Estrogen modulates the differential expression of cardiac myocyte chymase isoforms and diastolic function.

Chymases, a family of serine proteases with chymotryptic activity, play a significant role in cardiac angiotensin II (Ang II) formation from its substrate Ang-(1-12) in both human and rodent models. No studies, to date, have assessed the differences in enzymatic activity among these isoforms in Ang II formation, particularly in the cardiomyocyte (CM). Using PCR and DNA sequencing, we demonstrated that MCP-1, MCP-2, MCP-4, and MCP-5 mRNAs are expressed in the CM of both spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). While rMCP-1 and rMCP-5 gene transcripts were higher than that of other isoforms in both rat strains, WKY CM exhibits higher levels of rMCP-1 and rMCP-5 mRNAs compared to the SHR CM. Ovariectomy (OVX) increased the expression of rMCP-1 and rMCP-5 mRNAs in WKY. In SHR, OVX was associated with a blunted increase in rMCP-1 mRNA compared to OVX normotensive WKY. Chymase activity, measured as Ang II formation from Ang-(1-12), significantly correlated with rMCP-1 and rMCP-5 mRNA expression in both rat strains. Both rMCP-1 and rMCP-5 mRNA expressions were positively correlated with progressive diastolic dysfunction (increasing the ratio of early mitral inflow velocity-to-early mitral annular velocity, E/e') and expanding chamber dimensions or increasing left ventricular internal diameter end diastole. These data show rMCP-1 and rMCP-5 as the Ang II forming chymase isoforms participating in the loss of normal cardiac function due to OVX in rodents.

Respiratory Syncytial Virus Infection of Human Lung Fibroblasts Induces a Hyaluronan-Enriched Extracellular Matrix That Binds Mast Cells and Enhances Expression of Mast Cell Proteases.

Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.

Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth.

Intrauterine growth restriction (IUGR) is caused by insufficient remodeling of spiral arteries (SAs). The mechanism underlying the relevance of natural killer cells (NKs) and mast cells (MCs) for SA remodeling and its effects on pregnancy outcome are not well understood. We show that NK depletion arrested SA remodeling without affecting pregnancy. MC depletion resulted in abnormally remodeled SAs and IUGR. Combined absence of NKs and MCs substantially affected SA remodeling and impaired fetal growth. We found that α-chymase mast cell protease (Mcpt) 5 mediates apoptosis of uterine smooth muscle cells, a key feature of SA remodeling. Additionally, we report a previously unknown source for Mcpt5: uterine (u) NKs. Mice with selective deletion of Mcpt5+ cells had un-remodeled SAs and growth-restricted progeny. The human α-chymase CMA1, phylogenetic homolog of Mcpt5, stimulated the ex vivo migration of human trophoblasts, a pre-requisite for SA remodeling. Our results show that chymases secreted by uMCs and uNKs are pivotal to the vascular changes required to support pregnancy. Understanding the mechanisms underlying pregnancy-induced vascular changes is essential for developing therapeutic options against pregnancy complications associated with poor vascular remodeling.

Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

BACKGROUND: Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. METHODS AND RESULTS: Isolated LV fibroblasts taken from 4 and 12week ACF Sprague-Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase's role in autophagy induction. CONCLUSION: In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.

Induction of Interleukin-9-Producing Mucosal Mast Cells Promotes Susceptibility to IgE-Mediated Experimental Food Allergy.

Experimental IgE-mediated food allergy depends on intestinal anaphylaxis driven by interleukin-9 (IL-9). However, the primary cellular source of IL-9 and the mechanisms underlying the susceptibility to food-induced intestinal anaphylaxis remain unclear. Herein, we have reported the identification of multifunctional IL-9-producing mucosal mast cells (MMC9s) that can secrete prodigious amounts of IL-9 and IL-13 in response to IL-33, and mast cell protease-1 (MCPt-1) in response to antigen and IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induced MMC9 development that required T cells, IL-4, and STAT6 transcription factor, but not IL-9 signals. Mice ablated of MMC9 induction failed to develop intestinal mastocytosis, which resulted in decreased food allergy symptoms that could be restored by adoptively transferred MMC9s. Finally, atopic patients that developed food allergy displayed increased intestinal expression of Il9- and MC-specific transcripts. Thus, the induction of MMC9s is a pivotal step to acquire the susceptibility to IgE-mediated food allergy.

Low numbers of tryptase+ and chymase+ mast cells associated with reduced survival and advanced tumor stage in melanoma.

