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BACKGROUND: Mast cells (MCs) develop from a rare population of peripheral blood circulating MC progenitors (MCps). Here, we investigated whether the frequency of circulating MCps is altered in asthma patients sensitized to birch pollen during pollen season, compared to out of season. METHODS: Asthma patients were examined during birch pollen season in late April to early June (May), and out of season in November-January. Spirometry measurements, asthma and allergy-related symptoms, asthma control questionnaire (ACQ), and asthma control test (ACT) scores were assessed at both time points. The MCp frequency was determined by flow cytometry in ficoll-separated blood samples from patients with positive birch pollen-specific IgE, and analyzed in relation to basic and disease parameters. RESULTS: The frequency of MCps per liter of blood was higher in May than in November (p = .004), particularly in women (p = .009). Patients that reported moderate to severe asthma symptoms (<.0001), nose or eye symptoms (p = .02; p = .01), or reduced asthma control (higher ACQ, p = .01) had higher MCp frequency in May than those that did not report this. These associations remained significant after adjusting for sex and BMI. The change in asthma control to a lower ACT score in May correlated with an increase in MCp frequency in May (p = .006, rho = 0.46). CONCLUSIONS: The data suggest that the frequency of MCps increases in symptomatic patients with allergic asthma. Our results unravel a link between asthma symptoms and circulating MCps, and bring new insight into the impact of natural allergen exposure on the expansion of MCs.
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Asma , Betula , Humanos , Feminino , Mastócitos , Pólen , Asma/diagnóstico , Asma/epidemiologia , AlérgenosRESUMO
Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses.
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Asma , Mastócitos , Humanos , Cavalos/genética , Animais , Camundongos , Mastócitos/metabolismo , Triptases/genética , Triptases/metabolismo , Asma/genética , Asma/veterinária , Perfilação da Expressão Gênica , Líquido da Lavagem BroncoalveolarRESUMO
Mast cells are implicated in the innate proinflammatory immune defence against bacterial insult, but the mechanisms through which mast cells respond to bacterial encounter are poorly defined. Here, we addressed this issue and show that mast cells respond vividly to wild type Streptococcus equi by up-regulating a panel of proinflammatory genes and by secreting proinflammatory cytokines. However, this response was completely abrogated when the bacteria lacked expression of sagA, whereas the lack of a range of other potential virulence genes (seeH, seeI, seeL, seeM, hasA, seM, aroB, pyrC, and recA) had no effect on the amplitude of the mast cell responses. The sagA gene encodes streptolysin S, a lytic toxin, and we next showed that the wild type strain but not a sagA-deficient mutant induced lysis of mast cells. To investigate whether host cell membrane perturbation per se could play a role in the activation of the proinflammatory response, we evaluated the effects of detergent- and pneumolysin-dependent lysis on mast cells. Indeed, exposure of mast cells to sublytic concentrations of all these agents resulted in cytokine responses of similar amplitudes as those caused by wild type streptococci. This suggests that sublytic membrane perturbation is sufficient to trigger full-blown proinflammatory signalling in mast cells. Subsequent analysis showed that the p38 and Erk1/2 signalling pathways had central roles in the proinflammatory response of mast cells challenged by either sagA-expressing streptococci or detergent. Altogether, these findings suggest that sagA-dependent mast cell membrane perturbation is a mechanism capable of activating the innate immune response upon bacterial challenge.
Assuntos
Proteínas de Bactérias/metabolismo , Inflamação/metabolismo , Mastócitos/imunologia , Streptococcus equi/genética , Streptococcus equi/patogenicidade , Estreptolisinas/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Citocinas/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Estreptolisinas/genética , Estreptolisinas/farmacologia , Virulência/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Despite intense efforts to develop efficient therapeutic regimes for asthma, there is a large demand for novel treatment strategies in this disease. Here we evaluated the impact of monensin, a drug with potent anti-mast cell effects, in a mouse model of asthma. Allergic airway inflammation was induced by sensitization of mice with house dust mite (HDM) antigen, and effects of monensin on airway hyperreactivity and inflammatory parameters were studied. Following intraperitoneal administration, monensin did not suppress airway hyperreactivity but was shown to have anti-inflammatory properties, as manifested by reduced eosinophil- and lymphocyte infiltration into the airway lumen, and by suppressed inflammation of the lung tissue. After intranasal instillation, monensin exhibited similar anti-inflammatory effects as seen after intraperitoneal administration. Moreover, intranasally administered monensin was demonstrated to suppress goblet cell hyperplasia, and to cause a reduction in the expression of genes coding for key inflammatory markers. Further, monensin blocked mast cell degranulation in the airways of allergen-sensitized mice. Together, this study reveals that monensin has the capacity to suppress key pathological events associated with allergic airway inflammation.
