Targeting mast cells: a new way to treat endometriosis

Introduction: Endometriosis is a chronic estrogen-dependent inflammatory disease of unclear etiology that affects 15–20% of women of reproductive age. Efforts are now focusing on understanding new mechanisms involved in its physiopathology, like novel target pathways and different molecules. There is evidence that mast cells (MCs) play a role in this disease. This article summarizes recent achievements in preclinical studies and clinical activities investigating the role of MCs in endometriosis. Targeting MCs might offer new alternatives to treat this disease.

Areas covered: A systematic literature search was performed (PubMed, Cochrane Library and ClinicalTrials.gov) using the keywords ‘endometriosis and mast cells’. All relevant articles (34) found in PubMed were examined and their reference lists reviewed in order to pinpoint further studies for potential inclusion.

Expert opinion: Since endometriosis is a multifactorial disease, and considering that numbers of MCs and activated MCs were clearly increased in endometriotic lesions in both animals and humans, use of MC stabilizers and inhibitors may prove to be effective to treat endometriosis and its associated pain. However, more data from preclinical studies and clinical trials will help to better define the status of MCs in the treatment of this pathology.     

Pharmacological targeting of the KIT growth factor receptor: a therapeutic consideration for mast cell disorders

KIT is a member of the tyrosine kinase family of growth factor receptors which is expressed on a variety of haematopoietic cells including mast cells. Stem cell factor (SCF)-dependent activation of KIT is critical for mast cell homeostasis and function. However, when KIT is inappropriately activated, accumulation of mast cells in tissues results in mastocytosis. Such dysregulated KIT activation is a manifestation of specific activating point mutations within KIT, with the human D816V mutation considered as a hallmark of human systemic mastocytosis. A number of other activating mutations in KIT have recently been identified and these mutations may also contribute to aberrant mast cell growth. In addition to its role in mast cell growth, differentiation and survival, localized concentration gradients of SCF may control the targeting of mast cells to specific tissues and, once resident within these tissues, mast cell activation by antigen may also be amplified by SCF. Thus, KIT inhibitors may have potential application in multiple conditions linked to mast cells including systemic mastocytosis, anaphylaxis, and asthma. In this review, we discuss the role of KIT in the context of mast cells in these disease states and how recent advances in the development of inhibitors of KIT activity and function may offer novel therapies for the treatment of these disorders.     

Changes of phospholipase D activity of rat peritoneal mast cells in degranulation

IM: To study the changes of phospholipase D (PLD) activity of actively sensitized rat peritoneal mast cells (RPMC) in degranulation. METHODS: Degranulation of RPMC was determined by measurement of β-hexosaminidase release. PLD activity assay was carried out by measurement of PLD product, choline, with chemiluminescent oxidation of luminol. RESULTS: Actively sensitized RPMC challenged with ovalbumin (0.5-8 mg/L for 120 s, 4 mg/L for 15-120 s) resulted in significant activation of PLD accompanied with the increment of P-hexosaminidase release. PLD activity of sensitized RPMC was increased by more than 2-fold compared with that of unsensitized RPMC which contained low levels of PLD activity [(35±13) pmol choline/min in 1×106cells], but β-hexosaminidase releases of the sensitized cells were as low as spontaneous releases.     

Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

Exposure to arsenic is a global health concern. We previously documented an inhibitory effect of inorganic Arsenite on IgE‐mediated degranulation of RBL‐2H3 mast cells (Hutchinson et al., 2011; J. Appl. Toxicol. 31: 231–241). Mast cells are tissue‐resident cells that are positioned at the host–environment interface, thereby serving vital roles in many physiological processes and disease states, in addition to their well‐known roles in allergy and asthma. Upon activation, mast cells secrete several mediators from cytoplasmic granules, in degranulation. The present study is an investigation of Arsenite's molecular target(s) in the degranulation pathway. Here, we report that arsenic does not affect degranulation stimulated by either the Ca²⁺ ionophore A23187 or thapsigargin, which both bypass early signaling events. Arsenic also does not alter degranulation initiated by another non‐IgE‐mediated mast cell stimulant, the G‐protein activator compound 48/80. However, arsenic inhibits Ca²⁺ influx into antigen‐activated mast cells. These results indicate that the target of arsenic in the degranulation pathway is upstream of the Ca²⁺ influx. Phospho‐Syk and phospho‐p85 phosphoinositide 3‐kinase enzyme‐linked immunosorbent assays data show that arsenic inhibits early phosphorylation events. Taken together, this evidence indicates that the mechanism underlying arsenic inhibition of mast cell degranulation occurs at the early tyrosine phosphorylation steps in the degranulation pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Meliae cortex extract exhibits anti-allergic activity through the inhibition of Syk kinase in mast cells

