What happens to basophils and tryptase, LXA4 and CysLTs during aspirin desensitization?

INTRODUCTION: Aspirin desensitization (AD) is an effective treatment in patients with non-steroidal anti-inflammatory drugs (NSAID)-exacerbated respiratory disease (NERD) by providing inhibitory effect on symptoms and polyp recurrence. However, limited data is available on how AD works. We aimed to study comprehensively the mechanisms underlying AD by examining basophil activation (CD203c upregulation), mediator-releases of tryptase, CysLT, and LXA4, and LTB4 receptor expression for the first 3 months of AD. METHODS: The study was conducted in patients with NERD who underwent AD (group 1: n = 23), patients with NERD who received no desensitization (group 2: n = 22), and healthy volunteers (group 3, n = 13). All participants provided blood samples for flow cytometry studies (CD203c and LTB4 receptor), and mediator releases (CysLT, LXA4, and tryptase) for the relevant time points determined. RESULTS: All baseline parameters of CD203c and LTB4 receptor expressions, tryptase, CysLT, and LXA4 releases were similar in each group (p > 0.05). In group 1, CD203c started to be upregulated at the time of reactions during AD, and continued to be high for 3 months when compared to controls. All other study parameters were comparable with baseline and at the other time points in each group (p > 0.05). CONCLUSION: Although basophils are active during the first 3 months of AD, no releases of CysLT, tryptase or LXA4 exist. Therefore, our results suggest that despite active basophils, inhibition of mediators can at least partly explain underlying the mechanism in the first three months of AD.

Mast cells disrupt the duodenal mucosal integrity: Implications for the mechanisms of barrier dysfunction in functional dyspepsia.

BACKGROUND: Functional dyspepsia (FD) is a common functional gastrointestinal (GI) disorder, but its pathophysiology is poorly understood. Mast cells (MCs) may play a critical role in the development of FD. Therefore, the aim of this study was to investigate the effect of MCs on barrier function, tight junction (TJ) proteins and related signaling pathways. METHODS: The expression of the TJ proteins claudin-8, ZO-1 and occludin in biopsy tissues from seven FD patients and five controls was assessed. Based on the in vivo results, we further investigated the effect of (1) MC degranulation in a coculture model of Caco-2/RBL-2H3 cells and tryptase in Caco-2 monolayers, (2) MC degranulation in the presence or absence of a PAR-2 antagonist and (3) MC degranulation in the presence or absence of an ERK1/2 signaling pathway inhibitor. The epithelial integrity of Caco-2 cell monolayers was assessed by measuring the transepithelial electrical resistance (TEER). The expression of TJ proteins was evaluated by western blotting, QT-PCR and immunostaining. RESULTS: Epithelial claudin-8, ZO-1 and occludin protein expression were significantly reduced in tissues from FD patients compared with controls. MC degranulation and tryptase decreased the TEER and reduced the expression of TJ proteins in Caco-2 cell monolayers. A PAR-2 antagonist and an ERK1/2 signaling pathway inhibitor significantly reduced the effect of MC degranulation on the TEER and TJ protein expression in Caco-2 cell monolayers. CONCLUSIONS: MCs disrupt duodenal barrier function by modulating the levels of TJ proteins, and the PAR-2 and ERK1/2 signaling pathways may mediate the pathogenesis of FD.

Pretreatment with propofol restores intestinal epithelial cells integrity disrupted by mast cell degranulation in vitro.