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Mast cells are effector cells known to contribute to allergic airway disease. When activated, mast cells release a broad spectrum of inflammatory mediators, including the mast cell-specific protease carboxypeptidase A3 (CPA3). The expression of CPA3 in the airway epithelium and lumen of asthma patients has been associated with a Th2-driven airway inflammation. However, the role of CPA3 in asthma is unclear and therefore, the aim of this study was to investigate the impact of CPA3 for the development and severity of allergic airway inflammation using knockout mice with a deletion in the Cpa3 gene. We used the ovalbumin (OVA)- and house-dust mite (HDM) induced murine asthma models, and monitored development of allergic airway inflammation. In the OVA model, mice were sensitized with OVA intraperitoneally at seven time points and challenged intranasally (i.n.) with OVA three times. HDM-treated mice were challenged i.n. twice weekly for three weeks. Both asthma protocols resulted in elevated airway hyperresponsiveness, increased number of eosinophils in bronchoalveolar lavage fluid, increased peribronchial mast cell degranulation, goblet cell hyperplasia, thickening of airway smooth muscle layer, increased expression of IL-33 and increased production of allergen-specific IgE in allergen-exposed mice as compared to mocktreated mice. However, increased number of peribronchial mast cells was only seen in the HDM asthma model. The asthma-like responses in Cpa3-/- mice were similar as in wild type mice, regardless of the asthma protocol used. Our results demonstrated that the absence of a functional Cpa3 gene had no effect on several symptoms of asthma in two different mouse models. This suggest that CPA3 is dispensable for development of allergic airway inflammation in acute models of asthma in mice.
Assuntos
Asma , Mastócitos , Animais , Camundongos , Alérgenos/metabolismo , Líquido da Lavagem Broncoalveolar , Carboxipeptidases/metabolismo , Modelos Animais de Doenças , Inflamação/genética , Inflamação/metabolismo , Pulmão/metabolismo , Mastócitos/metabolismo , Camundongos Endogâmicos BALB C , Ovalbumina/metabolismoRESUMO
Equine asthma (EA) is a heterogenous, complex disease, with a significant negative impact on horse welfare and performance. EA and human asthma share fundamental similarities, making EA a useful model for studying the disease. One relevant sample type for investigating chronic lung inflammation is bronchoalveolar lavage fluid (BALF), which provides a snapshot of the immune cells present in the alveolar space. To investigate the immune cell landscape of the respiratory tract in horses with mild-to-moderate equine asthma (mEA) and healthy controls, single-cell RNA sequencing was conducted on equine BALF cells. We characterized the major immune cell populations present in equine BALF, as well as subtypes thereof. Interestingly, the most significantly upregulated gene discovered in cases of mEA was FKBP5, a chaperone protein involved in regulating the activity of the glucocorticoid receptor.
Assuntos
Asma , Doenças dos Cavalos , Animais , Asma/genética , Asma/veterinária , Líquido da Lavagem Broncoalveolar , Doenças dos Cavalos/genética , Cavalos , Sistema Respiratório , Transcriptoma , Regulação para CimaRESUMO
Introduction: Asthma is characterized by an imbalance between proteases and their inhibitors. Hence, an attractive therapeutic option could be to interfere with asthma-associated proteases. Here we exploited this option by assessing the impact of nafamostat, a serine protease inhibitor known to neutralize mast cell tryptase. Methods: Nafamostat was administered in a mouse model for asthma based on sensitization by house dust mite (HDM) extract, followed by the assessment of effects on airway hyperreactivity, inflammatory parameters and gene expression. Results: We show that nafamostat efficiently suppressed the airway hyperreactivity in HDM-sensitized mice. This was accompanied by reduced infiltration of eosinophils and lymphocytes to the airways, and by lower levels of pro-inflammatory compounds within the airway lumen. Further, nafamostat had a dampening impact on goblet cell hyperplasia and smooth muscle layer thickening in the lungs of HDM-sensitized animals. To obtain deeper insight into the underlying mechanisms, a transcriptomic analysis was conducted. This revealed, as expected, that the HDM sensitization caused an upregulated expression of numerous pro-inflammatory genes. Further, the transcriptomic analysis showed that nafamostat suppressed the levels of multiple pro-inflammatory genes, with a particular impact on genes related to asthma. Discussion: Taken together, this study provides extensive insight into the ameliorating effect of nafamostat on experimental asthma, and our findings can thereby provide a basis for the further evaluation of nafamostat as a potential therapeutic agent in human asthma.