The anti-allergic action of various Oriental medicinal herbs was investigated using in vitro and in vivo experimental models. Of these extracts, the ethanol extract of Meliae cortex (MC) exhibited the most potent activity in mast cells; its IC(50) values were 29+/-1.5 microg/ml for antigen stimulation and 57+/-3.4 microg/ml for thapsigargin stimulation. It inhibited compound-48/80-induced systemic anaphylaxis by 52.9% at a dose of 300 mg/kg in mice; it also inhibited the expression of the proinflammatory mediator TNF-alpha. With regard to its mechanism of action, MC suppressed the activating phosphorylation of Syk, a key enzyme in mast-cell signaling processes and that of Akt in a dose-dependent manner. It also inhibited the MAP kinase ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. Taken together, these results suggest that the anti-allergic activity of MC may be due to the inhibition of histamine secretion and cytokine expression through the Syk inhibition in mast cells.     

肾组织肥大细胞表型特点分析研究

目的 阐明各种肾病患者肾组织肥大细胞的表型特点.方法 选取包括糖尿病肾病、IgA肾病、急性间质性肾炎、慢性间质性肾炎、过敏性紫癜性肾炎、膜性肾病和狼疮性肾炎在内的肾病患者369例,正常供肾16例,以免疫组化方法检测肾活检标本中肥大细胞类胰蛋白酶和糜蛋白酶表达情况,对其进行分型分析.结果 双连续切片分析发现,绝大多数肥大细胞表现为类胰蛋白酶和糜蛋白酶染色双阳性,仅有少数表现为类胰蛋白酶或糜蛋白酶单阳性;“双夹心”连续切片分析法显示,所谓的类胰蛋白酶或糜蛋白酶单阳性肥大细胞实际上是类胰蛋白酶和糜蛋白酶双阳性;以类胰蛋白酶为标记分子和以糜蛋白酶为标记分子检测肾组织肥大细胞的结果比较没有显著区别.结论 类胰蛋白酶糜蛋白酶亚型(MCTC亚型)是正常个体和肾病患者肾组织肥大细胞的基本亚型.     

Saucerneol D inhibits eicosanoid generation and degranulation through suppression of Syk kinase in mast cells

Previously we reported that saucerneol D (SD), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis) suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. The aim of this study was to elucidate whether SD modulates the generation of other inflammatory mediators in activated mast cells. We investigated the effects of SD on cyclooxygenase-2 (COX-2)-dependent prostaglandin D₂ (PGD₂) and 5-lipoxygenase (5-LO)-dependent leukotriene C₄ (LTC₄) generations as well as degranulation in cytokine-stimulated mouse bone marrow-derived mast cells (BMMCs). Biochemical analyses of the cytokine-mediated signaling pathways showed that SD suppressed the phosphorylation of Syk kinase and multiple downstream signaling processes including phospholipase Cγ1 (PLCγ1)-mediated intracellular Ca²⁺ influx and activation of mitogen-activated protein kinases (MAPKs; including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH₂-terminal kinase (JNK) and p38) and the nuclear factor-κB (NF-κB) pathway. Taken together, the present study suggests that SD suppresses eicosanoid generation and degranulation through Syk-dependent pathway in BMMCs.     

Morin inhibits Fyn kinase in mast cells and IgE-mediated type I hypersensitivity response in vivo

Mast cells are responsible for IgE-mediated allergic responses. Although dietary flavonoid morin has been known to suppress mast cell activation, its in vivo anti-allergic activity and the underlying mechanisms remain are largely unknown. In this study, we determine whether morin suppresses IgE-mediated allergic responses in an animal model and its mechanism of action. Morin suppressed IgE-mediated PCA in mice (ED₅₀ 23.9 mg/kg) and inhibited degranulation and production of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-4 in antigen (Ag)-stimulated mast cells. The mechanism of action was a follows. Morin inhibited the activating phosphorylation of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT) in rat basophilic leukemia (RBL)-2H3 cells and bone marrow-derived mast cells (BMMCs). Akt and the mitogen-activated protein (MAP) kinases, p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK) were inhibited as well. In vitro kinase assay indicated that Fyn kinase, not Lyn and Syk, was inhibited by morin in a dose-dependent manner (IC₅₀ 5.7 μM). In conclusion, the results suggest that morin suppresses the IgE-mediated allergic response by primarily inhibiting Fyn kinase in mast cells.     