Propofol has been shown to against intestinal reperfusion injury when treated either before or after ischemia, during which mast cell could be activated. The aim of this study was to evaluate the role of propofol in restoring the intestinal epithelial cells integrity disrupted by mast cell activation or the released tryptase after activation in vitro. We investigated the effect of: (1) tryptase on Caco-2 monolayers in the presence of PAR-2 inhibitor or propofol, (2) mast cell degranulation in a Caco-2/LAD-2 co-culture model in the presence of propofol, and (3) propofol on mast cell degranulation. Epithelial integrity was detected using transepithelial resistance (TER) and permeability to fluorescein isothiocyanate (FITC)-dextran (the apparent permeability coefficient, Papp). The expression of junctional proteins zonula occludens-1 (ZO-1/TJP1) and occludin were determined using western blot analysis and immunofluorescence microscopy. The intracellular levels of reactive oxidative species (ROS) and Ca2+ were measured using flow cytometry. Tryptase directly enhanced intestinal barrier permeability as demonstrated by significant reductions in TER, ZO-1, and occludin protein expression and concomitant increases in Papp. The intestinal barrier integrity was restored by PAR-2 inhibitor but not by propofol. Meanwhile, mast cell degranulation resulted in epithelial integrity disruption in the Caco-2/LAD-2 co-culture model, which was dramatically attenuated by propofol. Mast cell degranulation caused significant increases in intracellular ROS and Ca(2+) levels, which were blocked by propofol and NAC. Propofol pretreatment can inhibit mast cell activation via ROS/Ca(2+) and restore the intestinal barrier integrity induced by mast cell activation, instead of by tryptase.

Aerosol-Administered Adelmidrol Attenuates Lung Inflammation in a Murine Model of Acute Lung Injury.

Acute lung injury (ALI) is a common and devastating clinical disorder with a high mortality rate and no specific therapy. The pathophysiology of ALI is characterized by increased alveolar/capillary permeability, lung inflammation, oxidative stress and structural damage to lung tissues, which can progress to acute respiratory distress syndrome (ARDS). Adelmidrol (ADM), an analogue of palmitoylethanolamide (PEA), is known for its anti-inflammatory and antioxidant functions, which are mainly due to down-modulating mast cells (MCs) and promoting endogenous antioxidant defense. The aim of this study is to evaluate the protective effects of ADM in a mice model of ALI, induced by intratracheal administration of lipopolysaccharide (LPS) at the dose of 5 mg/kg. ADM 2% was administered by aerosol 1 and 6 h after LPS instillation. In this study, we clearly demonstrated that ADM reduced lung damage and airway infiltration induced by LPS instillation. At the same time, ADM counteracted the increase in MC number and the expression of specific markers of MC activation, i.e., chymase and tryptase. Moreover, ADM reduced oxidative stress by upregulating antioxidant enzymes as well as modulating the Nf-kB pathway and the resulting pro-inflammatory cytokine release. These results suggest that ADM could be a potential candidate in the management of ALI.

RNA-seq characterization of histamine-releasing mast cells as potential therapeutic target of osteoarthritis.

OBJECTIVE: Mast cells in the osteoarthritis (OA) synovium correlate with disease severity. This study aimed to further elucidate the role of mast cells in OA by RNA-Seq analysis and pharmacological blockade of the activity of histamine, a key mast cell mediator, in murine OA. METHODS: We examined OA synovial tissues and fluids by flow cytometry, immunostaining, single-cell and bulk RNA-Seq, qPCR, and ELISA. Cetirizine, a histamine H1 receptor (H1R) antagonist, was used to treat the destabilization of the medial meniscus (DMM) mouse model of OA. RESULTS: Flow cytometry and immunohistology analysis of OA synovial cells revealed KIT+ FcεRI+ and TPSAB1+ mast cells. Single-cell RNA-Seq of OA synovial cells identified the expression of prototypical mast cell markers KIT, TPSAB1, CPA3 and HDC, as well as distinctive markers HPGD, CAVIN2, IL1RL1, PRG2, and CKLF, confirmed by bulk RNA-Seq and qPCR. A mast cell prototypical marker expression score classified 40 OA patients into three synovial pathotypes: mast cell-high, -medium, and -low. Additionally, we detected mast cell mediators including histamine, tryptase AB1, CPA3, PRG2, CAVIN2, and CKLF in OA synovial fluids. Elevated H1R expression was detected in human OA synovium, and treatment of mice with the H1 receptor antagonist cetirizine reduced the severity and OA-related mediators in DMM. CONCLUSION: Based on differential expression of prototypical and distinct mast cell markers, human OA joints can be stratified into mast cell-high, -medium, and -low synovial tissue pathotypes. Pharmacologic blockade of histamine activity holds the potential to improve OA disease outcome.

