人肥大细胞的IgE依赖性组胺和类胰蛋白酶分泌

目的　利用人结肠组织的肥大细胞和肥大细胞激活的体外研究系统评价人肥大细胞释放类胰蛋白酶和组胺的能力及其机制。方法　经酶悬浮的人结肠肥大细胞与抗IgE抗体共同培养后记录浓度相关曲线和时间关系曲线。类胰蛋白酶用酶联免疫吸附试验的方法测量 ,而组胺则由一种以玻璃纤维为基础的荧光比色法测量。结果　抗IgE抗体可引起浓度相关性的组胺和类胰蛋白酶释放 ,最大组胺和类胰蛋白酶分泌量分别比基础分泌量超出 2 .7和 2 .5倍以上。抗IgE抗体的作用从加样后 10s开始 ,6min后达高峰并至少持续 15min。百日咳毒素和代谢抑制剂能够分别抑制抗IgE抗体引起的组胺和类胰蛋白酶释放。百日咳毒素还能够减少自发性类胰蛋白酶释放。结论　人结肠肥大细胞在受到抗IgE抗体刺激时具有平行释放类胰蛋白酶和组胺的能力 ,这个过程与肥大细胞膜G蛋白偶联受体的激活有关 ,并消耗能量。肥大细胞自发性释放组胺和类胰蛋白酶的功能可能是通过不同的机制实现的。     

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

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

绵羊肥大细胞中类胰蛋白酶的证实

用甲苯胺蓝和阿尔辛蓝常规组织化学方法及特异性酶底物鉴定人肥大细胞类胰蛋白酶的酶组织化学技术，采用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体，（ＡＡ１、ＡＡ３和ＡＡ５）通过间接免疫过的经物酶技术对绵羊肥大细胞的组织化学特性进行研究，酶组织化学技术及免疫组织化学技术均首次证实了绵羊肥大细胞颗粒中含有类胰蛋白酶，且该酶可作为绵羊肥大细胞的特异性标志，对于绵羊肥大细胞的常规组织化学染色，Ｃａｒｎｏｙ液及中性缓门     

Listeria monocytogenes induces mast cell extracellular traps

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when β-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.     

Mast cell infiltrates in vulvodynia represent secondary and idiopathic mast cell hyperplasias

Mast cell infiltrates in tissues of vulvodynia are common, but they have not been characterized for criteria of neoplastic mast cell disease or correlated with patient's concomitant diseases associated with increased mast cells. Formalin‐fixed specimens of 35 patients with vulvodynia were evaluated immunohistochemically with antibodies to CD 3,4,8,20,117c and human mast cell tryptase, and for WHO‐criteria of neoplastic mastocytosis (>25% spindled mast cell, CD25 expression, point mutations of the c‐kit gene (D816V), and chronically elevated serum tryptase levels). Only 20/35 specimens showed a T‐lymphocyte dominant inflammatory infiltrate on HE‐stained sections, but all showed mast cells. 4/35 biopsies showed <10 mast cells/mm², 15/35 specimens 40–60 mast cells/mm² and 16/35 specimens >60 mast cells/mm² (average 80/mm²). Control tissue contained typically <10 mast cells/mm². Spindling, CD25‐expression, c‐kit gene mutations, or increased serum tryptase levels were not detected. 26/35 (74%) patients had concomitant autoimmune diseases, psoriasis, atopy, various allergies, preceding infections. Independent of the subtype of vulvodynia, the majority of mast cell rich biopsies with >40 mast cells/mm² were classified as a secondary mast cell disorder reflecting an activated immune system in 75% of vulvodynia patients. Patients with increased mast cells may benefit from medical therapy targeting mast cells.     

Development of a new,more sensitive immunoassay for human tryptase: Use in systemic anaphylaxis

Tryptase, a neutral protease, is selectively concentrated in the secretory granules of human mast cells, and its release into the circulation serves as a clinical marker of mast cell activation. The current study describes a new, more sensitive ELISA utilizing a newly developed, mouse monoclonal IgG1 antibody for capture called B12 and capable of detecting tryptase in normal plasma and serum. The greater sensitivity of the new immunoassay results in part from a greater portion of tryptase being detected. Mean levels of tryptase in serum from normal subjects from Richmond, Virginia (4.9 ng/ml;n=56), Munich, Germany (3.8 ng/ml;n=19), and Amersfoort, The Netherlands (1.9 ng/ml;n=8) were as indicated. In 62 subjects with ongoing allergic rhinitis, tryptase levels were no different in serum than for 19 normal controls, indicating that local mast cell activation is not necessarily reflected in the circulation. In 61 subjects sensitive to honey bee or yellow jacket venom by history, the 17 destined to have a severe, hypotensive response to a sting challenge had higher levels of tryptase at baseline than mild reactors, nonreactors, and controls, suggesting that baseline levels of tryptase may predict the severity of the clinical response to allergen in sensitive subjects.     

