Mast cell restricted mouse and human tryptase·heparin complexes hinder thrombin-induced coagulation of plasma and the generation of fibrin by proteolytically destroying fibrinogen

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.     

Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung

Human pulmonary mast cells (MCs) express tryptases alpha and beta I, and both granule serine proteases are exocytosed during inflammatory events. Recombinant forms of these tryptases were generated for the first time to evaluate their substrate specificities at the biochemical level and then to address their physiologic roles in pulmonary inflammation. Analysis of a tryptase-specific, phage display peptide library revealed that tryptase beta I prefers to cleave peptides with 1 or more Pro residues flanked by 2 positively charged residues. Although recombinant tryptase beta I was unable to activate cultured cells that express different types of protease-activated receptors, the numbers of neutrophils increased >100-fold when enzymatically active tryptase beta I was instilled into the lungs of mice. In contrast, the numbers of lymphocytes and eosinophils in the airspaces did not change significantly. More important, the tryptase beta I-treated mice exhibited normal airway responsiveness. Neutrophils did not extravasate into the lungs of tryptase alpha-treated mice. Thus, this is the first study to demonstrate that the two nearly identical human MC tryptases are functionally distinct in vivo. When MC-deficient W/W(v) mice were given enzymatically active tryptase beta I or its inactive zymogen before pulmonary infection with Klebsiella pneumoniae, tryptase beta I-treated W/W(v) mice had fewer viable bacteria in their lungs relative to zymogen-treated W/W(v) mice. Because neutrophils are required to combat bacterial infections, human tryptase beta I plays a critical role in the antibacterial host defenses of the lung by recruiting neutrophils in a manner that does not alter airway reactivity.     

The roles of mast cells in anticancer immunity

The tumor microenvironment (TME), which is composed of stromal cells such as endothelial cells, fibroblasts, and immune cells, provides a supportive niche promoting the growth and invasion of tumors. The TME also raises an immunosuppressive barrier to effective antitumor immune responses and is therefore emerging as a target for cancer immunotherapies. Mast cells (MCs) accumulate in the TME at early stages, and their presence in the TME is associated with poor prognosis in many aggressive human cancers. Some well-established roles of MCs in cancer are promoting angiogenesis and tumor invasion into surrounding tissues. Several mouse models of inducible and spontaneous cancer show that MCs are among the first immune cells to accumulate within and shape the TME. Although MCs and other suppressive myeloid cells are associated with poor prognosis in human cancers, high densities of intratumoral T effector (T(eff)) cells are associated with a favorable prognosis. The latter finding has stimulated interest in developing therapies to increase intratumoral T cell density. However, cellular and molecular mechanisms promoting high densities of intratumoral T(eff) cells within the TME are poorly understood. New evidence suggests that MCs are essential for shaping the immune-suppressive TME and impairing both antitumor T(eff) cell responses and intratumoral T cell accumulation. These roles for MCs warrant further elucidation in order to improve antitumor immunity. Here, we will summarize clinical studies of the prognostic significance of MCs within the TME in human cancers, as well as studies in mouse models of cancer that reveal how MCs are recruited to the TME and how MCs facilitate tumor growth. Also, we will summarize our recent studies indicating that MCs impair generation of protective antitumor T cell responses and accumulation of intratumoral T(eff) cells. We will also highlight some approaches to target MCs in the TME in order to unleash antitumor cytotoxicity.     

T cell proliferation by direct cross-talk between OX40 ligand on human mast cells and OX40 on human T cells: comparison of gene expression profiles between human tonsillar and lung-cultured mast cells

Mast cells (MCs) are the primary effector cells in allergic reactions and have also been found to activate T cells and to reside in close physical proximity to T cells. However, the molecular mechanisms involved in the MC-T cell interaction remain unclear. We hypothesized that human tonsillar MCs, which locate in the interfollicular areas, might interact with T cells. Thus, we first established a culture system of human tonsillar MCs and then compared gene expression profiles of tonsillar MCs with that of lung MCs before and after aggregation of FcepsilonRI by using high-density oligonucleotide probe arrays. Here we show that resting tonsillar MCs, when compared with lung MCs, revealed significantly higher expression levels for CC chemokines (CCL3 and 4), which recruit T cells, and for TNFR superfamilies (OX40 ligand and 4-1BB ligand), which induce proliferation of T cells. After aggregation of FcepsilonRI, not only tonsillar MCs but also lung MCs up-regulated the expression of these molecules. We confirmed that T cell proliferation is induced in direct cross-talk by the MC surface molecule OX40 ligand. These results suggest that human MCs may play important roles in adaptive immunity through the T cell responses.     

