Publications by authors named "Richard L Stevens"

44 Publications

Experimental Arthritis Is Dependent on Mouse Mast Cell Protease-5.

J Biol Chem 2017 03 13;292(13):5392-5404. Epub 2017 Feb 13.

From the Department of Infectious Diseases, Immunology, and Sexual Health, St. George Hospital, and the St. George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales 2217, Australia.

The constitutive heparin (HP) mast cells (MCs) in mice express mouse MC protease (mMCP)-5 and carboxypeptidase A (mMC-CPA). The amino acid sequence of mMCP-5 is most similar to that of human chymase-1, as are the nucleotide sequences of their genes and transcripts. Using a homologous recombination approach, a C57BL/6 mouse line was created that possessed a disrupted gene. The resulting mice were fertile and had no obvious developmental abnormality. Lack of mMCP-5 protein did not alter the granulation of the IL-3/IL-9-dependent mMCP-2 MCs in the jejunal mucosa of -infected mice. In contrast, the constitutive HP MCs in the tongues of mMCP-5-null mice were poorly granulated and lacked mMC-CPA protein. Bone marrow-derived MCs were readily developed from the transgenic mice using IL-3. Although these MCs contained high levels of mMC-CPA mRNA, they also lacked the latter exopeptidase. mMCP-5 protein is therefore needed to target translated mMC-CPA to the secretory granule along with HP-containing serglycin proteoglycans. Alternately, mMCP-5 is needed to protect mMC-CPA from autolysis in the cell's granules. Fibronectin was identified as a target of mMCP-5, and the exocytosis of mMCP-5 from the MCs in the mouse's peritoneal cavity resulted in the expression of metalloproteinase protease-9, which has been implicated in arthritis. In support of the latter finding, experimental arthritis was markedly reduced in mMCP-5-null mice relative to wild-type mice in two disease models.
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http://dx.doi.org/10.1074/jbc.M116.773416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392683PMC
March 2017

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

PLoS One 2016 16;11(3):e0151638. Epub 2016 Mar 16.

Department of Infectious Diseases, Immunology, and Sexual Health, St. George Hospital, and the St. George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151638PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794117PMC
July 2016

The multifaceted mast cell in inflammatory bowel disease.

Inflamm Bowel Dis 2014 Dec;20(12):2364-78

*Division of Gastroenterology, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts; †Division of Gastroenterology, University Hospital Zurich, Zurich, Switzerland; and ‡Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.

Mast cells (MCs) are tissue-resident immune cells that carry out protective roles against pathogens. In disease states, such as inflammatory bowel disease, these granulocytes release a diverse array of mediators that contribute to inflammatory processes. They also participate in wound repair and tissue remodeling. In this review, the composition of MCs and how their phenotypes can be altered during inflammation of the gastrointestinal tract is detailed. Animal and human clinical studies that have implicated the participation of MCs in inflammatory bowel disease are reviewed, including the contribution of the cell's mediators to clinical symptoms, stress-triggered inflammation, and fistula and strictures. Studies that have focused on negating the proinflammatory roles of MCs and their mediators in animal models suggest new targets for therapies for patients with inflammatory bowel disease.
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http://dx.doi.org/10.1097/MIB.0000000000000142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428674PMC
December 2014

Ras guanine nucleotide-releasing protein 4 is aberrantly expressed in the fibroblast-like synoviocytes of patients with rheumatoid arthritis and controls their proliferation.

Arthritis Rheumatol 2015 Feb;67(2):396-407

Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Objective: Ras guanine nucleotide-releasing protein 4 (RasGRP-4) is a calcium-regulated guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor not normally expressed in fibroblasts. While RasGRP-4-null mice are resistant to arthritis induced by anti-glucose-6-phosphate isomerase autoantibodies, the relevance of these findings to humans is unknown. We undertook this study to evaluate the importance of RasGRP-4 in the pathogenesis of human and rat arthritis.

Methods: Synovial tissue from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were evaluated immunohistochemically for the presence of RasGRP-4 protein. Fibroblast-like synoviocytes (FLS) were isolated from synovial samples, and expression of RasGRP-4 was evaluated by real-time quantitative reverse transcription-polymerase chain reaction analyses. The proliferation potency of FLS was evaluated by exposing the cells to a RasGRP-4-specific small interfering RNA (siRNA). Finally, the ability of RasGRP-4-specific siRNAs to hinder type II collagen-induced arthritis in rats was evaluated to confirm the importance of the signaling protein in the disease.

