Diverse effects of bacterial cell wall components on mast cell degranulation,cysteinyl leukotriene generation and migration

Nowadays there is more and more evidence that mast cells take part in antibacterial defence. Mast cells have the ability to kill bacteria via phagocytose‐dependent or phagocytose‐independent ways and express antimicrobial peptides that can directly kill pathogens at their site of entry. What is more, mast cells are capable of processing bacterial antigens for presentation through class I and II MHC molecules. Some data indicate that these cells can release various proinflammatory mediators in response to activation with bacteria and/or their products, however this information is still far from complete. Therefore, in this study we examined the ability of PGN from Staphylococcus aureus, LPS from Eschericha coli and LAM from Mycobacterium smegmatis to stimulate mature rat mast cell degranulation as well as cysteinyl LT generation. We also studied the influence of these bacterial components on mast cell migration. We found that PGN, LPS and LAM all failed to induce mast cell degranulation and histamine release. At the same time, activation of mast cells with these bacterial antigens resulted in generation and release of significant amounts of LT. Moreover, we documented that, even in the presence of laminin, none of the bacterial antigens used stimulated mast cell migration. However, PGN did induce migration of RANTES‐primed mast cells, and LPS did stimulate mast cell migratory response after priming with IL‐6. Our results show that PGN, LPS and LAM might be among the important bacterial antigens involved in mast cell activation during bacterial infection.     

Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis

TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.     

Mast-cell responses to pathogens

Mast cells have mainly been studied in the setting of allergic disease, but the importance of mast cells for host defence against several pathogens has now been well established. The location of mast cells, which are found closely associated with blood vessels, allows them to have a crucial sentinel role in host defence. The mast cell has a unique 'armamentarium' of receptor systems and mediators for responding to pathogen-associated signals. Studies of this intriguing immune-effector cell provide important insights into the complex mechanisms by which appropriate innate and acquired immune responses are initiated.     

Mast Cell Proteoglycans

Mast cells are versatile effector cells of the immune system, contributing to both innate and adaptive immunity toward pathogens but also having profound detrimental activities in the context of inflammatory disease. A hallmark morphological feature of mast cells is their large content of cytoplasmic secretory granules, filled with numerous secretory compounds, including highly negatively charged heparin or chondroitin sulfate proteoglycans of serglycin type. These anionic proteoglycans provide the basis for the strong metachromatic staining properties of mast cells seen when applying various cationic dyes. Functionally, the mast cell proteoglycans have been shown to have an essential role in promoting the storage of other granule-contained compounds, including bioactive monoamines and different mast cell-specific proteases. Moreover, granule proteoglycans have been shown to regulate the enzymatic activities of mast cell proteases and to promote apoptosis. Here, the current knowledge of mast cell proteoglycans is reviewed.     

The role of mast cells in asthma

While the role of mast cells in allergic reactions is unequivocal, their precise functions in asthma remain controversial. Mast cells uniquely populate all vascularized organs and tissues, including the upper and lower respiratory tree, even in healthy individuals. Histologic evidence suggests that asthma is accompanied by a mast cell hyperplasia in the inflamed mucosal epithelium and the adjacent smooth muscle. The mechanisms responsible for constitutive mast cell development have been partly elucidated. Moreover, both in vitro studies and in vivo disease models indicate that mast cells have a remarkably flexible program of gene expression, and this program can be drastically altered by the T-cell-derived Th2 cytokines relevant to asthma. Moreover, the role of mast cells in innate immunity is now firmly established, and the capacity for numerous microbial pathogens to initiate their activation in vitro and in vivo suggest mechanisms by which microbes could initiate disease exacerbations.     

FcεR(1)-Mediated Mast Cell Reactivity Is Amplified through Prolonged Toll-Like Receptor-Ligand Treatment

Background

Mast cell-derived mediators mediate several of the pathological features of asthma. Microbial infections induce asthma exacerbations in which the contribution of mast cells remains incomprehensible.

Principal Findings

In this study we have investigated the characteristic expression pattern of Toll-like receptors (TLRs) 1–9 and the effect of TLR ligand treatment on IgE-receptor mediated mast cell reactivity. For the studies we employed in vitro differentiated connective tissue like mast cells (CTLMC) and mucosal like mast cells (MLMC) from mice. Both phenotypes were treated for 24 h or 96 h with ligands for TLR1/2, TLR2/6, TLR3 and TLR4, before activation with IgE and antigen. Prolonged exposure (96 h) with TLR-ligands promoted mast cell reactivity following IgE-receptor activation. TLR4 activation with LPS generated the most pronounced effect, with an enhanced degranulation and secretion of leukotrienes, cytokines and chemokines, in both CTLMC and MLMC. The effect of LPS was mediated through a Myd88-dependent pathway and the increased effect involved JNK-dependent pathway.