The role of mast cells in cutaneous melanoma remains unclear. Tryptase and chymase are serine proteinases and major proteins in mast cell secretory granules. Therefore, this study aimed to investigate the presence of tryptase and chymase mast cells in benign and malignant cutaneous melanocytic lesions and in lymph node metastases of melanomas. The presence of positively stained mast cells was correlated with clinicopathological characteristics in invasive melanomas. Paraffin-embedded sections of 28 benign (13 intradermal, 10 compound, and five junctional nevi) and 26 dysplastic nevi, 15 in-situ melanomas, 36 superficially (pT1, Breslow's thickness<1 mm), and 49 deeply (pT4, Breslow's thickness>4 mm) invasive melanomas and 30 lymph node metastases were immunohistochemically stained for mast cell tryptase and chymase, and immunopositive cells were counted using the hotspot counting method. The mean count of tryptase and chymase mast cells was lower in invasive melanomas compared with in-situ melanomas and dysplastic and benign nevi. In deeply invasive melanomas, the difference was statistically significant compared with dysplastic nevi (P=0.003 for tryptase and P=0.009 for chymase) and in-situ melanomas (0.043 for tryptase). Low numbers of tryptase mast cells were associated with poor overall survival (P=0.031) in deeply invasive melanomas and with a more advanced stage (T1b, P=0.008) in superficially invasive melanomas. Low numbers of chymase mast cells were associated with microsatellites (P=0.017) in deeply invasive melanomas. The results suggest that these serine proteinases of mast cells may be protective in the pathogenesis of melanoma.

Chymase activities and survival in endotoxin-induced human chymase transgenic mice.

We examined the effects of overexpressed human chymase on survival and activity in lipopolysaccharide (LPS)-treated mice. Human chymase transgenic (Tg) and wild-type C57BL/6 (WT) mice were treated with LPS (0.03, 0.1 and 0.3 mg/day; intraperitoneal) for 2 weeks. Treatment with 0.03 mg LPS did not affect survival in either WT or Tg mice. WT mice were not affected by 0.1 mg/day of LPS, whereas 25% of Tg mice died. Survival of mice treated with 0.3 mg/day of LPS was 87.5% and 0% in WT and Tg, respectively. LPS-induced increases in chymase activity in the heart and skin were significantly greater in Tg than WT mice. These data suggest a possible contribution of human chymase activation to LPS-induced mortality.

In vivo visualization of uterine mast cells by two-photon microscopy.

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study of in vivo behavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, their in vivo localization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCs in vivo using double-transgenic C57BL/6J Mcpt5-Cre ROSA26-EYFP mice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1 (Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs live in utero during the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFP mice offers a novel and powerful in vivo approach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

Trichinella spiralis secreted enzymes regulate nucleotide-induced mast cell activation and release of mouse mast cell protease 1.

Extracellular nucleotides are important triggers of innate immunity, acting on a wide variety of cells via signaling through purinergic receptors. Mucosal mast cells contribute to expulsion of a number of gastrointestinal nematode parasites, and mouse mast cell protease 1 has been shown to have a critical role in clearance of Trichinella spiralis from the intestinal tract. We show here that adenosine, ADP, ATP, UDP, and UTP all stimulate calcium mobilization in bone marrow-derived mast cells with a mucosal phenotype. Secreted proteins from T. spiralis infective larvae inhibit nucleotide-induced mast cell activation, and that induced by ADP and UDP is specifically blocked by parasite secretory 5'-nucleotidase. Release of mouse mast cell protease 1 is stimulated by ADP and ATP. Both parasite secreted products and the 5'-nucleotidase inhibit ADP-induced release of mast cell protease, whereas that stimulated by ATP is partially inhibited by secreted products alone. This indicates that the 5'-nucleotidase contributes to but is not solely responsible for inhibition of nucleotide-mediated effects on mast cell function. Secretion of nucleotide-metabolizing enzymes by parasitic nematodes most likely evolved as a strategy for suppression of innate immune responses and is discussed in this context.

PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial protection against acute ischaemia-reperfusion injury: involvement of PI3K-Akt.