Results: Unexpectedly, RasGRP-4 protein was detected in the synovial hyperplastic lining, where proliferating FLS preferentially reside. FLS isolated from tissues obtained from a subpopulation of RA patients expressed much more RasGRP-4 than did FLS from examined OA patients. Moreover, the level of RasGRP-4 transcript was correlated with the FLS proliferation rate. The ability of cultured FLS to divide was diminished when they were treated with RasGRP-4-specific siRNAs. The intraarticular injection of RasGRP-4-specific siRNAs also dampened experimental arthritis in rats.

Conclusion: RasGRP-4 is aberrantly expressed in FLS and helps regulate their growth. This intracellular signaling protein is therefore a candidate target for dampening proliferative synovitis and joint destruction.
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http://dx.doi.org/10.1002/art.38924DOI Listing
February 2015

Ancient origin of mast cells.

Biochem Biophys Res Commun 2014 Aug 2;451(2):314-8. Epub 2014 Aug 2.

Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

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 >500million years ago, well before the development of adaptive immunity. The remarkable conservation of MCs throughout evolution is consistent with their importance in innate immunity.
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http://dx.doi.org/10.1016/j.bbrc.2014.07.124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145527PMC
August 2014

Importance of mast cell Prss31/transmembrane tryptase/tryptase-γ in lung function and experimental chronic obstructive pulmonary disease and colitis.

J Biol Chem 2014 Jun 12;289(26):18214-27. Epub 2014 May 12.

the Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115,

Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restricted protease of unknown function that is retained on the outer leaflet of the plasma membrane when MCs are activated. We determined the nucleotide sequences of the Prss31 gene in different mouse strains and then used a Cre/loxP homologous recombination approach to create a novel Prss31(-/-) C57BL/6 mouse line. The resulting animals exhibited no obvious developmental abnormality, contained normal numbers of granulated MCs in their tissues, and did not compensate for their loss of the membrane tryptase by increasing their expression of other granule proteases. When Prss31-null MCs were activated with a calcium ionophore or by their high affinity IgE receptors, they degranulated in a pattern similar to that of WT MCs. Prss31-null mice had increased baseline airway reactivity to methacholine but markedly reduced experimental chronic obstructive pulmonary disease and colitis, thereby indicating both beneficial and adverse functional roles for the tryptase. In a cigarette smoke-induced model of chronic obstructive pulmonary disease, WT mice had more pulmonary macrophages, higher histopathology scores, and more fibrosis in their small airways than similarly treated Prss31-null mice. In a dextran sodium sulfate-induced acute colitis model, WT mice lost more weight, had higher histopathology scores, and contained more Cxcl-2 and IL-6 mRNA in their colons than similarly treated Prss31-null mice. The accumulated data raise the possibility that inhibitors of this membrane tryptase may provide additional therapeutic benefit in the treatment of humans with these MC-dependent inflammatory diseases.
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http://dx.doi.org/10.1074/jbc.M114.548594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140286PMC
June 2014

Mast cell-restricted tetramer-forming tryptases and their beneficial roles in hemostasis and blood coagulation.

Immunol Allergy Clin North Am 2014 May 15;34(2):263-81. Epub 2014 Mar 15.

Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 1 Jimmy Fund Way, Smith Building, Boston, MA 02115, USA.

Tetramer-forming tryptase (hTryptase-β) was recently discovered to have a prominent role in preventing the internal accumulation of life-threatening fibrin deposits and fibrin-platelet clots. The anticoagulant activity of hTryptase-β is an explanation for the presence of hemorrhagic disorders in some patients with anaphylaxis or mastocytosis. The fragments of hFibrinogen formed by the proteolysis of this prominent protein by hTryptase-β could be used as biomarkers in the blood and/or urine for the identification and monitoring of patients with mast cell-dependent disorders. Recombinant hTryptase-β has potential to be used in clinical settings where it is desirable to inhibit blood coagulation.
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http://dx.doi.org/10.1016/j.iac.2014.01.001DOI Listing
May 2014

Mouse mast cell proteases 4 and 5 mediate epidermal injury through disruption of tight junctions.