Anti-histamine effects of dipotassium glycyrrhizinate on lung fibroblasts, implicating its therapeutic mechanism for pulmonary fibrosis.

OBJECTIVES: To examine the possible anti-histamine effects of dipotassium glycyrrhizinate (DG), a dipotassium salt of glycyrrhizic acid, on histamine-mediated lung fibroblast activation, differentiation and proliferation; to investigate the potential and underlying mechanisms for pulmonary fibrosis (PF) treatment. METHODS: Rat primary lung fibroblasts were extracted to establish cell models; histamine, DG and loratadine (LTD, a histamine receptor antagonist) were applied. Cell proliferation, migration and cell cycle were explored; intracellular signal proteins were detected; mitochondrial membrane potential was examined. KEY FINDINGS: The anti-histamine effects of DG were found in a similar pattern of LTD on lung fibroblasts. DG inhibited histamine-induced cell activation, proliferation and migration; DG altered histamine-mediated mitochondrial membrane potentials. DG reduced the histamine-induced PAR-2 (a tryptase receptor) expression to impair mast cell tryptase co-working. Histamine-induced expressions of MMP-2, FAK, TNF-α, P38, iNOS were decreased by DG, while Bax and caspase-3, P53 were increased by DG against histamine effects. Histamine drove cells from G0/G1 to S phases, whereas DG rested cells by inhibiting G0/G1 and G2/M phases. CONCLUSIONS: This study provided the evidences that DG can inhibit histamine-induced effects on lung fibroblasts and promote apoptosis of abnormally activated lung fibroblasts, implicating its potential therapeutic mechanisms against PF development, also for those histamine-related diseases.

Propofol pretreatment alleviates mast cell degranulation by inhibiting SOC to protect the myocardium from ischemia-reperfusion injury.

Propofol (PPF) has a protective effect on myocardial ischemia-reperfusion (I/R) injury (MIRI). The purpose of this study was to investigate whether the myocardial protective effect of propofol is related to the inhibition of mast cell degranulation and explore the possible mechanisms involved. Our in vivo results showed that compared with the sham group, cardiac function, infarct size, histopathological damage, apoptosis, and markers of myocardial necrosis were significantly increased in the ischemia-reperfusion group, and propofol pretreatment alleviated these effects. In the coculture system, propofol-treated mast cells reduced their tryptase activity, resulting in cardiomyocyte protective effects, such as decreased apoptosis of cardiomyocytes and decreased expression of myocardial necrosis markers. Finally, experimental results in vitro revealed that thapsigargin (TG) can increase mast cell degranulation, tryptase release, calcium ion concentration, and the expression of STIM1 and Orai1 induced by H/R, but propofol pretreatment can partially reverse the above effects. These results suggested that the cardioprotective effect of propofol is achieved in part by inhibiting calcium influx through store-operated Ca2+ channels (SOCs) and thus alleviating mast cell degranulation.

Plasma-Derived Exosomes in Chronic Spontaneous Urticaria Induce the Production of Mediators by Human Mast Cells.