Peroxisome proliferator-activated receptors (PPAR) regulate the expression of genes involved in lipid metabolism, energy production, and inflammation. Their role in ischaemia-reperfusion (I/R) is less clear, although research indicates involvement of PPARs in some forms of preconditioning. This study aimed to explore the effects of PPAR-α activation on the I/R injury and potential cardioprotective downstream mechanisms involved. Langendorff-perfused hearts of rats pretreated with the selective PPAR-α agonist WY-14643 (WY, pirinixic acid; 3 mg·(kg body mass)·day(-1); 5 days) were subjected to 30 min ischaemia - 2 h reperfusion with or without the phosphatidylinositol 3-kinase (PI3K)-Akt inhibitor wortmannin for the evaluation of functional (left ventricular developed pressure, LVDP) recovery, infarct size (IS), and reperfusion-induced arrhythmias. A 2-fold increase in baseline PPAR-α mRNA levels (qPCR) in the WY-treated group and higher post-I/R PPAR-α levels compared with those in untreated controls were accompanied by similar changes in the expression of PPAR-α target genes PDK4 and mCPT-1, regulating glucose and fatty acid metabolism, and by enhanced Akt phosphorylation. Post-ischaemic LVDP restoration in WY-treated hearts reached 60% ± 9% of the pre-ischaemic values compared with 24% ± 3% in the control hearts (P < 0.05), coupled with reduced IS and incidence of ventricular fibrillation that was blunted by wortmannin. Results indicate that PPAR-α up-regulation may confer preconditioning-like protection via metabolic effects. Downstream mechanisms of PPAR-α-mediated cardioprotection may involve PI3K-Akt activation.

Diabetes-associated angiotensin activation enhances liver metastasis of colon cancer.

We examined the effects of hyperglycemic conditions on liver metastasis of colorectal cancer (CRC). Angiotensin (A)-II increased growth, invasion, and anti-apoptotic survival in HT29 and CT26 cells. In contrast, angiotensinogen (ATG) increased these features in HT29 cells but not in CT26 cells. HT29 cells expressed A-II type 1 receptor, chymase, and rennin, whereas CT26 cells did not express renin. Renin expression and ATG-induced cell growth, invasion, and survival induced and increased as glucose concentration increased in HT29 cells and also CT26 cells. An inhibitor of renin or chymase abrogated A-II production in HT29 cells. Reduction of hepatic ATG production by cholesterol-conjugated antisense S-oligodeoxynucleotide suppressed liver metastasis of HT29 cells. An examination of 121 CRC patients showed that diabetes in CRC cases was associated with higher blood HbA1c, higher renin and A-II concentrations in the primary tumors, and higher incidence of liver metastasis than in nondiabetic cases. These results suggest that diabetes-associated angiotensin activation enhances liver metastasis of CRC and may therefore provide a possible target for antimetastatic therapy in CRC.

Microvascular density and mast cells in benign and malignant pheochromocytomas.

Pheochromocytomas, uncommon adrenal tumors, have an uncertain behavior. Recently, PASS criteria were proposed for differentiating between benign and malignant cases. These are not perfect, however. The aim of the study was to investigate angiogenesis and mast cell density in context of the clinical behavior and morphologic characteristics of pheochromocytomas. Mean intratumoral chymase positive cell count was 14.50 for malignant, 15.73 for benign cases; mean subcapsular chymase positive cell count was 12.50 for malignant, 11.27 for benign cases. Mean intratumoral tryptase positive cell count was 17.50 for malignant and 17.91 for benign cases; mean subcapsular tryptase positive cell count was 15.25 for malignant and 15.73 for benign cases. Mean intratumoral CD31 positive vessel count was 46.98 for malignant and 51.02 for benign cases; mean subcapsular CD31 positive vessel count was 44.86 for malignant and 39.81 for benign cases. Mean intratumoral CD105 positive vessel count was 37.84 for malignant and 35.95 for benign cases; mean subcapsular CD105 positive vessel count was 26.36 for malignant and 22.03 for benign cases. The differences between benign and malignant cases were not significant. All the vascular counts were correlated with mast cells counts. PASS index was inversely correlated with mast cell counts.

High density of tryptase-positive mast cells in patients with renal cell carcinoma on hemodialysis: correlation with expression of stem cell factor and protease activated receptor-2.

Patients on hemodialysis are at higher risk of renal cell carcinoma probably because of inflammatory and immune system disorders. The aim of this study was to clarify the pathologic roles of 2 phenotypes of mast cells, mast cell tryptase and mast cell chymase, and their correlation with stem cell factor and protease-activated receptor 2 in patients with renal cell carcinoma on hemodialysis. The densities of mast cell tryptase and mast cell chymase and expressions of stem cell factor and protease-activated receptor 2 were examined in 35 patients with hemodialysis-renal cell carcinoma and 39 with non-hemodialysis-renal cell carcinoma who were diagnosed and treated in our hospital. Protein expression was examined by immunohistochemistry. The proliferation index represented the number of Ki-67-positive cells. There were no significant differences in clinicopathologic features between the 2 groups. Mast cell tryptase densities in intratumoral (8.3 per high-power field) and peritumoral areas (8.7 per high-power field) were higher in hemodialysis-renal cell carcinoma than non-hemodialysis-renal cell carcinoma (2.7 and 5.3 per high-power field). No such significant correlations were detected in mast cell chymase. In hemodialysis-renal cell carcinoma, intratumoral mast cell tryptase density correlated with the proliferation index (P = .039 and P = .008, respectively) and also with stem cell factor and protease-activated receptor 2 expression. Our results emphasize the important roles of mast cell tryptase in cancer cell proliferation and recurrence in hemodialysis-renal cell carcinoma. Stem cell factor and protease-activated receptor 2 seem to up-regulate mast cell tryptase functions in these patients. The results suggest collaborative effects of stem cell factor, mast cell tryptase, and protease-activated receptor 2 on the malignant potential of hemodialysis-renal cell carcinoma.