J Immunol 2014 Mar 12;192(6):2812-20. Epub 2014 Feb 12.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;

We previously established a mast cell (MC)-dependent thermal injury model in mice with ulceration and scar formation that depended on nonredundant functions of mouse MC protease (mMCP)4 and mMCP5. We hypothesized that MC activation is an early event and now find by histology that exocytosis of granule contents occurred by 2 min after thermal injury in wild-type (WT) C57BL/6 mice and in the mMCP4- or mMCP5-deficient mice. The degranulation was equivalent for MCs in the dermis and hypodermis of all three strains, but only the WT mice showed an appreciable increase in epidermal thickness. There was no loss of total MCs, partially degranulated plus intact, during the 4 h of observation. By electron microscopy, MCs in all strains showed early zonal degranulation at 30 s with marked progression in magnitude by 120 s and no mitochondrial injury or cellular necrosis. Concomitantly there was an increase in intercellular spaces indicative of tight junction (TJ) disruption in WT mice but not in the mMCP4- or mMCP5-deficient strains. The desmosomes were intact in all strains. Immunodetection of the TJ protein claudin 4 in WT and mMCP5-deficient mice indicated a significant reduction after scald injury whereas mMCP4(-/-) mice showed no significant changes. Taken together, these findings reveal that a second-degree burn injury can initiate an immediate novel zonal degranulation of MCs throughout all skin layers and a disruption of the epidermal TJs dependent on the nonredundant presence of mMCP4 and mMCP5.
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http://dx.doi.org/10.4049/jimmunol.1301794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970423PMC
March 2014

Development of mast cells and importance of their tryptase and chymase serine proteases in inflammation and wound healing.

Adv Immunol 2014 ;122:211-52

Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA. Electronic address:

Mast cells (MCs) are active participants in blood coagulation and innate and acquired immunity. This review focuses on the development of mouse and human MCs, as well as the involvement of their granule serine proteases in inflammation and the connective tissue remodeling that occurs during the different phases of the healing process of wounded skin and other organs. The accumulated data suggest that MCs, their tryptases, and their chymases play important roles in tissue repair. While MCs initially promote healing, they can be detrimental if they are chronically stimulated or if too many MCs become activated at the same time. The possibility that MCs and their granule serine proteases contribute to the formation of keloid and hypertrophic scars makes them potential targets for therapeutic intervention in the repair of damaged skin.
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http://dx.doi.org/10.1016/B978-0-12-800267-4.00006-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115062PMC
May 2014

Mast cell-restricted, tetramer-forming tryptases induce aggrecanolysis in articular cartilage by activating matrix metalloproteinase-3 and -13 zymogens.

J Immunol 2013 Aug 24;191(3):1404-12. Epub 2013 Jun 24.

South Western Sydney Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia.

Mouse mast cell protease (mMCP)-6-null C57BL/6 mice lost less aggrecan proteoglycan from the extracellular matrix of their articular cartilage during inflammatory arthritis than wild-type (WT) C57BL/6 mice, suggesting that this mast cell (MC)-specific mouse tryptase plays prominent roles in articular cartilage catabolism. We used ex vivo mouse femoral head explants to determine how mMCP-6 and its human ortholog hTryptase-β mediate aggrecanolysis. Exposure of the explants to recombinant hTryptase-β, recombinant mMCP-6, or lysates harvested from WT mouse peritoneal MCs (PMCs) significantly increased the levels of enzymatically active matrix metalloproteinases (MMP) in cartilage and significantly induced aggrecan loss into the conditioned media, relative to replicate explants exposed to medium alone or lysates collected from mMCP-6-null PMCs. Treatment of cartilage explants with tetramer-forming tryptases generated aggrecan fragments that contained C-terminal DIPEN and N-terminal FFGVG neoepitopes, consistent with MMP-dependent aggrecanolysis. In support of these data, hTryptase-β was unable to induce aggrecan release from the femoral head explants obtained from Chloe mice that resist MMP cleavage at the DIPEN↓FFGVG site in the interglobular domain of aggrecan. In addition, the abilities of mMCP-6-containing lysates from WT PMCs to induce aggrecanolysis were prevented by inhibitors of MMP-3 and MMP-13. Finally, recombinant hTryptase-β was able to activate latent pro-MMP-3 and pro-MMP-13 in vitro. The accumulated data suggest that human and mouse tetramer-forming tryptases are MMP convertases that mediate cartilage damage and the proteolytic loss of aggrecan proteoglycans in arthritis, in part, by activating the zymogen forms of MMP-3 and MMP-13, which are constitutively present in articular cartilage.
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http://dx.doi.org/10.4049/jimmunol.1300856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3727140PMC
August 2013

A new short-term mouse model of chronic obstructive pulmonary disease identifies a role for mast cell tryptase in pathogenesis.

J Allergy Clin Immunol 2013 Mar 4;131(3):752-62. Epub 2013 Feb 4.

Priority Research Centre for Asthma and Respiratory Disease and Hunter Medical Research Institute, University of Newcastle, Newcastle, Australia.

Background: Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe.

Objectives: We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD.

Methods: Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice.

Results: After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages.