Mast cell activation and inflammatory mediators play central roles in the pathogenesis of chronic spontaneous urticaria (CSU). The factors that induce mast cell activation in CSU are still largely unknown. Exosomes (EXs) are extracellular vesicles that activate mast cells. In this study, we enriched the EXs derived from the plasma of healthy volunteers and that of patients with CSU without antihistamine sensitivity (i.e., CSU-derived EXs with antihistamine sensitivity) or resistance (i.e., CSU-derived EXs with antihistamine resistance) using ultracentrifugation. We then incubated these EXs with HMC-1 human mast cells. Notably, CSU-derived EXs with antihistamine sensitivity and CSU-derived EXs with antihistamine resistance increased tryptase-1 expression; histamine production; inflammatory mediator production; and toll-like receptor (TLR) 2, TLR4, and phosphorylated MAPK levels in HMC-1 cells. These effects were more significant in the group with CSU-derived EXs with antihistamine resistance than in the group with CSU-derived EXs with antihistamine sensitivity. TLR2, TLR4, and MAPK inhibitors (CC-401, TAK-715, and SCH772984, respectively) reduced CSU-derived EXs-Stimulated production of inflammatory mediators in HMC-1 cells. Overall, EXs in the plasma of patients with CSU were found to activate mast cells and elicit the production of multiple inflammatory mediators, partly through the TLR2, TLR4, and MAPK pathways. In addition, CSU-derived EXs with antihistamine resistance had more powerful mast cell‒activating and histamine-release abilities. Thus, these EXs may be involved in the pathogenesis of CSU with antihistamine resistance.

Mast cell tryptase enhances wound healing by promoting migration in human bronchial epithelial cells.

Epithelial damage and increase of intraepithelial mast cells (MC) are characteristics of asthma. The role of MC mediator tryptase and the protease-activated receptor-2 (PAR2) on epithelial wound healing is not fully investigated. Stimulation of bronchial epithelial cells (BECs) with tryptase promoted gap closure, migration and cellular speed compared to controls. Stimulated BECs had higher expression of migration marker CD151 compared to controls. Proliferation marker KI67 was upregulated in tryptase-stimulated BECs compared to controls. Treatment with PAR2 antagonist I-191 reduced gap closure, migration and cell speed compared to BECs stimulated with tryptase. We found that tryptase enhances epithelial wound healing by increased migration and proliferation, which is in part regulated via PAR2. Our data suggest that tryptase might be beneficial in tissue repair under baseline conditions. However, in a pathological context such as asthma with increased numbers of activated MCs, it might lead to epithelial remodeling and loss of function.

Mast Cell Protease 7 Promotes Angiogenesis by Degradation of Integrin Subunits.

Previous studies from our laboratory have shown that during angiogenesis in vitro, rmMCP-7 (recombinant mouse mast cell protease-7) stimulates endothelial cell spreading and induces their penetration into the matrix. The ability of rmMCP-7 to induce angiogenesis in vivo was assessed in the present study using a directed in vivo angiogenesis assay (DIVAA™). Vessel invasion of the angioreactor was observed in the presence of rmMCP-7 but was not seen in the control. Since integrins are involved in endothelial cell migration, the relationship between rmMCP-7 and integrins during angiogenesis was investigated. Incubation with rmMCP-7 resulted in a reduction in the levels of integrin subunits αv and ß1 on SVEC4-10 endothelial cells during angiogenesis in vitro. Furthermore, the degradation of integrin subunits occurs both through the direct action of rmMCP-7 and indirectly via the ubiquitin/proteasome system. Even in the presence of a proteasome inhibitor, incubation of endothelial cells with rmMCP-7 induced cell migration and tube formation as well as the beginning of loop formation. These data indicate that the direct degradation of the integrin subunits by rmMCP-7 is sufficient to initiate angiogenesis. The results demonstrate, for the first time, that mMCP-7 acts in angiogenesis through integrin degradation.

Effect of tryptase on mouse brain microvascular endothelial cells via protease-activated receptor 2.

BACKGROUND: Mast cells (MCs), the 'first responders' in brain injury, are able to disrupt the blood-brain barrier (BBB), but the underlying mechanism is not well understood. Tryptase is the most abundant MC secretory product. Protease-activated receptor 2 (PAR-2) has been identified as a specific receptor for tryptase, which is abundantly expressed in brain microvascular endothelial cells. The BBB comprises brain microvascular endothelial cells that display specialised molecular properties essential for BBB function and integrity. Therefore, the purpose of the present study was to investigate the effects of tryptase on mouse brain microvascular endothelial cell line bEnd3 and its potential mechanisms of action. METHODS: Induction of mouse brain microvascular endothelial cell activation by tryptase was examined. Then, mouse brain microvascular endothelial cells were pretreated with a PAR-2 antagonist and stimulated with tryptase. Cellular activation, proinflammatory cytokine production, expression of PAR-2, Toll-like receptors (TLRs) and mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-kappa B) phosphorylation were assessed. RESULTS: Tryptase upregulated the production of VCAM-1, MMPs (MMP9 and MMP2), TLR4 and TNF-α and downregulated the expression of the tight junction proteins occludin and claudin-5 in mouse brain microvascular endothelial cell. Among the MAPK and NF-kappa B pathway, ERK and NF-kappa B were activated by tryptase. All of these effects could be eliminated by the PAR-2 inhibitor. CONCLUSION: Based on our findings, we conclude that tryptase can trigger brain microvascular endothelial cell activation and proinflammatory mediator release. These findings may further clarify the involvement and mechanism of tryptase in BBB disruption.

Mast cells and mast cell tryptase enhance migration of human lung fibroblasts through protease-activated receptor 2.

BACKGROUND: Mast cells may activate fibroblasts and contribute to remodeling processes in the lung. However, the mechanism behind these actions needs to be further investigated. Fibroblasts are major regulators of on-going remodeling processes. Protease activated receptor 2 (PAR2) expressed by fibroblasts may be activated by serine proteases, such as the mast cell mediator tryptase. The objective in this study was to investigate the effects of mast cells and specifically mast cell tryptase on fibroblast migration and the role of PAR2 activation. METHODS: Human lung fibroblasts (HFL-1) were cultured together with human peripheral blood-derived mast cells or LAD2 mast cells and stimulated with either conditioned medium from LAD2 cells or tryptase. Analyses of immunological stimulation of mast cells by IgE/anti IgE in the co-culture system were also performed. The importance of PAR2 activation by mast cells and mast cell tryptase for the migratory effects of fibroblasts was investigated by pre-treatment with the PAR2 antagonist P2pal-18S. The expression of PAR2 was analyzed on fibroblasts and mast cells. RESULTS: The migratory capacity of HFL-1 cells was enhanced by blood-derived mast cells (p < 0.02), LAD2 cells (p < 0.001), conditioned medium (p < 0.05) and tryptase (p < 0.006). P2pal-18S decreased the induced migration caused by mast cells (p < 0.001) and tryptase (p < 0.001) and the expression of PAR2 was verified in HFL-1 cells. Mast cells immunologically stimulated with IgE/Anti IgE had no further effects on fibroblast migration. CONCLUSIONS: Mast cells and the mast cell mediator tryptase may have crucial roles in inducing lung fibroblast migration via PAR-2 activation, which may contribute to remodeling processes in chronic lung diseases.

Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

BACKGROUND: Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. OBJECTIVES: To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. METHODS: We have injected recombinant human ßII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. RESULTS: Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. CONCLUSIONS: Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2.

Protease activated-receptor 2 is necessary for neutrophil chemorepulsion induced by trypsin, tryptase, or dipeptidyl peptidase IV.