Assuntos
Antiasmáticos , Asma , Eosinofilia , Humanos , Animais , Camundongos , Antiasmáticos/uso terapêutico , Eosinófilos/metabolismo , Citocinas/metabolismo , Asma/metabolismo , Pulmão/metabolismo , Eosinofilia/metabolismo , Expressão GênicaRESUMO
Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.
Assuntos
Apoptose/fisiologia , Membranas Intracelulares/metabolismo , Mastócitos/metabolismo , Proteoglicanas/metabolismo , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Ativação Enzimática/fisiologia , Mastócitos/citologia , Camundongos , Camundongos Knockout , Permeabilidade , Proteoglicanas/genética , Vesículas Secretórias/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin,a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation,whereas serglycin −/− MCs completely lacked this ability.Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist,which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex.Moreover, IL-13 degradation was abrogated in MC-CPA −/−MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein.Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.
Assuntos
Interleucina-13/metabolismo , Mastócitos/fisiologia , Proteoglicanas/metabolismo , Serina Proteases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Carboxipeptidases A/genética , Carboxipeptidases A/imunologia , Carboxipeptidases A/metabolismo , Degranulação Celular , Células Cultivadas , Deleção de Genes , Heparina/imunologia , Heparina/metabolismo , Antagonistas de Heparina/farmacologia , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Interleucina-13/imunologia , Camundongos , Peritônio/citologia , Proteoglicanas/genética , Proteoglicanas/imunologia , Proteólise , Serina Proteases/imunologia , Inibidores de Serina Proteinase/farmacologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/imunologiaRESUMO
Mast cells (MCs) are currently receiving increased attention among the scientific community, largely because of the recent identification of crucial functions for MCs in a variety of disorders. However, it is in many cases not clear exactly how MCs contribute in the respective settings. MCs express extraordinarily high levels of a number of proteases of chymase, tryptase, and carboxypeptidase A type, and these are stored in high amounts as active enzymes in the MC secretory granules. Hence, MC degranulation leads to the massive release of fully active MC proteases, which probably have a major impact on any condition in which MC degranulation occurs. Indeed, the recent generation and evaluation of mouse strains lacking individual MC proteases have indicated crucial contributions of these to a number of different disorders. MC proteases may thus account for many of the effects ascribed to MCs and are currently emerging as promising candidates for treatment of MC-driven disease. In this review, we discuss these findings.
Assuntos
Infecções/imunologia , Inflamação/imunologia , Mastócitos/enzimologia , Mastócitos/imunologia , Peptídeo Hidrolases/metabolismo , Animais , Humanos , Peptídeo Hidrolases/imunologiaRESUMO
It is widely established that mast cells (MCs) have a harmful role in asthma, for example by secreting various proinflammatory substances stored within their secretory granule. However, in this study, we show that one of the substances stored within MC granule, chymase, in fact has a protective role in allergic airway inflammation, indicating that MCs may possess both harmful and protective activities in connection with this type of disease. Wild-type (WT) mice and mice lacking mouse MC protease 4 (mMCP-4), a chymase that is functionally homologous to human chymase, were sensitized and challenged with OVA, followed by the assessment of airway physiology and inflammatory parameters. Our results show that the airway hyperresponsiveness was significantly higher in mMCP-4(-/-) as compared with WT mice. Moreover, the degree of lung tissue inflammation was markedly higher in mice lacking mMCP-4 than in WT controls. Histological analysis revealed that OVA sensitization/challenge resulted in a marked increased in the thickness of the smooth muscle cell (SMC) layer and, notably, that the degree of SMC layer thickening was more pronounced in mMCP-4(-/-) animals than in WT controls, thus indicating that chymase may have an effect on airway SMCs. In support of this, mMCP-4-positive MCs were located in the close vicinity of the SMC layer, mainly in the upper airways, and mMCP-4 was shown to be the major chymase expressed in these MCs. Taken together, our results indicate that chymase present in the upper airways protects against allergic airway responses, possibly by regulating SMCs.