Conclusion: A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.
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http://dx.doi.org/10.1016/j.jaci.2012.11.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060894PMC
March 2013

Interleukin-33 primes mast cells for activation by IgG immune complexes.

PLoS One 2012 11;7(10):e47252. Epub 2012 Oct 11.

Department of Medicine, Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Mast cells (MCs) are heterogeneous cells whose phenotype is modulated by signals received from the local microenvironment. Recent studies have identified the mesenchymal-derived cytokine IL-33 as a potent direct activator of MCs, as well as regulator of their effector phenotype, and have implicated this activity in the ability of mast cells to contribute to murine experimental arthritis. We explored the hypothesis that IL-33 enables participation of synovial MCs in murine K/BxN arthritis by promoting their activation by IgG immune complexes. Compared to wild-type (WT) control mice, transgenic animals lacking the IL-33 receptor ST2 exhibited impaired MC-dependent immune complex-induced vascular permeability (flare) and attenuated K/BxN arthritis. Whereas participation of MCs in this model is mediated by the activating IgG receptor FcγRIII, we pre-incubated bone marrow-derived MCs with IL-33 and found not only direct induction of cytokine release but also a marked increase in FcγRIII-driven production of critical arthritogenic mediators including IL-1β and CXCL2. This "priming" effect was associated with mRNA accumulation rather than altered expression of Fcγ receptors, could be mimicked by co-culture of WT but not ST2(-/-) MCs with synovial fibroblasts, and was blocked by antibodies against IL-33. In turn, WT but not ST2(-/-) MCs augmented fibroblast expression of IL-33, forming a positive feedback circuit. Together, these findings confirm a novel role for IL-33 as an amplifier of IgG immune complex-mediated inflammation and identify a potential MC-fibroblast amplification loop dependent on IL-33 and ST2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047252PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469528PMC
April 2013

The translational repressor T-cell intracellular antigen-1 (TIA-1) is a key modulator of Th2 and Th17 responses driving pulmonary inflammation induced by exposure to house dust mite.

Immunol Lett 2012 Aug 15;146(1-2):8-14. Epub 2012 Apr 15.

Department of Medicine, Harvard Medical School, and Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA 02115, USA.

T-cell intracellular antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1(-/-)) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1(-/-) mice also had higher levels of Df-specific IgE and IgG(1) in serum and ex vivo restimulated Tia-1(-/-) lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.
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http://dx.doi.org/10.1016/j.imlet.2012.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407291PMC
August 2012

Ras guanine nucleotide-releasing protein-4 (RasGRP4) involvement in experimental arthritis and colitis.

J Biol Chem 2012 Jun 16;287(24):20047-55. Epub 2012 Apr 16.

Department of Pulmonary Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

RasGRP4 (Ras guanine nucleotide-releasing protein-4) is an intracellular, calcium-regulated guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor expressed in mast cells (MCs) and their progenitors. To study the function of this signaling protein in inflammatory disorders, a homologous recombination approach was used to create a RasGRP4-null C57BL/6 mouse line. The resulting transgenic animals had normal numbers of MCs in their tissues that histochemically and morphologically resembled those in WT C57BL/6 mice. MCs could also be generated from RasGRP4-null mice by culturing their bone marrow cells in IL-3-enriched conditioned medium. Despite these data, the levels of the transcripts that encode the proinflammatory cytokines IL-1β and TNF-α were reduced in phorbol 12-myristate 13-acetate-treated MCs developed from RasGRP4-null mice. Although inflammation was not diminished in a Dermatophagoides farinae-dependent model of allergic airway disease, dextran sodium sulfate-induced colitis was significantly reduced in RasGRP4-null mice relative to similarly treated WT mice. Furthermore, experimental arthritis could not be induced in RasGRP4-null mice that had received K/BxN mouse serum. The latter findings raise the possibility that the pharmacologic inactivation of this intracellular signaling protein might be an effective treatment for arthritis or inflammatory bowel disease.
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http://dx.doi.org/10.1074/jbc.M112.360388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370188PMC
June 2012

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

J Biol Chem 2012 Mar 10;287(11):7834-44. Epub 2012 Jan 10.

Department of Medicine, Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

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.
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http://dx.doi.org/10.1074/jbc.M111.325712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318743PMC
March 2012

Mast cell tryptase deficiency attenuates mouse abdominal aortic aneurysm formation.

Circ Res 2011 May 14;108(11):1316-27. Epub 2011 Apr 14.

Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA.

Rationale: Mast cells (MCs) contribute to the formation of abdominal aortic aneurysms (AAAs) by producing biologically active mediators. Tryptase is the most abundant MC granule protein and participates in MC activation, protease maturation, leukocyte recruitment, and angiogenesis-all processes critical to AAA pathogenesis.