Mastocytosis and related disorders

Mastocytosis represents a heterogeneous group of disorders characterized by an abnormal accumulation of mast cells in one or more organ systems. Mastocytosis is further divided into different subtypes according to the sites of involvement, laboratory findings, and degree of organ impairment. Cutaneous mastocytosis is diagnosed in the presence of skin involvement and absence of extracutaneous disease, and is most commonly seen in the pediatric population. Systemic mastocytosis, the disease form most commonly seen in adults, is characterized by the presence of multifocal, compact (dense) mast cell aggregates in the bone marrow or other extracutaneous organs. The mast cells may display atypical, often spindle-shape morphology and/or aberrant CD2 and/or CD25 expression. Elevation of serum tryptase and/or presence of KIT D816V mutation are other common findings. Systemic mastocytosis is further divided into different subtypes based on a combination of clinical features and laboratory findings. Recent studies have indicated that CD30 is frequently expressed in aggressive systemic mastocytosis and mast cell leukemia but infrequently in indolent systemic mastocytosis, and may be a useful marker for distinguishing these subtypes of systemic mastocytosis from one another. A group of related myeloid disorders, collectively termed myelomastocytic overlap syndromes, may pose diagnostic difficulty because of their significant clinical and pathologic overlap with systemic mastocytosis, and these will also be discussed in this review.     

肺动脉高压大鼠T型和TC型肥大细胞的改变

目的:建立SD大鼠高肺血流并用野百合碱注射所致肺动脉高压模型,观察肺组织中肥大细胞及肥大细胞两种亚型的变化,探讨肥大细胞在肺动脉高压中的机制。方法:大鼠腹主动脉-下腔静脉造瘘,术后7天一次性腹腔注射野百合碱60mg/kg,制造肺动脉高压模型,35天后测定肺动脉压,取肺组织进行免疫组织化学染色,观察肥大细胞的变化,HE染色观察肺组织一般病理变化。结果:HE染色显示肺组织中动脉管壁明显增粗,有新生血管形成。平均肺动脉压力模型组较对照组升高(P<0.05);右心室/(左心室+室间隔)模型组较对照组增加(P<0.05),肥大细胞总数及类糜蛋白酶阳性的肥大细胞模型组较对照组增多(P<0.05)。结论:腹主动脉-下腔静脉造瘘合并野百合碱腹腔注射成功的建立了肺动脉高压的动物模型,肥大细胞在肺组织的聚集以及活化、肥大细胞亚型的改变可能是肺动脉高压形成的机制之一。     

Human mast cells express functional TrkA and are a source of nerve growth factor

Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions. Despite reports that rodent mast cells proliferate in the presence of nerve growth factor (NGF), human mast cells reportedly do not respond to this factor. To determine if human mast cells express the NGF receptors, TrkA tyrosine receptor and the low affinity NGF receptor (LNGFR), we first analyzed the mRNA expression by RT-PCR of TrkA and LNGFR in a human mast cell line (HMC-1) and in human mast cells cultured in the presence of stem cell factor. Both HMC-1 and cultured human mast cells were found to express TrkA but not LNGFR. TrkA protein was demonstrated by Western blot analysis of HMC-1 lysates. Using flow cytometric analysis and mast cell tryptase as a mast cell marker, both HMC-1 cells and cultured human mast cells were shown to co-express tryptase and TrkA. Treatment of mast cells with NGF resulted in phosphorylation of TrkA on tyrosine residues as detected by immunoblotting with an antiphosphotyrosine antibody. Furthermore, NGF induced the immediate early gene c-fos in HMC-1 cells. HMC-1 cells and cultured human mast cells were also found to express NGF mRNA, and conditioned medium from HMC-1 cells stimulated neurite outgrowth from chicken embryonic sensory ganglia in culture. This effect was blocked by anti-NGF. Thus, mast cells express functional TrkA and synthesize NGF, suggesting a mechanism by which NGF may act as an autocrine factor for human mast cells, and by which mast cells and nerves may interact.     