Assuntos
Hipersensibilidade/imunologia , Inflamação/imunologia , Mastócitos/imunologia , Serina Endopeptidases/imunologia , Animais , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Hipersensibilidade/enzimologia , Inflamação/enzimologia , Pulmão/imunologia , Pulmão/patologia , Mastócitos/citologia , Mastócitos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/fisiologia , Ovalbumina/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Serglycin (SG) proteoglycan consists of a small core protein to which glycosaminoglycans of chondroitin sulfate or heparin type are attached. SG is crucial for maintaining mast cell (MC) granule homeostasis through promoting the storage of various basic granule constituents, where the degree of chondroitin sulfate/heparin sulfation is essential for optimal SG functionality. However, the regulation of the SG core protein expression and of the various chondroitin sulfate/heparin sulfotransferases during MC differentiation and activation are poorly understood. Here we addressed these issues and show that expression of the SG core protein, chondroitin 4-sulfotransferase (C4ST)-1, and GalNAc(4S)-6-O-sulfotransferase (GalNAc4S6ST) are closely linked to MC maturation. In contrast, the expression of chondroitin 6-sulfotransferase correlated negatively with MC maturation. The expression of N-deacetylase/N-sulfotransferase (NDST)-2, a key enzyme in heparin synthesis, also correlated strongly with MC maturation, whereas the expression of the NDST-1 isoform was approximately equal at all stages of maturation. MC activation by either calcium ionophore or IgE ligation caused an up-regulated expression of the SG core protein, C4ST-1, and GalNAc4S6ST, accompanied by increased secretion of chondroitin sulfate as shown by biosynthetic labeling experiments. In contrast, NDST-2 was down-regulated after MC activation, suggesting that MC activation modulates the nature of the glycosaminoglycan chains attached to the SG core protein. Taken together, these data show that MC maturation is associated with the expression of a distinct signature of genes involved in SG proteoglycan synthesis, and that MC activation modulates their expression.
Assuntos
Regulação da Expressão Gênica/imunologia , Mastócitos/citologia , Mastócitos/metabolismo , Proteoglicanas/biossíntese , Sulfotransferases/biossíntese , Proteínas de Transporte Vesicular/biossíntese , Amidoidrolases/biossíntese , Amidoidrolases/genética , Animais , Diferenciação Celular/imunologia , Sulfatos de Condroitina/metabolismo , Grânulos Citoplasmáticos/metabolismo , Expressão Gênica , Heparina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
Mastitis is an inflammatory condition of the mammary tissue and represents a major problem for the dairy industry worldwide. The present study was undertaken to study how experimentally induced acute bovine mastitis affects inflammatory parameters and changes in the metabolome. To this end, we induced experimental mastitis in nine cows by intramammary infusion of 100 µg purified Escherichia coli lipopolysaccharide (LPS) followed by kinetic assessments of cytokine responses (by enzyme-linked immunosorbent assay), changes in the metabolome (assessed by nuclear magnetic resonance), clinical parameters (heat, local pain perception, redness, swelling, rectal temperature, clot formation, and color changes in the milk), and milk somatic cell counts, at several time points post LPS infusion. Intramammary LPS infusion induced clinical signs of mastitis, which started from 2 h post infusion and had returned to normal levels within 24-72 h. Milk changes were seen with a delay compared with the clinical signs and persisted for a longer time. In parallel, induction of IL-6 and TNF-α were seen in milk, and there was also a transient elevation of plasma IL-6 whereas plasma TNF-α was not significantly elevated. In addition, a robust increase in CCL2 was seen in the milk of LPS-infused cows, whereas G-CSF, CXCL1, and histamine in milk were unaffected. By using a metabolomics approach, a transient increase of plasma lactose was seen in LPS-induced cows. In plasma, significant reductions in ketone bodies (3-hydroxybutyrate and acetoacetate) and decreased levels of short-chain fatty acids, known to be major products released from the gut microbiota, were observed after LPS infusion; a profound reduction of plasma citrate was also seen. Intramammary LPS infusion also caused major changes in the milk metabolome, although with a delay in comparison with plasma, including a reduction of lactose. We conclude that the LPS-induced acute mastitis rapidly affects the plasma metabolome and cytokine induction with similar kinetics as the development of the clinical signs, whereas the corresponding effects in milk occurred with a delay.