Objective: To test the hypothesis that tryptase participates directly in AAA formation.

Methods And Results: Immunohistochemistry demonstrated enhanced tryptase staining in media and adventitia of human and mouse AAA lesions. Serum tryptase levels correlated significantly with the annual expansion rate of AAA before (r = 0.30, P = 0.003) and after (r = 0.29, P = 0.005) adjustment for common AAA risk factors in a patient follow-up study, and associated with risks for later surgical repair or overall mortality before (P = 0.009, P = 0.065) and after (P = 0.004, P = 0.001) the adjustment. Using MC protease-6-deficient mice (Mcpt6(-/-)) and aortic elastase perfusion-induced experimental AAAs, we proved a direct role of this tryptase in AAA pathogenesis. Whereas all wild-type (WT) mice developed AAA at 14 or 56 days postperfusion, Mcpt6(-/-) mice were fully protected. AAA lesions from Mcpt6(-/-) mice had fewer inflammatory and apoptotic cells, and lower chemokine levels, than did those from WT mice. MC from WT mice restored reduced AAA lesions and lesion inflammatory cell content in MC-deficient Kit(W-sh/W-sh) mice, but those prepared from Mcpt6(-/-) mice did not. Mechanistic studies demonstrated that tryptase deficiency affected endothelial cell (EC) chemokine and cytokine expression, monocyte transmigration, smooth-muscle cell apoptosis, and MC and AAA lesion cysteinyl cathepsin expression and activities.

Conclusions: This study establishes the direct participation of MC tryptase in the pathogenesis of experimental AAAs, and suggests that levels of this protease can serve as a novel biomarker for abdominal aortic expansion.
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http://dx.doi.org/10.1161/CIRCRESAHA.111.243758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108141PMC
May 2011

Essential role for mast cell tryptase in acute experimental colitis.

Proc Natl Acad Sci U S A 2011 Jan 20;108(1):290-5. Epub 2010 Dec 20.

Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.

Patients with inflammatory bowel disease (IBD) have increased numbers of human tryptase-β (hTryptase-β)-positive mast cells (MCs) in the gastrointestinal tract. The amino acid sequence of mouse mast cell protease (mMCP)-6 is most similar to that of hTryptase-β. We therefore hypothesized that this mMCP, or the related tryptase mMCP-7, might have a prominent proinflammatory role in experimental colitis. The dextran sodium sulfate (DSS) and trinitrobenzene sulfonic acid (TNBS) colitis models were used to evaluate the differences between C57BL/6 (B6) mouse lines that differ in their expression of mMCP-6 and mMCP-7 with regard to weight loss, colon histopathology, and endoscopy scores. Microarray analyses were performed, and confirmatory real-time PCR, ELISA, and/or immunohistochemical analyses were carried out on a number of differentially expressed cytokines, chemokines, and matrix metalloproteinases (MMPs). The mMCP-6-null mice that had been exposed to DSS had significantly less weight loss as well as significantly lower pathology and endoscopy scores than similarly treated mMCP-6-expressing mice. This difference in colitis severity was confirmed endoscopically in the TNBS-treated mice. Evaluation of the distal colon segments revealed that numerous proinflammatory cytokines, chemokines that preferentially attract neutrophils, and MMPs that participate in the remodeling of the ECM were all markedly increased in the colons of DSS-treated WT mice relative to untreated WT mice and DSS-treated mMCP-6-null mice. Collectively, our data show that mMCP-6 (but not mMCP-7) is an essential MC-restricted mediator in chemically induced colitis and that this tryptase acts upstream of many of the factors implicated in IBD.
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http://dx.doi.org/10.1073/pnas.1005758108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017166PMC
January 2011

The inflammatory response after an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5.

J Immunol 2010 Dec 12;185(12):7681-90. Epub 2010 Nov 12.

Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.

A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.
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http://dx.doi.org/10.4049/jimmunol.1002803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058366PMC
December 2010

Synovial fibroblasts promote the expression and granule accumulation of tryptase via interleukin-33 and its receptor ST-2 (IL1RL1).

J Biol Chem 2010 Jul 28;285(28):21478-86. Epub 2010 Apr 28.

Department of Medicine, Division of Rheumatology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

A characteristic feature of tissue resident human mast cells (MCs) is their hTryptase-beta-rich cytoplasmic granules. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-beta, 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-alpha or interleukin (IL)-1beta. 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.
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http://dx.doi.org/10.1074/jbc.M110.114991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898446PMC
July 2010

Critical role of mast cell chymase in mouse abdominal aortic aneurysm formation.