Compared to neutrophil chemoattractants, relatively little is known about the mechanism neutrophils use to respond to chemorepellents. We previously found that the soluble extracellular protein dipeptidyl peptidase IV (DPPIV) is a neutrophil chemorepellent. In this report, we show that an inhibitor of the protease activated receptor 2 (PAR2) blocks DPPIV-induced human neutrophil chemorepulsion, and that PAR2 agonists such as trypsin, tryptase, 2f-LIGRL, SLIGKV, and AC55541 induce human neutrophil chemorepulsion. Several PAR2 agonists in turn block the ability of the chemoattractant fMLP to attract neutrophils. Compared to neutrophils from male and female C57BL/6 mice, neutrophils from male and female mice lacking PAR2 are insensitive to the chemorepulsive effects of DPPIV or PAR2 agonists. Acute respiratory distress syndrome (ARDS) involves an insult-mediated influx of neutrophils into the lungs. In a mouse model of ARDS, aspiration of PAR2 agonists starting 24 h after an insult reduce neutrophil numbers in the bronchoalveolar lavage (BAL) fluid, as well as the post-BAL lung tissue. Together, these results indicate that the PAR2 receptor mediates DPPIV-induced chemorepulsion, and that PAR2 agonists might be useful to induce neutrophil chemorepulsion.

Tryptase potentiates enteric nerve activation by histamine and serotonin: Relevance for the effects of mucosal biopsy supernatants from irritable bowel syndrome patients.

BACKGROUND: We previously showed that mucosal biopsy supernatants from irritable bowel syndrome patients activated neurons despite low concentrations of tryptase, histamine, and serotonin which individually would not cause spike discharge. We studied the potentiating responses between these mediators on excitability of enteric neurons. METHODS: Calcium-imaging was performed using the calcium-sensitive dye Fluo-4 AM in human submucous plexus preparations from 45 individuals. Histamine, serotonin, and tryptase were applied alone and in combinations to evaluate nerve activation which was assessed by analyzing increase in intracellular Ca2+ ([Ca2+ ]i ), the proportion of responding neurons and the product of both defined as Ca-neuroindex (NI). Protease activated receptor (PAR) 2 activating peptide, PAR2 antagonist and the serine protease-inhibitor FUT-175 were used to particularly investigate the role of proteases. KEY RESULTS: Histamine or serotonin (1 µmol/L each) evoked only few small responses (median NI [25%/75%]: 0 [0/148]; 85 [0/705] respectively). Their combined application evoked statistically similar responses (216 [21/651]). Addition of the PAR2 activator tryptase induced a significantly higher Ca-NI (1401 [867/4075]) compared to individual application of tryptase or to coapplied histamine and serotonin. This synergistic potentiation was neither mimicked by PAR2 activating peptide nor reversed by the PAR2 antagonist GB83, but abolished by FUT-175. CONCLUSIONS & INFERENCES: We observed synergistic potentiation between histamine, serotonin, and tryptase in enteric neurons, which is mediated by proteolytic activity rather than PAR2 activation. This explained neuronal activation by a cocktail of these mediators despite their low concentrations and despite a relatively small PAR2-mediated response in human submucous neurons.

[Tryptase inhibits cell apoptosis through upregulating PAR-2 and Rho kinase in rheumatoid arthritis synovial fibroblasts].

Objective To investigate the regulatory effects of tryptase on protease-activated receptors 2 (PAR-2), Rho signal pathway and apoptosis of MH7A rheumatoid arthritis synovial fibroblasts. Methods In MH7A cells, the expression of PAR-2 was measured by flow cytometry; cell apoptosis was examined by annexin V- FITC/PI staining combined with flow cytometry; the expression of Rho kinase was detected by Pull-down assay and Western botting. Results Tryptase upregulated the expression of PAR-2 in MH7A cells, and suppressed Fas-mediated apoptosis of MH7A cells in a dose-dependent manner. Meanwhile, PAR-2 inhibitor, FSLLRY-NH2 significantly reduced anti-apoptotic effects of tryptase in MH7A cells, which was related with the increase of activated Rho kinase expression. Conclusion Tryptase plays a role in resistance to the apoptosis of MH7A cells through raising PAR-2 and activating Rho kinase.

Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors.

Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7) in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization.

Trypsin, Tryptase, and Thrombin Polarize Macrophages towards a Pro-Fibrotic M2a Phenotype.

