Gunnar Pejler

Approaches for Analyzing the Roles of Mast Cells and Their Proteases In Vivo

The roles of mast cells in health and disease remain incompletely understood. While the evidence that mast cells are critical effector cells in IgE-dependent anaphylaxis and other acute IgE-mediated allergic reactions seems unassailable, studies employing various mice deficient in mast cells or mast cell-associated proteases have yielded divergent conclusions about the roles of mast cells or their proteases in certain other immunological responses. Such controversial results call into question the relative utility of various older versus newer approaches to ascertain the roles of mast cells and mast cell proteases in vivo. This review discusses how both older and more recent mouse models have been used to investigate the functions of mast cells and their proteases in health and disease. We particularly focus on settings in which divergent conclusions about the importance of mast cells and their proteases have been supported by studies that employed different models of mast cell or mast cell protease deficiency. We think that two major conclusions can be drawn from such findings: (1) no matter which models of mast cell or mast cell protease deficiency one employs, the conclusions drawn from the experiments always should take into account the potential limitations of the models (particularly abnormalities affecting cell types other than mast cells) and (2) even when analyzing a biological response using a single model of mast cell or mast cell protease deficiency, details of experimental design are critical in efforts to define those conditions under which important contributions of mast cells or their proteases can be identified.

Cloning, structural analysis, and expression of the pig IgE ε chain

Abstractu2003As a step in the evolutionary studies of immunoglobulin E (IgE) and for the purpose of developing new reagents that will facilitate a more detailed analysis of IgE-mediated inflammatory reactions in a large animal model, we here present the cloning of the ε chain of IgE in the domestic pig (Sus scrufa). A partial cDNA clone for the ε chain of pig IgE was isolated by polymerase chain reaction (PCR) amplification using degenerate primers directed against conserved regions in the second (CH2) and the fourth (CH4) constant domains of IgE. cDNA derived from mRNA isolated from the spleen and lymph nodes of a pig actively sensitized with a protein extract from the nematode Ascaris suum was used as template. Screening of a spleen cDNA library with the partial cDNA clone as probe resulted in isolation of a clone that contained the entire coding region. The nucleotide sequence was determined and was found to conform with the previously identified mammalian ε-chain sequences. The highest degree of similarity was found to sheep IgE. A DNA construct encoding a baculovirus signal sequence, a histidine hexapeptide, and the CH2-CH3-CH4 domains of the pig IgE ε chain was obtained by PCR amplification. The construct was ligated into the baculovirus expression vector pVL1392. Infection of High Five insect cells with recombinant baculovirus resulted in expression and secretion of a soluble 6 × His-CH2-CH3-CH4 protein product.

The Role of Mast Cells in Bacterial Infection.

Mast cells (MCs) are particularly abundant at host-environment interfaces, such as skin and intestinal mucosa. Because of their location, it has been hypothesized that MCs can act as sentinel cells that sense microbial attacks and initiate a protective immune response. Several studies have suggested that animals deficient in MCs exhibit a worsened pathology in various experimental models of bacterial infection. However, other studies have indicated that MCs under certain circumstances may have a detrimental impact on bacterial disease, and there are also recent studies indicating that MCs are dispensable for the clearance of bacterial pathogens. Herein, we review thexa0current knowledge of the role of MCs in bacterial infection.

Distorted Secretory Granule Composition in Mast Cells with Multiple Protease Deficiency

Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

Proteome analysis of mast cell releasates reveals a role for chymase in the regulation of coagulation factor XIIIA levels via proteolytic degradation

Background: Mast cells are significantly involved in IgE‐mediated allergic reactions; however, their roles in health and disease are incompletely understood. Objective: We aimed to define the proteome contained in mast cell releasates on activation to better understand the factors secreted by mast cells that are relevant to the contribution of mast cells in diseases. Methods: Bone marrow–derived cultured mast cells (BMCMCs) and peritoneal cell–derived mast cells were used as “surrogates” for mucosal and connective tissue mast cells, respectively, and their releasate proteomes were analyzed by mass spectrometry. Results: Our studies showed that BMCMCs and peritoneal cell–derived mast cells produced substantially different releasates following IgE‐mediated activation. Moreover, we observed that the transglutaminase coagulation factor XIIIA (FXIIIA) was one of the most abundant proteins contained in the BMCMC releasates. Mast cell–deficient mice exhibited increased FXIIIA plasma and activity levels as well as reduced bleeding times, indicating that mast cells are more efficient in their ability to downregulate FXIIIA than in contributing to its amounts and functions in homeostatic conditions. We found that human chymase and mouse mast cell protease‐4 (the mouse homologue of human chymase) had the ability to reduce FXIIIA levels and function via proteolytic degradation. Moreover, we found that chymase deficiency led to increased FXIIIA amounts and activity, as well as reduced bleeding times in homeostatic conditions and during sepsis. Conclusions: Our study indicates that the mast cell protease content can shape its releasate proteome. Moreover, we found that chymase plays an important role in the regulation of FXIIIA via proteolytic degradation.

Tryptase-catalyzed core histone truncation: a novel epigenetic regulatory mechanism in mast cells.