Characterization of vascular mural cells during zebrafish development

Development and maturation of the nascent cardiovascular system requires the recruitment of mural cells (MCs) around the vascular tree in a process called vascular myogenesis. Understanding the origin and development of vascular MCs has been hampered by difficulties in observing these cells in vivo and performing defined genetic and experimental manipulations in available model organisms. Here, we investigate the origin of vascular MCs using molecular and genetic tools in zebrafish. We show that vascular MCs are present around the lateral dorsal aortae (LDA) and anterior mesenteric arteries (AMA) of developing animals, and that they also contribute to the outflow tract of the developing heart and ventral aorta (VA). Genetic data indicate that the vascular MCs of the LDA and AMA do not arise from blood or endothelial progenitors but from other derivatives of the lateral plate mesoderm. We further show that zebrafish vascular MCs share many of the morphological, molecular and functional characteristics of vascular smooth muscle cells and pericytes found in higher vertebrates. These data establish the zebrafish as a useful cellular and genetic model to study vascular myogenesis as well as tumor angiogenesis and other MC-associated diseases.     

Mast cell tryptases: examination of unusual characteristics by multiple sequence alignment and molecular modeling.

Tryptases are trypsin-like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M. Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three-dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active-site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline-rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin-binding sequence, the tryptases have 10-13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn-linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site-directed mutagenesis.     

Identification of a subgroup of glycosylphosphatidylinositol-anchored tryptases

The tryptase locus on mouse chromosome 17A3.3 contains 13 genes that encode enzymatically active serine proteases with different tissue expression profiles and substrate specificities. Mouse mast cell protease (mMCP) 6, mMCP-7, mMCP-11/protease serine member S (Prss) 34, tryptase 6/Prss33, tryptase ε/Prss22, implantation serine protease (Isp) 1/Prss28, and Isp-2 are constitutively exocytosed enzymes. We now demonstrate that tryptase 5/Prss32, pancreasin/Prss27, and testis serine protease-1 are inserted into plasma membranes via glycosylphosphatidylinositol (GPI) anchors analogous to Prss21, and that these serine proteases can be released from the cell’s surface by a phosphatidylinositol-specific phospholipase C. These data suggest that the C-terminal residues play key roles in determining where tryptases compartmentalize in cells. GPI-anchored proteins are targeted to lipid rafts. Thus, our identification of a number of GPI-anchored tryptases whose genes reside at mouse chromosome 17A3.3 also implicates important biological functions for this new family of serine proteases on the surfaces of cells.     

Pathophysiological Roles of Gap Junction in Glomerular Mesangial Cells

Glomerular mesangial cells (MCs) are specialized vascular smooth muscle cells that play a critical role in the control of glomerular hemodynamics. One of the intriguing features of MCs is their extraordinary abundance in gap junctions (GJs). It has long been speculated that GJs may bridge MCs together and provide the mesangium with the characteristics of a functional syncytium. Accumulating scientific evidence supports this idea. GJs are reported to be critically involved in important physiological processes like tubuloglomerular feedback and glomerular filtration. In addition, GJs are implicated in the control of many cellular processes of MCs, including growth, differentiation and survival. This article summarizes the current knowledge on the roles of GJs in glomerular pathophysiology.     

Ancient origin of mast cells

The sentinel roles of mammalian mast cells (MCs) in varied infections raised the question of their evolutionary origin. We discovered that the test cells in the sea squirt Ciona intestinalis morphologically and histochemically resembled cutaneous human MCs. Like the latter, C. intestinalis test cells stored histamine and varied heparin·serine protease complexes in their granules. Moreover, they exocytosed these preformed mediators when exposed to compound 48/80. In support of the histamine data, a C. intestinalis-derived cDNA was isolated that resembled that which encodes histidine decarboxylase in human MCs. Like heparin-expressing mammalian MCs, activated test cells produced prostaglandin D₂ and contained cDNAs that encode a protein that resembles the synthase needed for its biosynthesis in human MCs. The accumulated morphological, histochemical, biochemical, and molecular biology data suggest that the test cells in C. intestinalis are the counterparts of mammalian MCs that reside in varied connective tissues. The accumulated data point to an ancient origin of MCs that predates the emergence of the chordates >500 million years ago, well before the development of adaptive immunity. The remarkable conservation of MCs throughout evolution is consistent with their importance in innate immunity.     