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Transplantation of microencapsulated islets may reduce hyperglycemia in the absence of immunosuppression. However, the efficiency of microencapsulated islet transplantation is low, requiring more islets to achieve normoglycemia than in vascularized islet transplantation. Exendin-4 (a glucagon-like receptor agonist) has been previously shown to improve islet transplantation outcome in rodents. We investigated whether this treatment would enhance the function of microencapsulated islets in vitro and in vivo. Encapsulated or naked islets were cultured with or without exendin-4 for 72 h. To test in vitro function, insulin release and glucose oxidation rates were measured in the absence or presence of exendin-4. In addition, in vivo function of a minimal mass of 350 microencapsulated islets was assessed by syngeneic transplantation into the peritoneal cavity of alloxan-diabetic mice. Glucose oxidation rates of microencapsulated islets were improved by 72-h pretreatment with exendin-4. Insulin release was increased both acutely after glucose stimulation and over a 40-h culture period by the presence of exendin-4. Transplantation outcome of microencapsulated islets cultured with exendin-4 was initially improved, but by day 7 there were no differences compared with control cultured microencapsulated islets. Culture of microencapsulated islets with exendin-4 increases glucose oxidation and insulin release rates, but the increased function seen in vitro was not enough to improve the long term outcome in a transplantation model.
Assuntos
Hipoglicemiantes/farmacologia , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/efeitos dos fármacos , Peptídeos/farmacologia , Peçonhas/farmacologia , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Exenatida , Glucose/metabolismo , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , OxirreduçãoRESUMO
Mast cells contain large amounts of fully active proteases that are stored in complex with serglycin proteoglycan in their secretory granules. Upon degranulation, such serglycin:protease complexes are released to the extracellular space and can potentially have an impact on the local inflammatory reaction, either through direct effects of serglycin proteoglycan or through effects mediated by its bound proteases. The objective of this study was to address this scenario by investigating the possibility that serglycin-associated proteases can regulate levels of pro-inflammatory cytokines. Indeed, we show here that activated cultured peritoneal mast cells from wild type mice efficiently reduced the levels of exogenously administered IL-6 and IL-17A, whereas serglycin-deficient mast cells lacked this ability. Furthermore, our data suggest that the reduction of IL-6 and IL-17A concentrations is due to proteolytic degradation mediated by serglycin-dependent serine proteases. Moreover, we show that activated mast cells have the capacity to release IL-6 and that the levels of this cytokine in supernatants were markedly higher in cultures of serglycin-deficient versus serglycin-sufficient mast cells, suggesting that serglycin-dependent serine proteases also participate in the regulation of endogenously produced IL-6. In summary, although the general consensus is that mast cells have a pathogenic impact on inflammatory settings, this study identifies a role for a mast cell-derived serglycin:serine protease axis in down-regulating levels of major inflammatory cytokines. These findings support the notion that mast cells could have a dual role in inflammatory settings, by both being able to secrete pathogenic compounds and being able to regulate their levels after release.
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Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre- R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre- R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre- R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.
Assuntos
Neoplasias Pulmonares/secundário , Mastócitos/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/patologia , Animais , Líquido da Lavagem Broncoalveolar , Quimases/metabolismo , Transição Epitelial-Mesenquimal , Genótipo , Inflamação , Pulmão/metabolismo , Masculino , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de SinaisRESUMO
Heparanase is an endo-ß-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa-/-) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa-/- and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa-/- mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.
Assuntos
Glucuronidase/metabolismo , Hipersensibilidade/enzimologia , Hipersensibilidade/patologia , Pulmão/enzimologia , Pulmão/patologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Deleção de Genes , Glucuronidase/antagonistas & inibidores , Humanos , Imunização , Inflamação/patologia , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ovalbumina , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/patologia , Testes de Função RespiratóriaRESUMO
Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf-/- mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma.