). CONCLUSION Active EoE exhibits the presence of EETs. Indications of epithelial barrier defects in association with epithelial cytokines are also present which may have contributed to the activation of eosinophils. The formation of EETs could serve as a firewall against the invasion of pathogens.

Basophils exhibit antibacterial activity through extracellular trap formation.

Basophils are primarily associated with immunomodulatory functions in allergic diseases and parasitic infections. Recently, it has been demonstrated that both activated human and mouse basophils can form extracellular DNA traps (BETs) containing mitochondrial DNA and granule proteins. In this report, we provide evidence that, in spite of an apparent lack of phagocytic activity, basophils can kill bacteria through BET formation.

Chlorophyll Breakdown in Senescent Arabidopsis Leaves. Characterization of Chlorophyll Catabolites and of Chlorophyll Catabolic Enzymes Involved in the Degreening Reaction

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.

Mimicking respiratory phosphorylation using purified enzymes

The enzymes of oxidative phosphorylation are a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

Adenosine 5'-Phosphosulfate Sulfotransferase and Adenosine 5'-Phosphosulfate Reductase Are Identical Enzymes

Adenosine 5′-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M r of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K m = 6.5 μM) and not adenosine 3′-phosphate 5′-phosphosulfate as sulfonyl donor. TheV max of recombinant Lemna APS sulfotransferase (40 μmol min−1 mg protein−1) was about 10 times higher than the previously published V max of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO3 2−. Bound sulfite in the form ofS-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO3 2− was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

BACKGROUND The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth. METHODOLOGY/PRINCIPAL FINDINGS We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth. CONCLUSIONS/SIGNIFICANCE We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^