Increased plasma chymase concentration and mast cell chymase expression in venous neointimal lesions of patients with CKD and ESRD.

The underlying inflammatory component of chronic kidney disease may predispose blood vessels to intimal hyperplasia (IH), which is the primary cause of dialysis access failure. We hypothesize that vascular pathology and markers of IH formation are antecedent to arteriovenous (AV) fistula creation. Blood, cephalic, and basilic vein segments were collected from predialysis chronic kidney disease (CKD) patients with no previous AV access and patients with end-stage renal disease (ESRD). Immunohistochemistry was performed with antibodies against mast cell chymase, transforming growth factor-beta (TGF-ß) and interleukin-6 (IL-6), which cause IH. Plasma chymase was measured by ELISA. IH was present in 91% of CKD and 75% of ESRD vein segments. Chymase was abundant in vessels with IH, with the greatest expression in intima and medial layers, and virtually absent in the controls. Chymase colocalized with TGF-ß1 and IL-6. Plasma chymase concentration was elevated up to 33-fold in patients with CKD versus controls and was associated with increased chymase in vessels with IH. We show that chymase expression in vessels with IH corresponds with plasma chymase concentrations. As chymase inhibition attenuates IH in animal models, and we find chymase is highly expressed in IH lesions of patients with CKD and ESRD, we speculate that chymase inhibition could have therapeutic value in humans.

Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis.

IL-17 and IL-23 are known to be absolutely central to psoriasis pathogenesis because drugs targeting either cytokine are highly effective treatments for this disease. The efficacy of these drugs has been attributed to blocking the function of IL-17-producing T cells and their IL-23-induced expansion. However, we demonstrate that mast cells and neutrophils, not T cells, are the predominant cell types that contain IL-17 in human skin. IL-17(+) mast cells and neutrophils are found at higher densities than IL-17(+) T cells in psoriasis lesions and frequently release IL-17 in the process of forming specialized structures called extracellular traps. Furthermore, we find that IL-23 and IL-1ß can induce mast cell extracellular trap formation and degranulation of human mast cells. Release of IL-17 from innate immune cells may be central to the pathogenesis of psoriasis, representing a fundamental mechanism by which the IL-23-IL-17 axis mediates host defense and autoimmunity.

Mast cells infiltrate the esophageal smooth muscle in patients with eosinophilic esophagitis, express TGF-ß1, and increase esophageal smooth muscle contraction.

BACKGROUND: Increased numbers of mast cells are present in the esophageal epithelium in patients with eosinophilic esophagitis (EE). However, mast cell infiltration into the esophageal lamina propria (LP) and smooth muscle (SM) and the effects of their products on SM function has not been determined. OBJECTIVE: We investigated mast cell localization and characterization in esophageal SM, the functional significance of mast cell TGF-ß1 expression to contraction of human esophageal smooth muscle (HESM) cells in vitro, and the effect of topical corticosteroids on the number of tryptase-positive (MC(T)) and chymase-positive (MC(C)) mast cells in patients with EE. METHODS: MC(T)- and MC(C)-positive mast cell numbers were quantitated in the epithelium, the LP before and after topical corticosteroid therapy, and the muscularis mucosa in patients with EE and control subjects by using immunohistology. Double immunofluorescence was used to assess mast cell production of TGF-ß1. The ability of TGF-ß1 to influence HESM cell contractility was assessed in vitro. RESULTS: In the SM in patients with EE, significantly increased numbers of MC(T)- and TGF-ß1-positive cells (but only low levels of eosinophils) were detected compared with those seen in control subjects. MC(T) expressed TGF-ß1, which increased the contractility of cultured primary HESM cells in vitro. Topical corticosteroid therapy in patients with EE significantly reduced epithelial MC(T) numbers but not LP tryptase-chymase-positive mast cell numbers. CONCLUSIONS: MC(T) numbers, rather than eosinophil numbers, are increased in the SM in patients with EE, express TGF-ß1, and increase the contractility of HESM cells in vitro. As such, mast cells localized to SM in patients with EE might modulate esophageal contractility.