Circulation 2009 Sep 31;120(11):973-82. Epub 2009 Aug 31.

Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Background: Mast cell chymase may participate in the pathogenesis of human abdominal aortic aneurysm (AAA), yet a direct contribution of this serine protease to AAA formation remains unknown.

Methods And Results: Human AAA lesions had high numbers of chymase-immunoreactive mast cells. Serum chymase level correlated with AAA growth rate (P=0.009) in a prospective clinical study. In experimental AAA produced by aortic elastase perfusion in wild-type (WT) mice or those deficient in the chymase ortholog mouse mast cell protease-4 (mMCP-4) or deficient in mMCP-5 (Mcpt4(-/-), Mcpt5(-/-)), Mcpt4(-/-) but not Mcpt5(-/-) had reduced AAA formation 14 days after elastase perfusion. Even 8 weeks after perfusion, aortic expansion in Mcpt4(-/-) mice fell by 50% compared with that of the WT mice (P=0.0003). AAA lesions in Mcpt4(-/-) mice had fewer inflammatory cells and less apoptosis, angiogenesis, and elastin fragmentation than those of WT mice. Although Kit(W-sh/W-sh) mice had protection from AAA formation, reconstitution with mast cells from WT mice, but not those from Mcpt4(-/-) mice, partially restored the AAA phenotype. Mechanistic studies suggested that mMCP-4 regulates expression and activation of cysteine protease cathepsins, elastin degradation, angiogenesis, and vascular cell apoptosis.

Conclusions: High chymase-positive mast cell content in human AAA lesions, greatly reduced AAA formation in Mcpt4(-/-) mice, and significant correlation of serum chymase levels with human AAA expansion rate suggests participation of mast cell chymase in the progression of human and mouse AAA.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.109.849679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757146PMC
September 2009

Stress in the ER leads to inflammatory bowel disease.

Inflamm Bowel Dis 2009 Nov;15(11):1765-6

Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.

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http://dx.doi.org/10.1002/ibd.20969DOI Listing
November 2009

Mast cells contribute to autoimmune inflammatory arthritis via their tryptase/heparin complexes.

J Immunol 2009 Jan;182(1):647-56

Department of Medicine, Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis, the contribution of MCs to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase/heparin complexes have proinflammatory activity, and significant amounts of human tryptase beta (hTryptase-beta) are present in rheumatoid arthritis synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-beta, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase/heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/BxN mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease-activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-beta/heparin or mMCP-6/heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase/heparin complexes in the K/BxN mouse arthritis model and connect our mouse findings with rheumatoid arthritis pathophysiology.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610352PMC
http://dx.doi.org/10.4049/jimmunol.182.1.647DOI Listing
January 2009

The mouse mast cell-restricted tetramer-forming tryptases mouse mast cell protease 6 and mouse mast cell protease 7 are critical mediators in inflammatory arthritis.

Arthritis Rheum 2008 Aug;58(8):2338-46

South Western Sydney Clinical School, University of New South Wales, Sydney, NSW, Australia.

Objective: Increased numbers of mast cells (MCs) that express beta tryptases bound to heparin have been detected in the synovium of patients with rheumatoid arthritis (RA). The corresponding tryptases in mice are mouse MC protease 6 (mMCP-6) and mMCP-7. Although MCs have been implicated in RA and some animal models of arthritis, no direct evidence for a MC-restricted tryptase in the pathogenesis of inflammatory arthritis has been shown. We created transgenic mice that lack heparin and different combinations of mMCP-6 and mMCP-7, to evaluate the roles of MC-restricted tryptase-heparin complexes in an experimental model of arthritis.

Methods: The methylated bovine serum albumin/interleukin-1beta (mBSA/IL-1beta) experimental protocol was used to induce inflammatory monarthritis in different mouse strains. Mice were killed at the time of peak disease on day 7, and histochemical methods were used to assess joint pathology.

Results: Arthritis was induced in the knee joints of mBSA/IL-1beta-treated mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(+) C57BL/6 mice, and numerous activated MCs that had exocytosed the contents of their secretory granules were observed in the diseased mice. In contrast, arthritis was markedly reduced in heparin-deficient mice and in mMCP-6(-)/mMCP-7(-) C57BL/6 mice.