Background Mast cells are key effector cells in allergic reactions. When activated to degranulate, they release a plethora of bioactive compounds from their secretory granules, including mast cell‐restricted proteases such as tryptase. In a previous study, we showed that tryptase, in addition to its intragranular location, can be found within the nuclei of mast cells where it truncates core histones at their N‐terminal ends. Objective Considering that the N‐terminal portions of the core histones constitute sites for posttranslational modifications of major epigenetic impact, we evaluated whether histone truncation by tryptase could have an impact on epigenetic events in mast cells. Methods Mast cells were cultured from wild‐type and tryptase null mice, followed by an assessment of their profile of epigenetic histone modifications and their phenotypic characteristics. Results We show that tryptase truncates nucleosomal histone 3 and histone 2B (H2B) and that its absence results in accumulation of the epigenetic mark, lysine 5‐acetylated H2B. Intriguingly, the accumulation of lysine 5‐acetylated H2B was cell age–dependent and was associated with a profound upregulation of markers of non–mast cell lineages, loss of proliferative control, chromatin remodeling as well as extensive morphological alterations. Conclusions These findings introduce tryptase‐catalyzed histone clipping as a novel epigenetic regulatory mechanism, which in the mast cell context may be crucial for maintaining cellular identity. Graphical abstract Figure. No Caption available.

Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu+ is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2−/− mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2−/− mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

Damnacanthal inhibits IgE receptor-mediated activation of mast cells.

Damnacanthal, an anthraquinone obtained from the noni fruit (Morinda citrifolia L.), has been described to possess anti-cancer and anti-inflammatory properties. Since mast cells are key players in various inflammatory conditions as well as in cancer, we considered the possibility that the biological actions of damnacanthal, at least partly, could be due to effects on mast cells. Many of the biological activities of mast cells are mediated by IgE receptor cross-linking, which results in degranulation with release of preformed granule mediators, as well as de novo synthesis and release of additional compounds. Here we show that damnacanthal has profound inhibitory activity on mast cell activation through this pathway. The release of the granule compounds beta-hexosaminidase and tryptase release was completely abrogated by damnacanthal at doses that were non-toxic to mast cells. In addition, damnacanthal inhibited activation-dependent pro-inflammatory gene induction, as well as cytokine/chemokine release in response to mast cell stimulation. The mechanism underlying damnacanthal inhibition was linked to impaired phosphorylation of Syk and Akt. Furthermore, damnacanthal inhibited mast cell activation in response to calcium ionophore A23187. Altogether, the data presented here demonstrate that damnacanthal inhibits mast cell activation induced by different stimuli and open a new window for the use of this compound as a mast cell stabilizer.

Mast Cells and MCPT4 Chymase Promote Renal Impairment after Partial Ureteral Obstruction

Obstructive nephropathy constitutes a major cause of pediatric renal progressive disease. The mechanisms leading to disease progression are still poorly understood. Kidney fibrotic lesions are reproduced using a model of partial unilateral ureteral obstruction (pUUO) in newborn mice. Based on data showing significant mast cell (MC) infiltration in patients, we investigated the role of MC and murine MCPT4, a MC-released chymase, in pUUO using MC- (Wsh/sh), MCPT4-deficient (Mcpt4−/−), and wild-type (WT) mice. Measurement of kidney length and volume by magnetic resonance imaging (MRI) as well as postmortem kidney weight revealed hypotrophy of operated right kidneys (RKs) and compensatory hypertrophy of left kidneys. Differences between kidneys were major for WT, minimal for Wsh/sh, and intermediate for Mcpt4−/− mice. Fibrosis development was focal and increased only in WT-obstructed kidneys. No differences were noticed for local inflammatory responses, but serum CCL2 was significantly higher in WT versus Mcpt4−/− and Wsh/sh mice. Alpha-smooth muscle actin (αSMA) expression, a marker of epithelial–mesenchymal transition (EMT), was high in WT, minimal for Wsh/sh, and intermediate for Mcpt4−/− RK. Supernatants of activated MC induced αSMA in co-culture experiments with proximal tubular epithelial cells. Our results support a role of MC in EMT and parenchyma lesions after pUUO involving, at least partly, MCPT4 chymase. They confirm the importance of morphologic impairment evaluation by MRI in pUUO.

Mastitis Pathogens with high Virulence in a Mouse Model Produce a Distinct cytokine Profile In Vivo

Mastitis is a serious medical condition of dairy cattle. Here, we evaluated whether the degree of virulence of mastitis pathogens in a mouse model can be linked to the inflammatory response that they provoke. Clinical isolates of Staphylococcus aureus (S.u2009aureus) (strain 556 and 392) and Escherichia coli (E. coli) (676 and 127), and laboratory control strains [8325-4 (S. aureus) and MG1655 (E. coli)], were injected i.p. into mice, followed by the assessment of clinical scores and inflammatory parameters. As judged by clinical scoring, E. coli 127 exhibited the largest degree of virulence among the strains. All bacterial strains induced neutrophil recruitment. However, whereas E.u2009coli 127 induced high peritoneal levels of CXCL1, G-CSF, and CCL2, strikingly lower levels of these were induced by the less virulent bacterial strains. High concentrations of these compounds were also seen in blood samples taken from animals infected with E.u2009coli 127, suggesting systemic inflammation. Moreover, the levels of CXCL1 and G-CSF, both in the peritoneal fluid and in plasma, correlated with clinical score. Together, these findings suggest that highly virulent clinical mastitis isolates produce a distinct cytokine profile that shows a close correlation with the severity of the bacterial infection.