Beta-adrenoceptor-mediated inhibition of mediator release from human peripheral blood-derived mast cells

1. Mast cells cultured from human peripheral blood have been used as a cell model for functional studies of human mast cells, particularly human lung mast cells. However, the beta-adrenoceptor subtype expressed by these cultured cells has not been identified. The aim of the present study was to characterize pharmacologically the beta-adrenoceptors involved in the suppression of IgE-mediated release of mediators, including histamine, prostaglandin (PG) D2 and leukotriene (LT) C4 from cultured mast cells. 2. Mast cells were cultured from mast cell progenitors isolated from peripheral blood in the presence of 200 ng/mL stem cell factor and 50 ng/mL interleukin-6. Mast cells were sensitized with human myeloma IgE, treated with beta-adrenoceptor agonists or antagonist and then challenged with anti-human IgE. The release of histamine, PGD2 and LTC4 from mast cells was determined. 3. Both isoprenaline and salbutamol inhibited anti-IgE-induced release of histamine, PGD2 and LTC4 from cultured mast cells in a dose-dependent manner. Isoprenaline was a more potent inhibitor than salbutamol. The pD2 values for the inhibition of the release of histamine, PGD2 and LTC4 were 7.37 +/- 0.12, 8.38 +/- 0.23, 8.85 +/- 0.23, respectively, for isoprenaline and 6.96 +/- 0.12, 7.65 +/- 0.36, 7.91 +/- 0.64, respectively, for salbutamol. The selective beta3-adrenoceptor agonist BRL-37344 failed to affect anti-IgE-induced histamine release from cultured mast cells. 4. The selective beta2-adrenoceptor antagonist ICI 118 551 (108 mol/L) strongly reversed the concentration-dependent suppression of histamine release by isoprenaline and salbutamol; however, the selective beta1-adrenoceptor antagonist atenolol (106 mol/L) did not have any effect. 5. These results indicate that both isoprenaline and salbutamol act at beta2-adrenoceptors to suppress IgE-mediated mediator release from cultured human mast cells.     

Targeting CD44 in mast cell regulation

Importance of the field: Accumulating evidence suggests that mast cells are involved in a wide variety of immune responses including chronic inflammation, immune tolerance and tumor immunity. Mast cells originate from hematopoietic stem cells and undergo terminal differentiation in the tissues, in which they are ultimately resident. Heterogeneity of tissue mast cells is, therefore, one of the key concepts for a better understanding of various immune responses.

Areas covered in this review: This review describes the candidate genes involved in regulation of cutaneous mast cell differentiation, with a particular attention to CD44, which is the primary receptor for hyaluronan.

What the reader will gain: CD44 is involved in various aspects of cutaneous inflammation. Regarding mast cells, CD44 is upregulated upon differentiation and maturation of mast cells, and plays a critical role in regulation of cutaneous mast cell number. Since both degradation and decrease of hyaluronan are often observed upon chronic inflammation, CD44 might be involved in modulation of local immune responses through regulation of cutaneous mast cell functions.

Take home message: Understanding of cutaneous immune responses should require clarification of local mast cell functions, a part of which is regulated by extracellular matrix components and their membrane receptors.     

Masitinib in treatment of pancreatic cancer

Introduction: Pancreatic cancer continues to have high mortality despite the development of many chemotherapeutic agents. The 5-year relative survival for stage IV patients is less than 3%. This urgent unmet need warrants the development of novel and active therapeutic agents, which focus both on targeting cancer cells and modifying the microenvironment of cancer cells.

Areas covered: In this article, the authors review the development of masitinib, a novel tyrosine kinase inhibitor of numerous targets, including c-Kit, PDGFR and FGFR. This review covers its development from the bench to clinical trials assessing its potential in pancreatic cancer.