Synovial Fibroblasts Promote the Expression and Granule Accumulation of Tryptase via Interleukin-33 and Its Receptor ST-2 (IL1RL1)

A characteristic feature of tissue resident human mast cells (MCs) is their hTryptase-β-rich cytoplasmic granules. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and we have shown that this tetramer-forming tryptase has beneficial roles in innate immunity but adverse roles in inflammatory disorders like experimental arthritis. Because the key tissue factors that control tryptase expression in MCs have not been identified, we investigated the mechanisms by which fibroblasts mediate the expression and granule accumulation of mMCP-6. Immature mouse bone marrow-derived MCs (mBMMCs) co-cultured with fibroblast-like synoviocytes (FLS) or mouse 3T3 fibroblasts markedly increased their levels of mMCP-6. This effect was caused by an undefined soluble factor whose levels could be increased by exposing FLS to tumor necrosis factor-α or interleukin (IL)-1β. Gene expression profiling of mBMMCs and FLS for receptor·ligand pairs of potential relevance raised the possibility that IL-33 was a sought after fibroblast-derived factor that promotes tryptase expression and granule maturation via its receptor IL1RL1/ST2. MCs lacking IL1RL1 exhibited defective fibroblast-driven tryptase accumulation, whereas recombinant IL-33 induced mMCP-6 mRNA and protein accumulation in wild-type mBMMCs. In agreement with these data, synovial MCs from IL1RL1-null mice exhibited a marked reduction in mMCP-6 expression. IL-33 is the first factor shown to modulate tryptase expression in MCs at the mRNA and protein levels. We therefore have identified a novel pathway by which mesenchymal cells exposed to inflammatory cytokines modulate the phenotype of local MCs to shape their immune responses.     

Focus on mast cells in the tumor microenvironment: Current knowledge and future directions

Mast cells (MCs) are crucial cells participating in both innate and adaptive immune processes that play important roles in protecting human health and in the pathophysiology of various diseases, such as allergies, cardiovascular diseases, and autoimmune diseases. In the context of tumors, MCs are a non-negligible population of immune cells in the tumor microenvironment (TME). In most tumor types, MCs accumulate in both the tumor tissue and the surrounding tissue. MCs interact with multiple components of the TME, affecting TME remodeling and the tumor cell fate. However, controversy persists regarding whether MCs contribute to tumor progression or trigger an anti-tumor immune response. This review focuses on the context of the TME to explore the specific properties and functions of MCs and discusses the crosstalk that occurs between MCs and other components of the TME, which affect tumor angiogenesis and lymphangiogenesis, invasion and metastasis, and tumor immunity through different mechanisms. We also anticipate the potential role of MCs in cancer immunotherapy, which might expand upon the success achieved with existing cancer therapies.     

Mast cells, angiogenesis, and tumour growth

Accumulation of mast cells (MCs) in tumours was described by Ehrlich in his doctoral thesis. Since this early account, ample evidence has been provided highlighting participation of MCs to the inflammatory reaction that occurs in many clinical and experimental tumour settings. MCs are bone marrow-derived tissue-homing leukocytes that are endowed with a panoply of releasable mediators and surface receptors. These cells actively take part to innate and acquired immune reactions as well as to a series of fundamental functions such as angiogenesis, tissue repair, and tissue remodelling. The involvement of MCs in tumour development is debated. Although some evidence suggests that MCs can promote tumourigenesis and tumour progression, there are some clinical sets as well as experimental tumour models in which MCs seem to have functions that favour the host. One of the major issues linking MCs to cancer is the ability of these cells to release potent pro-angiogenic factors. This review will focus on the most recent acquisitions about this intriguing field of research. This article is part of a Special Issue entitled: Mast cells in inflammation.     