Attenuation of experimental murine colitis by acanthoic acid from Acanthopanax koreanum.

Acanthoic acid (AA) is a pimaradiene diterpene isolated from Acanthopanax koreanum. We examined the effect of AA in dextran sulfate sodium (DSS)-induced colitis. AA (100 mg/kg or 300 mg/kg) was administered p.o. daily for 7 days. AA significantly inhibited Disease Activity Index, histological score, and myeloperoxidase activity. Furthermore, AA markedly suppressed the protein expression of TNF-alpha, COX-2, NF-kappaB and chymase as well as the mRNA expression of TNF-alpha and COX-2. These results suggest that AA exerts beneficial effects in experimental colitis, and therefore we propose that this compound may have therapeutic implications for ulcerative colitis.

Transcriptional regulation of mouse mast cell protease-2 by interleukin-15.

Mast cells (MCs) play a critical role in innate and adaptive immunity through the release of cytokines, chemokines, lipid mediators, biogenic amines, and proteases. We recently showed that the activities of MC proteases are transcriptionally regulated by intracellularly retained interleukin-15 (IL-15), and we provided evidence that this cytokine acts as a specific regulator of mouse mast cell protease-2 (mMCP-2). Here, we show that in wild-type bone marrow-derived mast cells (BMMCs) IL-15 inhibits mMCP-2 transcription indirectly by inducing differential expression and mMCP-2 promoter binding of the bifunctional transcription factors C/EBPbeta and YY1. In wild-type BMMCs, C/EBPbeta expression predominates over YY1 expression, and thus C/EBPbeta preferentially binds to the mMCP-2 promoter. In IL-15-deficient BMMCs, the opposite is found: YY1 expression predominates and binds to the mMCP-2 promoter at the expense of C/EBPbeta. Hypertranscription of the mMCP-2 gene in IL-15-deficient BMMCs is associated with histone acetylation and, intriguingly, with methylation of non-CpG dinucleotides within the MCP-2 promoter. This suggests a novel model of cytokine-controlled protease transcription: non-CpG methylation maintains a chromosomal domain in an "open" configuration that is permissive for gene expression.

Basophils preferentially express mouse Mast Cell Protease 11 among the mast cell tryptase family in contrast to mast cells.

Tryptases and chymases are the major proteins stored and secreted by mast cells, and they have various biological functions. However, the nature of proteases produced by basophils has been poorly characterized, particularly in mice. mMCP-11 is the most recently discovered mast cell tryptase in mice and was originally identified as Prss34, which is transcribed in some mast cell-like cell lines and at the early stage in the culture of BMMC with IL-3. Curiously, Prss34 is preferentially expressed in the BM and spleen among normal tissues in contrast to other mast cell tryptases. Therefore, it remains elusive what types of cells express mMCP-11 in vivo. Here, we show that mMCP-11 is highly expressed by primary basophils and to a much lesser extent, by some mast cells. Prss34 transcripts were detected abundantly in primary and cultured basophils and very weakly in peritoneal mast cells or cultured BMMC. Conversely, transcripts for mMCP-6 and mMCP-7 tryptases were preferentially expressed by cultured and peritoneal mast cells but not basophils. We established a mMCP-11-specific mAb and showed that mMCP-11 proteins are indeed expressed by primary basophils and those infiltrating the affected tissues during allergic inflammation and parasitic infections. Some primary mast cells also expressed mMCP-11 proteins, albeit at a much lower level. Thus, basophils rather than mast cells are the major source of mMCP-11. This is the first study to demonstrate that mouse basophils produce a trypsin-like protease.

Sphingosine-1-phosphate induces development of functionally mature chymase-expressing human mast cells from hematopoietic progenitors.

Mast cells (MCs) play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. MCs developed from cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MC(T)s), the phenotype predominant in the lung. MC progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. S1P accelerated the development of cord blood-derived MCs (CB-MCs) and strikingly increased the numbers of MC-expressing chymase. These MCs have functional Fc epsilonRIs, and similar to skin MC(TC)s that express both tryptase and chymase, also express CD88 and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MC(TC)s, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs. These results suggest crucial roles for S1P in regulating development of human MCs and their functions and reveal a complex interplay between macrophages and MC progenitors in the development of mature human MCs.