Expert opinion: While masitinib has not shown an increase in overall survival (OS) or progression free survival (PFS) compared to the current standard of care in patients with pancreatic adenocarcinoma, masitinib may have a role in decreasing inflammation related to those patients with increased pain scores with pancreatic adenocarcinoma. If we have the tools to identify accurate subgroups of patients who may benefit from particular therapies, this agent may be of benefit to these patients. Indeed, if more sophisticated biomarkers and the identification of patient subgroups are better explained, the authors believe that masitinib will become part of the armamentarium against pancreatic adenocarcinoma.     

类糜蛋白酶抑制剂对人结肠肥大细胞组胺释放的影响

目的　研究类糜蛋白酶抑制剂对人结肠肥大细胞释放组胺的影响。方法　用酶消化人结肠组织并分离细胞成份。激发过程在LP4试管中、37℃条件下完成。组胺水平用以玻璃纤维为基础的荧光方法测定。结果　类糜蛋白酶抑制剂ZIGPFM、TPCK和α1 抗胰蛋白酶无明显刺激人类结肠肥大细胞释放组胺的作用。 3种类糜蛋白酶抑制剂均可以剂量依赖性方式抑制抗IgE抗体诱导的组胺释放 ,最大浓度的ZIGPFM(1mmol·L-1)、TPCK(80mmol·L-1)和α1 抗胰蛋白酶(30mmol·L-1)可分别抑制 37%、2 6 %和 36 8%的组胺释放。在 37℃条件下同结肠细胞预培养 2 0min与无预培养相比 ,ZIGPFM和TPCK抑制抗IgE抗体诱导的组胺释放的作用略有增强。 3种类糜蛋白酶抑制剂均可以剂量依赖性方式抑制CI诱导的组胺释放 ,最大抑制范围在 2 3 6 %～ 35 %之间。在 37℃条件下同结肠细胞预培养 2 0min与无预培养相比 ,TPCK对CI诱导的组胺释放的抑制作用略有增强 ,但ZIGPFM则无此特点。结论　我们发现了类糜蛋白酶抑制剂可抑制人结肠肥大细胞IgE依赖性和非依赖性组胺释放 ,提示类糜蛋白酶抑制剂可能有抗炎症性肠病的作用 ,可研究开发     

肥大细胞在免疫反应中的调节作用

肥大细胞（MC）起源于造血干细胞,包括黏膜MC和结缔组织MC两个亚群。机体中MC不仅可作为效应细胞,也可作为免疫调节细胞起作用。它可通过MHC分子对抗原进行加工、递呈,也可产生化学因子募集T细胞。在免疫反应中,MC一方面通过直接接触或产生的TNF-α、蛋白酶类正向调节免疫应答,另一方面也可通过分泌IL-10等分子负向调节免疫应答。     

地塞米松抑制肥大细胞脱颗粒及其机制初探

目的：研究糖皮质激素对Ⅰ型超敏反应中的肥大细胞脱颗粒反应是否有直接作用。方法：利用流式细胞仪技术观察地塞米松对RBL-2H3细胞(大鼠嗜碱粒细胞)脱颗粒程度的影响，利用western blot分析：ERK1／ERK2信号的改变。结果：地塞米松预先作用于RBL-2H3细胞，使AnnexinV标记的细胞阳性率较致敏原活化组明显下降，MAPK活性明显降低。结论：地塞米松抑制肥大细胞脱颗粒反应，其机制与MAPK活性有关。     

Twenty-first century mast cell stabilizers

Mast cell stabilizing drugs inhibit the release of allergic mediators from mast cells and are used clinically to prevent allergic reactions to common allergens. Despite the relative success of the most commonly prescribed mast cell stabilizer, disodium cromoglycate, in use for the preventative treatment of bronchial asthma, allergic conjunctivitis and vernal keratoconjunctivitis, there still remains an urgent need to design new substances that are less expensive and require less frequent dosing schedules. In this regard, recent developments towards the discovery of the next generation of mast cell stabilizing drugs has included studies on substances isolated from natural sources, biological, newly synthesized compounds and drugs licensed for other indications. The diversity of natural products evaluated range from simple phenols, alkaloids, terpenes to simple amino acids. While in some cases their precise mode of action remains unknown it has nevertheless sparked interest in the development of synthetic derivatives with improved pharmacological properties. Within the purely synthetic class of inhibitors, particular attention has been devoted to the inhibition of important signalling molecules including spleen TK and JAK3. The statin class of cholesterol-lowering drugs as well as nilotinib, a TK inhibitor, are just some examples of clinically used drugs that have been evaluated for their anti-allergic properties. Here, we examine each approach under investigation, summarize the test data generated and offer suggestions for further preclinical evaluation before their therapeutic potential can be realized.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1     

Advanced glycation end products (AGEs) activate mast cells

BACKGROUND AND PURPOSE

Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells.