Melanocyte destruction and repigmentation in vitiligo: A model for nerve cell damage and regrowth

Melanocytes (MCs) are melanin-producing cells of the skin that are derived from neural crest cells. Vitiligo vulgaris is a common depigmentation disorder resulting from the destruction of functional MCs in the affected skin. The three prevailing pathomechanisms of vitiligo are the immune hypothesis, the neural hypothesis and the autocytotoxic hypothesis. None of these mechanisms has been conclusively proven. Melanoblasts (MBs) in the outer root sheath of the hair follicles are the reservoir cells for repigmentation. Recovery from vitiligo is initiated by activation and proliferation of these MBs, followed by upward migration to the nearby epidermis that forms perifollicular pigmentation islands. Migration, proliferation and differentiation of MCs and MBs are regulated by keratinocyte-derived factors and some coat color genes. Any therapy for vitiligo must explain not only the repopulation of MCs but also their functional development. In patients with vitiligo, MCs are destroyed in the skin, the eyes, and possibly the ears. However, the concept of vitiligo as a systemic disease will be clearly established only when the mechanisms involved in vitiligo are identified. Recent advances in the fields of neural crest cell culture and molecular genetics have opened new perspectives in the understanding of vitiligo. Not only will this result in better treatments for vitiligo patients, but possibly will also provide a key to triggering nerve cell regrowth in other nervous diseases.     

Commensal Bacteria Lipoteichoic Acid Increases Skin Mast Cell Antimicrobial Activity against Vaccinia Viruses

Mast cells (MCs) are considered sentinels in the skin and mucosa. Their ability to release antimicrobial peptides, such as cathelicidin, protects against bacterial infections when the epithelial barrier is breached. We recently described that MCs defend against bacterial and viral infections through the release of cathelicidin during degranulation. In this study, we hypothesize that cathelicidin expression is induced in MCs by the activation of TLR2 from bacterial products (lipoteichoic acid) produced by commensal bacteria at the epithelial surface. Our research shows that signaling through TLR2 increases the production and expression of cathelicidin in mast cells, thereby enhancing their capacity to fight vaccinia virus. MCs deficient in cathelicidin were less efficient in killing vaccinia virus after lipoteichoic acid stimulation than wild-type cells. Moreover, the activation of TLR2 increases the MC recruitment at the skin barrier interface. Taken together, our findings reveal that the expression and control of antimicrobial peptides and TLR signaling on MCs are key in fighting viral infection. Our findings also provide new insights into the pathogenesis of skin infections and suggest potential roles for MCs and TLR2 ligands in antiviral therapy.     

CD117+ Dendritic and Mast Cells Are Dependent on RasGRP4 to Function as Accessory Cells for Optimal Natural Killer Cell-Mediated Responses to Lipopolysaccharide

Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an evolutionarily conserved calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor. While an important intracellular signaling protein for CD117⁺ mast cells (MCs), its roles in other immune cells is less clear. In this study, we identified a subset of in vivo-differentiated splenic CD117⁺ dendritic cells (DCs) in wild-type (WT) C57BL/6 mice that unexpectedly contained RasGRP4 mRNA and protein. In regard to the biologic significance of these data to innate immunity, LPS-treated splenic CD117⁺ DCs from WT mice induced natural killer (NK) cells to produce much more interferon-γ (IFN-γ) than comparable DCs from RasGRP4-null mice. The ability of LPS-responsive MCs to cause NK cells to increase their expression of IFN-γ was also dependent on this intracellular signaling protein. The discovery that RasGRP4 is required for CD117⁺ MCs and DCs to optimally induce acute NK cell-dependent immune responses to LPS helps explain why this signaling protein has been conserved in evolution.     