For both wound healing and the formation of a fibrotic lesion, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes and pro-fibrotic M2a macrophages, which together with fibroblasts form scar tissue. Monocytes can also differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages. The proteases thrombin, which is activated during blood clotting, and tryptase, which is released by activated mast cells, potentiate fibroblast proliferation and fibrocyte differentiation, but their effect on macrophages is unknown. Here we report that thrombin, tryptase, and the protease trypsin bias human macrophage differentiation towards a pro-fibrotic M2a phenotype expressing high levels of galectin-3 from unpolarized monocytes, or from M1 and M2 macrophages, and that these effects appear to operate through protease-activated receptors. These results suggest that proteases can initiate scar tissue formation by affecting fibroblasts, fibrocytes, and macrophages.

The intestinal barrier in irritable bowel syndrome: subtype-specific effects of the systemic compartment in an in vitro model.

BACKGROUND: Irritable bowel syndrome (IBS) is a disorder with multifactorial pathophysiology. Intestinal barrier may be altered, especially in diarrhea-predominant IBS (IBS-D). Several mediators may contribute to increased intestinal permeability in IBS. AIM: We aimed to assess effects of tryptase and LPS on in vitro permeability using a 3-dimensional cell model after basolateral cell exposure. Furthermore, we assessed the extent to which these mediators in IBS plasma play a role in intestinal barrier function. MATERIALS AND METHODS: Caco-2 cells were grown in extracellular matrix to develop into polarized spheroids and were exposed to tryptase (10 - 50 mU), LPS (1 - 50 ng/mL) and two-fold diluted plasma samples of 7 patients with IBS-D, 7 with constipation-predominant IBS (IBS-C) and 7 healthy controls (HC). Barrier function was assessed by the flux of FITC-dextran (FD4) using live cell imaging. Furthermore, plasma tryptase and LPS were determined. RESULTS: Tryptase (20 and 50 mU) and LPS (6.25 - 50 ng/mL) significantly increased Caco-2 permeability versus control (all P< 0.05). Plasma of IBS-D only showed significantly elevated median tryptase concentrations (7.1 [3.9 - 11.0] vs. 4.2 [2.2 - 7.0] vs. 4.2 [2.5 - 5.9] µg/mL; P<0.05) and LPS concentrations (3.65 [3.00 - 6.10] vs. 3.10 [2.60-3.80] vs. 2.65 [2.40 - 3.40] EU/ml; P< 0.05) vs. IBS-C and HC. Also, plasma of IBS-D increased Caco-2 permeability versus HC (0.14450 ± 0.00472 vs. 0.00021 ± 0.00003; P < 0.001), which was attenuated by selective inhibition of tryptase and LPS (P< 0.05). CONCLUSION: Basolateral exposure of spheroids to plasma of IBS-D patients resulted in a significantly increased FD4 permeation, which was partially abolished by selective inhibition of tryptase and LPS. These findings point to a role of systemic tryptase and LPS in the epithelial barrier alterations observed in patients with IBS-D.

A brief exposure to tryptase or thrombin potentiates fibrocyte differentiation in the presence of serum or serum amyloid p.

A key question in both wound healing and fibrosis is the trigger for the initial formation of scar tissue. To help form scar tissue, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes, but fibrocyte differentiation is strongly inhibited by the plasma protein serum amyloid P (SAP), and healthy tissues contain very few fibrocytes. In wounds and fibrotic lesions, mast cells degranulate to release tryptase, and thrombin mediates blood clotting in early wounds. Tryptase and thrombin are upregulated in wound healing and fibrotic lesions, and inhibition of these proteases attenuates fibrosis. We report that tryptase and thrombin potentiate human fibrocyte differentiation at biologically relevant concentrations and exposure times, even in the presence of concentrations of serum and SAP that normally completely inhibit fibrocyte differentiation. Fibrocyte potentiation by thrombin and tryptase is mediated by protease-activated receptors 1 and 2, respectively. Together, these results suggest that tryptase and thrombin may be an initial trigger to override SAP inhibition of fibrocyte differentiation to initiate scar tissue formation.