EXPERIMENTAL APPROACHES

Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured.

KEY RESULTS

Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G_i-proteins and intracellular Ca²⁺ increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls.

CONCLUSIONS AND IMPLICATIONS

Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.     

聚山梨酯80刺激肥大细胞RBL-2H3脱颗粒作用的评价

目的：研究不同类别及厂家的聚山梨酯80直接刺激肥大细胞RBL-2H3脱颗粒的作用,为建立完善的注射用辅料引发类过敏反应的筛选评价体系提供依据。方法：以不同剂量的聚山梨酯80处理RBL-2H3细胞,测定β-氨基己糖苷酶释放量,并通过MTT法进一步研究有脱颗粒作用的受试物对RBL-2H3细胞的毒性作用。结果：8种聚山梨酯80样品具有不同程度的直接刺激RBL-2H3细胞脱颗粒的作用,且呈浓度依赖性,其中上海试剂采购供应站提供的受试物作用极强,威尔制药口服级受试物作用较强,威尔制药A、威尔制药B、威尔制药注射级、威尔制药4个受试物作用相近,2个进口品种的作用略弱。在产生直接刺激RBL-2H3细胞脱颗粒作用的浓度下,上海试剂采购供应站的聚山梨酯80有明显的细胞毒性,并呈浓度依赖性。结论：聚山梨酯80能够直接刺激RBL-2H3细胞脱颗粒,其作用强度不同反映了产品的质量差异;上海试剂采购供应站提供的聚山梨酯80直接刺激RBL-2H3细胞脱颗粒的作用可能由其细胞毒性引起。     

Therapeutic prospect of Syk inhibitors

Background: The non-receptor spleen tyrosine kinase (Syk; EC 2.7.10.2) is involved in signal transduction in a variety of cell types. In particular, it is a key mediator of immune receptors signaling in host inflammatory cells (B cells, mast cells, macrophages and neutrophils), important for both allergic and antibody-mediated autoimmune diseases. Deregulated Syk kinase activity also allows growth factor-independent proliferation and transforms bone marrow-derived pre-B cells that are able to induce leukemia. Consequently, the development of Syk kinase inhibitors could conceivably treat these disorders and so they have became a major focus in the pharmaceutical and biotech industry. Objective: In this review, we analyze the structure and role of Syk kinase, the use of small molecules, interacting with ATP-binding site, as inhibitors of kinase activity and finally the potential of using inhibitors of Syk kinase expression to attenuate pathological conditions. Conclusion: Syk kinase inhibition is suggested as a powerful tool for the therapy of different pathologies.     

Long-term cultures of mast cells: a new model for studying the allergic response

Mast cells are the key cells of allergic reactions and probably play also a role in chronic inflammatory reactions resulting in fibrosis. Although their biochemical and functional properties have been extensively investigated, several studies have been hampered by the absence of a tissue culture system to keep these cells alive and functionally active for long periods of time. Recently we have developed an in vitro system in which rat peritoneal mast cells are cocultured with 3T3 mouse skin derived fibroblasts (MC/3T3). Under these tissue culture conditions, mast cells do not proliferate and maintain their viability and functional activity for more than a month. This system allowed us to carry out long-term studies on the functional properties of mast cells. We have found that mast cells activated both by IgE-dependent and IgE-independent stimuli survive, and slowly replenish their histamine content. After a non-immunological challenge mast cells retain their full potential to release histamine upon a repeated similar challenge. In contrast, immunologically challenged mast cells become partially unresponsive to a similar activation event for up to 3 weeks. By exploiting this long-term culture system we described a novel type of mast cell activation induced by cytokines. The onset of this activation is slow and its course appears to be chronic-continuous, very different from the classical anaphylactic type activation that is completed within few minutes. Since MC/3T3 release higher amounts of histamine, this system is a sensitive tool to investigate the antiallergic properties of various drugs. By employing MC/3T3 cultures we were able to show that the gold salt auranofin inhibits histamine release from mast cells stimulated by different secretagogues. In addition, salbutamol inhibited histamine release from repeatedly challenged mast cells; and nedocromil sodium was effective in preventing mast cell activation when incubated for a week with MC/3T3.