Novel Insight into the in vivo Function of Mast Cell Chymase: Lessons from Knockouts and Inhibitors.

Mast cells are now recognized as key players in diverse pathologies, but the mechanisms by which they contribute in such settings are only partially understood. Mast cells are packed with secretory granules, and when they undergo degranulation in response to activation the contents of the granules are expelled to the extracellular milieu. Chymases, neutral serine proteases, are the major constituents of the mast cell granules and are hence released in large amounts upon mast cell activation. Following their release, chymases can cleave one or several of a myriad of potential substrates, and the cleavage of many of these could potentially have a profound impact on the respective pathology. Indeed, chymases have recently been implicated in several pathological contexts, in particular through studies using chymase inhibitors and by the use of chymase-deficient animals. In many cases, chymase has been shown to account for mast cell-dependent detrimental effects in the respective conditions and is therefore emerging as a promising drug target. On the other hand, chymase has been shown to have protective roles in other pathological settings. More unexpectedly, chymase has also been shown to control certain homeostatic processes. Here, these findings are reviewed.

Chymase-Cre; Mcl-1fl/fl Mice Exhibit Reduced Numbers of Mucosal Mast Cells.

Mast cells (MCs) are considered as key effector cells in the elicitation of allergic symptoms, and they are essential players in innate and adaptive immune responses. In mice, two main types of MCs have been described: connective tissue MCs (CTMCs) and mucosal MCs (MMCs). However, little is known about the biological functions of MMCs, which is due to the lack of suitable models to investigate MMCs in vivo. Here, we aimed to generate a mouse model selectively deficient in MMCs. It has been previously described that Cre expressed under the control of the baboon α-chymase promotor is predominantly localized in MMCs. Therefore, we mated α-chymase-Cre transgenic mice with mice bearing a floxed allele of the myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 encodes for an intracellular antiapoptotic factor in MCs; hence, a selective reduction in MMCs was expected. Our results show that this new mouse model contains markedly reduced numbers of MMCs in mucosal tissues, whereas numbers of CTMCs are normal. Thus, Chm-Cre; Mcl-1fl/fl mice are a useful tool for the investigation of the pathophysiological functions of MMCs in vivo.

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.

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

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 mMCP-5 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 Trichinella spiralis-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.

The role of mouse mast cell proteases in the proliferative phase of wound healing in microdeformational wound therapy.

BACKGROUND: Stored in the secretory granules of cutaneous mouse mast cells are mouse mast cell proteases (mMCP-4, -5, and -6). Using transgenic mouse lines that lacked these enzymes, it was shown that mMCP-4 and mMCP-5 modulate the outcome of burn-induced skin injury. Whether or not these proteases also play a role in the repair of surgically damaged skin, with or without microdeformational wound therapy, remains to be determined. METHODS: Wild-type C57BL/6 mice and transgenic C57BL/6 mouse lines lacking mMCP-4, -5, or -6 were subjected to surgical wounding of their skin. Wounds were splinted with a stabilizing patch, and the mice received either microdeformational wound therapy (n = 5) or occlusive dressing (n = 5) for 7 days. Wound healing parameters were assessed in the proliferative phase. RESULTS: Cell proliferation in the wounded wild-type mice receiving microdeformational wound therapy was 60 ± 3 percent. Cell proliferation was only 35 ± 5 percent, 25 ± 5 percent, and 45 ± 4 percent for the treated mMCP-4-, mMCP-5-, and mMCP-6-null mice, respectively (p = 0.005). Blood vessel sprouting was higher in the control mice with microdeformational wound therapy (170 ± 40 vessels/high-power field) compared with mouse mast cell protease 6-null mice with microdeformational wound therapy (70 ± 20 vessels/high-power field; p = 0.005), and higher in the control mice with occlusive dressing (110 ± 30 vessels/high-power field) compared with mMCP-4-null mice with occlusive dressing (50 ± 20 vessels/high-power field; p = 0.01). Qualitatively, the granulation tissue of all the protease-deficient groups receiving microdeformational wound therapy was disrupted. CONCLUSION: Results suggest that mouse mast cell proteases 4, 5, and 6 are mediators of the critical role mast cells play in microdeformational wound therapy in the proliferative phase of healing.

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

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.

Mast cell chymase protects against renal fibrosis in murine unilateral ureteral obstruction.

Mast cell release of chymase is important in tissue remodeling and may participate in inflammation leading to fibrosis and organ failure. Here we analyzed the function of chymase in unilateral ureteral obstruction, an established accelerated model of renal tubulointerstitial fibrosis. Mice deficient in mouse mast cell protease 4 (mMCP4), the functional counterpart of human chymase, had increased obstruction-induced fibrosis when compared to wild-type mice indicating a protective effect of mMCP4. Engraftment of mast cell-deficient Kit(Wsh/Wsh) mice with wild type, but not mMCP4-deficient mast cells, restored protection confirming the role of mMCP4. Kidneys of mMCP4-deficient mice had higher levels of renal tubular damage, interstitial fibrosis, collagen deposition, increased α-smooth muscle actin, and decreased E-cadherin expression compared to the kidneys of wild-type mice. Further analysis showed an elevated inflammatory response in mMCP4-deficient mice with increased levels of kidney-infiltrating macrophages and T cells and local profibrotic TGF-ß1 and CCL2. Granulated and degranulated mast cells and mMCP4 were mainly found in the kidney capsule, respectively, before and after ureteral obstruction. Analysis of mMCP4 substrates showed that it mediates its anti-fibrotic actions by degrading interstitial deposits of fibronectin, a known promoter of inflammatory cell infiltration and adhesion. Thus, mast cell released mMCP4 has anti-fibrotic potential in acutely induced obstructive nephropathy.

Impairment of intestinal barrier and secretory function as well as egg excretion during intestinal schistosomiasis occur independently of mouse mast cell protease-1.

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1(-/-)). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1(-/-)mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1(-/-)mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1(-/-)mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.

Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism.

Altered intestinal barrier function is postulated to be a central predisposing factor to intestinal diseases, including inflammatory bowel diseases and food allergies. However, the mechanisms involved in maintaining homeostatic intestinal barrier integrity remain undefined. In this study, we demonstrate that mice deficient in mast cells (Kit(W-sh/W-sh) [Wsh]) or mast cell chymase (Mcpt4(-/-)) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Altered intestinal barrier function was linked to decreased intestinal epithelial cell migration along the villus/crypt axis, altered intestinal morphology, and dysregulated claudin-3 crypt expression. Remarkably, engraftment of Wsh mice with WT but not Mcpt4(-/-) mast cells restored intestinal epithelial cell migration, morphology, and intestinal epithelial barrier function. Collectively, these findings identify a mechanism by which mast cells regulate homeostatic intestinal epithelial migration and barrier function.