Mast cell alpha and beta tryptases changed rapidly during primate speciation and evolved from gamma-like transmembrane peptidases in ancestral vertebrates

Human mast cell tryptases vary strikingly in secretion, catalytic competence, and inheritance. To explore the basis of variation, we compared genes from a range of primates, including humans, great apes (chimpanzee, gorilla, orangutan), Old- and New-World monkeys (macaque and marmoset), and a prosimian (galago), tracking key changes. Our analysis reveals that extant soluble tryptase-like proteins, including alpha- and beta-like tryptases, mastins, and implantation serine proteases, likely evolved from membrane-anchored ancestors because their more deeply rooted relatives (gamma tryptases, pancreasins, prostasins) are type I transmembrane peptidases. Function-altering mutations appeared at widely separated times during primate speciation, with tryptases evolving by duplication, gene conversion, and point mutation. The alpha-tryptase Gly(216)Asp catalytic domain mutation, which diminishes activity, is present in macaque tryptases, and thus arose before great apes and Old World monkeys shared an ancestor, and before the alphabeta split. However, the Arg(-3)Gln processing mutation appeared recently, affecting only human alpha. By comparison, the transmembrane gamma-tryptase gene, which anchors the telomeric end of the multigene tryptase locus, changed little during primate evolution. Related transmembrane peptidase genes were found in reptiles, amphibians, and fish. We identified soluble tryptase-like genes in the full spectrum of mammals, including marsupial (opossum) and monotreme (platypus), but not in nonmammalian vertebrates. Overall, our analysis suggests that soluble tryptases evolved rapidly from membrane-anchored, two-chain peptidases in ancestral vertebrates into soluble, single-chain, self-compartmentalizing, inhibitor-resistant oligomers expressed primarily by mast cells, and that much of present numerical, behavioral, and genetic diversity of alpha- and beta-like tryptases was acquired during primate evolution.     

Novel mast cell lines with enhanced proliferative and degranulative abilities established from temperature-sensitive SV40 large T antigen transgenic mice

Mast cells (MCs) play crucial roles in innate immunity to parasitic and bacterial infections as well as in hypersensitivity, such as the induction and exacerbation of allergy and autoimmune diseases. The regulatory mechanisms for MC development and effector functions are of great interest for developing novel therapeutic strategies against such disorders. Here we report the establishment of novel, immortalized MC lines from bone marrow (BM) cells of a temperature-sensitive mutant of SV40 large T antigen-transgenic mice (termed SVMCs). BM cells from tsSV40LT mice were cultured in the presence of interleukin (IL)-3 for 3 weeks, and then subjected to limiting dilution and single-cell cloning, yielding 27 independent MC clones, three of which were subjected to further analysis. On culture with nerve growth factor, stem cell factor and IL-3, these SVMC clones showed morphologic and biochemical changes from mucosal MC-like to connective-tissue MC-like phenotypes. These SVMC lines exhibited a significantly enhanced proliferation rate, and a higher responsiveness to the high affinity Fc receptor for IgE-mediated intracellular calcium mobilization and degranulation than those of BM-derived cultured MCs. These cell lines should facilitate studies on the mechanisms for the development, differentiation and effector functions of MCs in health and diseases.     

Mast cell accumulation in glioblastoma with a potential role for stem cell factor and chemokine CXCL12

Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf-/- mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma.     

Formation of enzymatically active, homotypic, and heterotypic tetramers of mouse mast cell tryptases. Dependence on a conserved Trp-rich domain on the surface

Mouse mast cell protease (mMCP) 6 and mMCP-7 are homologous tryptases stored in granules as macromolecular complexes with heparin and/or chondroitin sulfate E containing serglycin proteoglycans. When pro-mMCP-7 and pseudozymogen forms of this tryptase and mMCP-6 were separately expressed in insect cells, all three recombinant proteins were secreted into the conditioned medium as properly folded, enzymatically inactive 33-kDa monomers. However, when their propeptides were removed, mMCP-6 and mMCP-7 became enzymatically active and spontaneously assumed an approximately 150-kDa tetramer structure. Heparin was not required for this structural change. When incubated at 37 degrees C, recombinant mMCP-7 progressively lost its enzymatic activity in a time-dependent manner. Its N-linked glycans helped regulate the thermal stability of mMCP-7. However, the ability of this tryptase to form the enzymatically active tetramer was more dependent on a highly conserved Trp-rich domain on its surface. Although recombinant mMCP-6 and mMCP-7 preferred to form homotypic tetramers, these tryptases readily formed heterotypic tetramers in vitro. This latter finding indicates that the tetramer structural unit is a novel way the mast cell uses to assemble varied combinations of tryptases.