Conclusion: MC-derived tryptase-heparin complexes play important roles in mBSA/IL-1beta-induced arthritis. Because mMCP-6 and mMCP-7 can compensate for each other in this disease model, the elimination of both tryptases is necessary to reveal the prominent roles of these serine proteases in joint inflammation and destruction. Our data suggest that the inhibition of MC-restricted tryptases could have therapeutic potential in the treatment of RA.
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http://dx.doi.org/10.1002/art.23639DOI Listing
August 2008

The Diacylglycerol-dependent translocation of ras guanine nucleotide-releasing protein 4 inside a human mast cell line results in substantial phenotypic changes, including expression of interleukin 13 receptor alpha2.

J Biol Chem 2008 Jan 16;283(3):1610-1621. Epub 2007 Nov 16.

Department of Medicine, University of New South Wales, and Department of Immunology, Allergy, and Infectious Diseases, St. George Hospital, 2 South St. Centre, New South Wales 2217, Australia. Electronic address:

Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell (MC)-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/phorbol ester receptor. An RasGRP4-defective variant of the human MC line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled RasGRP4 for monitoring the movement of this protein inside MCs after exposure to phorbol 12-myristate 13-acetate (PMA), and for evaluating the protein's ability to control gene expression. RasGRP4 resided primarily in the cytosol. After exposure to PMA, RasGRP4 quickly translocated to the inner leaflet of the cell's plasma membrane. 15-30 min later, this signaling protein translocated from the plasma membrane to other intracellular sites. The translocation of RasGRP4 from the cytosol to its varied membrane compartments was found to be highly dependent on Phe(548) in the protein's C1 DAG/PMA-binding domain. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and c-kit/CD117 was lost from the cell's surface. Transcript-profiling approaches revealed that RasGRP4 profoundly regulated the expression of hundreds of genes in HMC-1 cells. For example, the expression of the transcript that encodes the interleukin (IL) 13 receptor IL-13Ralpha2 increased 61- to 860-fold in RasGRP4-expressing HMC-1 cells. A marked increase in IL-13Ralpha2 protein levels also was found. The accumulated data suggest RasGRP4 translocates to varied intracellular compartments via its DAG/PMA-binding domain to regulate signaling pathways that control gene and protein expression in MCs, including the cell's ability to respond to IL-13.
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http://dx.doi.org/10.1074/jbc.M707042200DOI Listing
January 2008

Defective expression of Ras guanyl nucleotide-releasing protein 1 in a subset of patients with systemic lupus erythematosus.

J Immunol 2007 Oct;179(7):4890-900

Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Dysregulation of Ras guanyl nucleotide-releasing protein 1 (RasGRP1) in mice results in a systemic lupus erythematosus (SLE)-like disorder. We therefore looked for defective isoforms and/or diminished levels of human RasGRP1 in a cohort of SLE patients. PBMCs were collected from twenty healthy individuals and thirty-two patients with SLE. mRNA was isolated and five RasGRP1 cDNAs from each subject were sequenced. T cell lysates from healthy controls and SLE patients also were evaluated for their levels of RasGRP1 protein. The accumulated data led to the identification of 13 new splice variants of the human RasGRP1 gene. Not only did our SLE patients have increased levels and types of these defective transcripts relative to normal individuals, two SLE patients were identified whose PBMCs and T cells contained very little, if any, functional RasGRP1 mRNA and protein. The presence of aberrantly spliced RasGRP1 transcripts also was correlated with lower levels of RasGRP1 protein in the patients' T cells. The lack of the normal isoform of RasGRP1 in some SLE patients and the increased prevalence of defective isoforms of RasGRP1 in others raise the possibility that dysregulation of this signaling protein contributes to the development of autoimmunity in a subset of SLE patients.
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http://dx.doi.org/10.4049/jimmunol.179.7.4890DOI Listing
October 2007

Mast cell-restricted tryptases: structure and function in inflammation and pathogen defense.

J Biol Chem 2007 Jul 15;282(29):20785-9. Epub 2007 May 15.

Department of Rheumatology, Liverpool Hospital and South Western Sydney Clinical School, University of New South Wales, Sydney, Australia, 2052.

Mast cells (MCs) are highly specialized immune cells present in mammals and in lower organisms that predate the development of adaptive immunity. The strong evolutionary pressure to retain MCs for >500 million years suggests critical roles for these cells in our survival. In support of this conclusion, no human has been identified to date that lacks MCs, despite the adverse roles of MCs in systemic anaphylaxis and varied inflammatory disorders. MCs express numerous lineage-restricted neutral proteases, and four members of the chromosome 17A3.3 family of tryptases are preferentially expressed in mouse MCs. The anatomical location of MCs at host-environment interfaces has raised the possibility that some of these enzymes are evolutionally conserved because they are needed for combating infectious organisms. Here we review recent insights into the structure and function of MC tryptases in inflammation and host defense against bacteria and other infectious organisms.
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http://dx.doi.org/10.1074/jbc.R700017200DOI Listing
July 2007

Protease-proteoglycan complexes of mouse and human mast cells and importance of their beta-tryptase-heparin complexes in inflammation and innate immunity.