Conclusion

We find that prolonged exposure of mast cells to pathogens/TLR-ligands modulates their effector responses by priming them for increased release of several inflammatory mediators when subsequently activated by IgE-receptors. These data suggest that infections might exaggerate the severity of allergic reactions such as in asthma, by enhancing mediator release from mast cells.     

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin

Mast cells respond to pathogens and allergens by secreting a vast array of preformed and newly synthesized mediators, including enzymes, vasoactive amines, lipid mediators, cytokines and chemokines, thereby affecting innate and adaptive immune responses and pathogenesis. Here, we present evidence that skin-, but not lung-associated primary mast cells as well as in vitro-differentiated bone marrow-derived mast cells (BMMC) express granzyme (gzm) B, but not gzmA or perforin (perf). GzmB is associated with cytoplasmic granules of BMMC and secreted after Fcepsilon-receptor-mediated activation. BMMC from wild type but not gzmB-deficient mice cause cell death in susceptible adherent target cells, indicating that the perf-independent cytotoxicity of BMMC is executed by gzmB. Furthermore, gzmB induces a disorganization of endothelial cell-cell contacts. The data suggest that activated mast cells contribute, via secreted gzmB, to cell death, increased vascular permeability, leukocyte extravasation and subsequent inflammatory processes in affected tissues.     

Development of a mast cell-based biosensor

A mast cell-based biosensor has been developed to enable the use of these cells in numerous applications including pharmaceutical screening, environmental monitoring, clinical diagnosis and homeland security. Rat basophilic leukemia (RBL) mast cells offer excellent potential for biosensor applications because they are robust and undergo a dramatic exocytotic response within minutes of antigen addition. To monitor mast cell activation, fluorescent dyes were loaded into the cells and used as indicators of alkalinization of secretory granules, calcium fluxes or generation of reactive oxygen species. These fluorescence assays efficiently measure activation of antigen-stimulated RBL mast cells, detecting the antigen with picomolar sensitivity. To demonstrate the utility of this mast cell-based biosensor for detection of microbial pathogens, an IgE chimeric protein was created by fusing the Fc region of the IgE antibody to CD14, a receptor for lipopolysaccharide. This chimeric protein has the capacity to bind to Escherichia coli and Listeria monocytogenes and also to IgE receptors on the mast cells, thereby stimulating a signaling response to bacteria. RBL mast cells labeled with the calcium indicator Fluo-4 are shown to be responsive to E. coli, only when sensitized with the chimeric protein, thus demonstrating a highly versatile biosensor for bacterial contamination.     

Dengue virus selectively induces human mast cell chemokine production

Severe dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1alpha, and MIP-1beta, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P < 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1alpha, or MIP-1beta response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.     

Increased dermal mast cell prevalence and susceptibility to development of basal cell carcinoma in humans

Exposure to ultraviolet B (UVB) radiation (280-320 nm) is the primary etiologic factor associated with the development of basal cell carcinoma (BCC). The outgrowth of these keratinocyte-derived skin lesions is enhanced by the ability of UVB to impair an immune response that would otherwise eliminate them. Studies in a range of inbred mouse strains as well as mast cell-depleted mice reconstituted with mast cell precursors support a functional link between histamine-staining dermal mast cells and the extent of susceptibility to UVB-induced systemic immunomodulation. Humans, like mouse strains, display variations in dermal mast cell prevalence. In a study of Danish and South Australian BCC patients and control subjects, one 4-mm punch biopsy of non-sun-exposed buttock skin was sampled from each participant. This skin site was investigated to avoid any changes in mast cell prevalence caused by sun exposure. Two sections (4 microm) per biopsy were immunohistochemically stained for detection of histamine-containing dermal mast cells. Computer-generated image analysis evaluated dermal mast cell prevalence in both sections by quantifying the total number of mast cells according to the total dermal area (expressed as mast cells per square millimeter). This technique enabled us to detect heterogeneity of dermal mast cell prevalence in buttock skin between individuals and provided evidence of an association between high dermal mast cell prevalence and BCC development in two diverse populations. We hypothesize that mast cells function in humans, as in mouse strains, by initiating immunosuppression following UV irradiation and, thereby, allowing a permissive environment for the development of BCC. Thus, a high dermal mast cell prevalence as demonstrable in buttock skin is a significant predisposing factor for development of BCC in humans.     

Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACT

Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.     

Protein content, dry mass and chemical composition of individual mast cells related to body growth.

Recently developed quantitative microscopical techniques were used to study relations between body growth and protein content as well as dry mass of individual mast cells. Since previous studies had shown an age-related increase of mast cell content of 5-hydroxytryptamine (5-HT) and heparin, these mast cell components were also included in the present study. The cells were obtained from the peritoneal cavity of rats aged 44--269 days (body weights 189--610 g). All studied mast cell parameters showed an increase that was related to the growth of the animals. The dry mass increased 60%, protein 50%, heparin 50% but 5-HT increased as much as 260% during the studied growth period. There was a mutual and linear correlation between all studied mast cell parameters. Population studies, based on large scale measurements of individual mast cells from young and adult rats, were made. These studies showed that histograms of 5-HT content, protein content and dry mass of individual mast cells were skewed with a tail towards higher values and approximately lognormal. On the other hand, the frequency distribution of heparin content of individual mast cells was approximately normal.     

Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells

Mast cells play a critical role in host defense against bacterial infection. Murine mast cells produce cytokines in response to bacterial peptidoglycan and LPS via Toll-like receptor (TLR) TLR2- and TLR4-dependent mechanisms. The expression of TLRs by human mast cells and responses to known TLR activators was examined. Human mast cells expressed mRNA for TLR1, TLR2, and TLR6 but not TLR4. Bacterial peptidoglycan and yeast zymosan were potent inducers of GM-CSF and IL-1beta and also induced substantial short-term cysteinyl leukotriene generation. In contrast, a synthetic triacylated lipopeptide induced short-term degranulation but failed to induce cysteinyl leukotriene production. The TLR4 activator Escherichia coli LPS did not induce a GM-CSF, IL-1beta leukotriene, or degranulation response. These data demonstrate highly selective production of different classes of mast cell mediators in response to distinct TLR activators of potential importance to the host response to bacterial or fungal pathogens.     

Distribution, density and heterogeneity of canine mast cells and influence of fixation techniques

 The present study was carried out to determine the physiological distribution of mast cell numbers and types in the dog according to tissue location, staining and fixation methods. Tissue samples from stomach, duodenum, lung, lymph node, skin and uterus were evaluated. Samples were fixed in formalin as well as in Carnoy’s fluid. The average number of mast cells was determined using a metachromatic staining method. Protease content of mast cells was examined with a double enzyme-immunohistochemical staining technique, using a histochemical reaction for chloroacetate esterase to detect chymase activity and an immunohistochemical staining method for the detection of tryptase. Canine mast cells can be subdivided into formalin-sensitive and -resistant mast cells. Three subtypes were identified according to their content of the mast cell-specific proteases tryptase (T) and chymase (C): T-, TC- and C-mast cells. Significant differences regarding the distribution of mast cell subtypes as well as the influence of the fixation method can be observed. This underlines the fact that data regarding mast cell heterogeneity from other species, obtained by different fixation methods, are not comparable. This fact has to be taken into consideration when evaluating mast cell subtypes under pathological conditions. Accepted: 29 January 1998     

Protein content,dry mass and chemical composition of individual mast cells related to body growth

Summary Recently developed quantitative microscopical techniques were used to study relations between body growth and protein content as well as dry mass of individual mast cells. Since previous studies had shown an age-related increase of mast cell content of 5-hydroxytryptamine (5-HT) and heparin, these mast cell components were also included in the present study. The cells were obtained from the peritoneal cavity of rats aged 44–269 days (body weights 189–610 g). All studied mast cell parameters showed an increase that was related to the growth of the animals. The dry mass increased 60%, protein 50%, heparin 50% but 5-HT increased as much as 260% during the studied growth period. There was a mutual and linear correlation between all studied mast cell parameters. Population studies, based on large scale measurements of individual mast cells from young and adult rats, were made. These studies showed that histograms of 5-HT content, protein content and dry mass of individual mast cells were skewed with a tail towards higher values and approximately lognormal. On the other hand, the frequency distribution of heparin content of individual mast cells was approximately normal.Supported by grants from the Swedish Medical Research Council, Project no 2235     

Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (FcεRI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with FcεRI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders.     

Morphological and functional characteristics of peritoneal mast cells from young rats

To study why neonatal and young rats are resistant to the effects of some secretagogues, such as compound 48/80 and 2.5-S nerve growth factor, we examined peritoneal mast cells from 14–15-day-old rats (young rats) and compared them to peritoneal mast cells from adults. Peritoneal mast cells from young rats contain approximately one-tenth of the amount of histamine observed in adult peritoneal mast cells. However, both cell populations contained similar low levels of the mucosal mast cell-associated protease rat mast cell protease II. Histochemical analysis of peritoneal mast cells from young rats using safranin O and berberine sulphate suggested that only a portion of the granules of these cells contained heparin. At an ultrastructural level the young rat peritoneal mast cell contains relatively few granules. The majority of mast cells from young rats have a bilobed or indented nucleus which is only rarely observed in adult cells. Functionally, the young rat peritoneal mast cell demonstrates a significantly reduced histamine release in response to the connective tissue mast cellspecific secretagogues compound 48/80 and 2.5-S nerve growth factor. In contrast, the percent histamine release in response to the neurotransmitter substance P, which degranulates both connective tissue mast cells and intestinal mucosal mast cells, was similar in the adult cells and the young rat cells. This study demonstrates substantial differences between the young rat and adult peritoneal mast cells which may explain the ability of very young animals to withstand large doses of certain secretagogues.