Serum total tryptase levels are increased in patients with active chronic urticaria

Background We have demonstrated previously mast cell histamine release upon incubation with chronic urticaria (CU) sera, presumably by degranulation. Objective To explore total and mature tryptase in order to assess whether any increase in total tryptase levels is due in part to mast cell degranulation or to mast cell burden. We also wanted to explore differences between the autoimmune groups called idiopathic (serum unable to activate basophils), and to correlate total and mature tryptase levels with different urticaria features. Methods We measured total and mature tryptase serum levels in 81 CU patients, 16 atopic donors and 21 healthy control sera. We assessed autoimmunity by measuring the CD63 expression in normal basophil donors upon incubation with CU sera. Results We found significantly higher levels of total tryptase in the sera of CU patients (6.6 ±4.1 μg/L) than in sera from healthy non‐atopic subjects (4.4 ±2.8 μg/L) and from atopic subjects (4.5 ±1.7 μg/L). Mature tryptase levels were undetectable (<1 ng/mL). Total tryptase levels in the autoimmune urticaria group were significantly higher (9.8 ±5.4 μg/L) than the idiopathic urticaria group (4.4 ±2.2 μg/L). A significant difference in total tryptase was found between symptomatic patients (7.3 ±4.1 μg/L) compared with asymptomatic ones (5.7 ±4.1 μg/L) at the time of venesection. No difference was found in mature tryptase levels either. Conclusion Total elevated tryptase levels are not accompanied by an elevated mature tryptase levels, as might be expected if the serum levels reflected mast cell degranulation. Cite this as: M. Ferrer, J. M. Nuñez‐Córdoba, E. Luquin, C. E. Grattan, J. M. De la Borbolla, M. L. Sanz, L. B. Schwartz, Clinical & Experimental Allergy, 2010 (40) 1760–1766.     

Tryptase in type I hypersensitivity

Tryptase is currently the main mast cell biomarker available in medical practice. Tryptase determination is a quantitative test performed in serum or plasma for the diagnosis, stratification, and follow-up of mast cell–related conditions. The continuous secretion of monomeric α and β protryptases forms the baseline tryptase level. Transient, activation-induced release of tryptase is known as acute tryptase. Because mast cells are tissue-resident cells, the detection of an acute tryptase release in the bloodstream is protracted, with a delay of 15 to 20 minutes after the onset of symptoms and a peak at approximately 1 hour. Constitutive release of tryptase is a marker of mast cell number and activity status, whereas transient release of mature tryptase is a marker of mast cell degranulation. Although consensual as a concept, the application of this statement in clinical practice has only been clarified since 2020. For baseline tryptase to be used as a biomarker, reference values need to be established. In contrast, defining a transient increase using acute tryptase can only be achieved as a function of the baseline status.     

Tryptase and histamine release during aspirin-induced respiratory reactions

The involvement of mast cells in the pathogenesis of aspirin (ASA)-induced respiratory reactions was investigated by measuring serum levels of tryptase, a neutral protease that is a specific marker of mast cell activation. ASA challenges were performed in 17 ASA-sensitive patients with asthma and rhinosinusitis, and tryptase and histamine levels were measured in their venous blood samples. In three subjects who experienced moderate to severe respiratory reactions extending to the skin and/or gastrointestinal tract, marked elevations of tryptase levels in postreaction serum samples (peak levels, 51.9 and 40.0 ng/ml) were discovered in two of these three subjects, and a small elevation of tryptase occurred in the serum of the third subject (3.1 ng/ml peak). Plasma histamine levels in postreaction samples were significantly elevated over baseline values in all three subjects (delta mean plasma histamine, 238 pg/ml versus 56 pg/ml for the remaining 14 subjects; p less than 0.04). In the remaining 14 subjects, who experienced similar respiratory reactions without extrapulmonary symptoms during aspirin challenge, changes in tryptase and histamine levels were not observed.     

Is unrecognized anaphylaxis a cause of sudden unexpected death?