Immunol Rev 2007 Jun;217:155-67

Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, MA, USA.

Approximately 50% of the weight of a mature mast cell (MC) consists of varied neutral proteases stored in the cell's secretory granules ionically bound to serglycin proteoglycans that contain heparin and/or chondroitin sulfate E/diB chains. Mouse MCs express the exopeptidase carboxypeptidase A3 and at least 15 serine proteases [designated as mouse MC protease (mMCP) 1-11, transmembrane tryptase/tryptase gamma/protease serine member S (Prss) 31, cathepsin G, granzyme B, and neuropsin/Prss19]. mMCP-6, mMCP-7, mMCP-11/Prss34, and Prss31 are the four members of the chromosome 17A3.3 family of tryptases that are preferentially expressed in MCs. One of the challenges ahead is to understand why MCs express so many different protease-proteoglycan macromolecular complexes. MC-like cells that contain tryptase-heparin complexes in their secretory granules have been identified in the Ciona intestinalis and Styela plicata urochordates that appeared approximately 500 million years ago. Because sea squirts lack B cells and T cells, it is likely that MCs and their tryptase-proteoglycan granule mediators initially appeared in lower organisms as part of their innate immune system. The conservation of MCs throughout evolution suggests that some of these protease-proteoglycan complexes are essential to our survival. In support of this conclusion, no human has been identified that lacks MCs. Moreover, transgenic mice lacking the beta-tryptase mMCP-6 are unable to combat a Klebsiella pneumoniae infection effectively. Here we summarize the nature and function of some of the tryptase-serglycin proteoglycan complexes found in mouse and human MCs.
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http://dx.doi.org/10.1111/j.1600-065X.2007.00525.xDOI Listing
June 2007

The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections.

J Biol Chem 2007 Jul 23;282(29):20809-15. Epub 2007 Apr 23.

Department of Pulmonary Medicine, The University of Texas M. D. Anderson Cancer Center and Center for Lung Inflammation and Infection, Institute for Biosciences and Technology, Houston, Texas 77030, USA.

Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity.
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http://dx.doi.org/10.1074/jbc.M611842200DOI Listing
July 2007

Mice deficient in heparan sulfate 6-O-sulfotransferase-1 exhibit defective heparan sulfate biosynthesis, abnormal placentation, and late embryonic lethality.

J Biol Chem 2007 May 3;282(21):15578-88. Epub 2007 Apr 3.

Institute for Molecular Science of Medicine and Laboratory Animal Research Center, Aichi Medical University, Nagakute, Aichi 480-1195, Japan.

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N- and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an approximately 50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO(4)) and HexA-GlcNSO(3)(6SO(4)) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues.
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http://dx.doi.org/10.1074/jbc.M607434200DOI Listing
May 2007

RasGRP4 in mast cell signalling and disease susceptibility.

Novartis Found Symp 2005 ;271:54-68; discussion 68-77, 95-9

Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MAL 02115, USA.

The nucleotide sequences of the mouse, rat and human cDNAs and genes that encode the fourth member of the Ras guanine nucleotide releasing protein (RasGRP) family of signalling proteins have been deduced. RasGRP4 is a mast cell-restricted, cation-dependent, guanine nucleotide exchange factor (GEF). It is also a diacylglycerol (DAG)/phorbol ester receptor that plays a prominent role in dictating which protease and eicosanoid mediators are expressed in rodent and human mast cell lines. RasGRP4 appears to act downstream of the tyrosine kinase receptor c-Kt/CD117 and upstream of the basic-helix-loop-helix-leucine zipper transcription factor MITE Allelic variants of RasGRP4 have been identified, as have functionally different isoforms that are the result of variable splicing of its gene. Earlier gene-linkage studies revealed a site on chromosome 7A3-B1 that controls intrinsic airway reactivity to methacholine in backcrossed C3H/HeJ and A/J mice. The 18-exon mouse RasGRP4 gene resides on chromosome 7A3-B1, and recent studies revealed that the mast cells developed from the hyporesponsive C3H/HeJ mouse strain preferentially produce a defective isoform of RasGRP4. These and other data suggest that RasGRP4 is of critical importance in mast cell development and that the expression of abnormal isoforms of the protein can lead to mast cell dysfunction.
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June 2006