Background: Serum tryptase levels reflect mast cell activation and correlate with anaphylactic reactions. Elevated post-mortem serum tryptase levels have been found in witnessed fatal anaphylaxis. Objective: This study was designed to examine whether or not unwitnessed anaphylaxis may be a hitherto unrecognized cause of sudden unexplained death. Methods: Mast cell tryptase was measured by immunoassay in 68 post-mortem sera remaining from a previous study which reported elevated venom-specific IgE antibodies in 22 (23%) of 94 victims of sudden unexpected death. Autopsies were performed in all cases. The cause of death was independently reported by pathologists unfamiliar with the nature of this study. Results: Serum tryptase levels were elevated (> 10ng/mL) in nine of 68 cases. The levels could not be predicted from the clinical circumstances surrounding death. Sera from four individuals contained both elevated tryptase and previously reported elevated venom-specific IgE. Conlusions: We conclude that mast cell activation may accompany up to 13% of sudden unexpected deaths in adults. Measurement of both tryptase and specific IgE antibody levels in post-mortem sera from persons experiencing sudden, unexpected death may identify a small subset of cases due to clinically unrecognized fatal anaphylaxis, including those due to insect stings.     

Quantification of mast cells in the uterine cervix of women infected with human immunodeficiency virus

The objective of the study was to identify mastocytosis in the chorionic epithelium of the uterine cervix in HIV-infected and non–HIV-infected women in autopsy specimens using histochemistry and immunohistochemistry techniques. Sixteen cervical tissue specimens were collected, of which 10 (62.50%) were from HIV-infected women. Histochemical and immunohistochemical techniques were used to evaluate mast cell density using Giemsa stain and anti-mast cell tryptase and anti-mast cell chymase antibodies, respectively. The study of the sheets and counting of mast cells with blue (Giemsa) or brown staining (anti-mast cell tryptase or chymase antibodies) were performed by 3 examiners, and 10 consecutive fields were examined under a light microscope at 400× magnification. A significant difference was found in mast cell density in the chorionic epithelium of the cervix in HIV-infected compared with non–HIV-infected women. The present study may contribute to the characterization of genital mucosa abnormalities and help better understand the potential role of mast cells in HIV-infected women.     

Increased mast cell tryptase in sudden infant death — anaphylaxis,hypoxia or artefact?

BACKGROUND: Increased concentrations of mast cell tryptase in post mortem blood have frequently been observed in sudden infant deaths but the cause of this has not yet been clarified. OBJECTIVE: The aim was to evaluate factors (immunological, morphological and anamnestic data) behind the observed increase in mast cell tryptase in sudden infant deaths with elevated tryptase. METHODS: Mast cell tryptase and total immunoglobulin (Ig) E were measured in post mortem sera from 44 infants younger than 1.5 years. Radioallergosorbent tests were performed for possible allergens (mixture for relevant food allergens, Phadiatop and latex). IgG subclasses, IgM, and complement factors (C3, C4 and factor B) were measured with radial immunodiffusion. Mast cells, labelled with antibodies against mast cell tryptase, were counted in the lungs and heart. The circumstances of death and medical history of the deceased infant and family were obtained through police and hospital records. RESULTS: In 40% of the SIDS cases tryptase was elevated (>10 microg/L). Total IgE in serum was increased in 33% compared with clinical reference values but showed no association with mast cell tryptase. RAST tests were positive in three cases. In one of these cases both tryptase and total IgE were elevated. The only variable that was associated with high tryptase values was prone position at death (P < or = 0.05 ). Allergy or asthma in the family were alleged in 50% of the cases, but was not associated with elevated tryptase or IgE. Children with elevated total IgE also displayed high concentrations of IgG1 and IgG2. Infants who died in the spring had significantly higher IgE than the others (P < or = 0.05). CONCLUSION: The results do not support the hypothesis that the elevated tryptase concentrations in sudden infant death are caused by allergy. The association between prone position at death and elevated tryptase could hypothetically be explained by mast cell degranulation due to, for example, a hypoxic stimulus in these infants.     

Effects of interleukin (IL)-13 on immediate-early response gene expression,phenotype and differentiation of human mast cells. Comparison with IL-4

The recently cloned interleukin 13 (IL-13) shares most investigated biological activities on B lymphocytes and monocytes with IL-4. In this study we investigated the potential role of IL-13 in regulating human mast cell activities. The effects of IL-13 on the expression of an immediate-early response gene (c-fos), proliferation, expression of mast cell-associated cell surface antigen (CD54 and Kit), and in vitro differentiation of human mast cells, were investigated. We compared the effect of IL-13 with that of IL-4. Both IL-13 and IL-4 induced expression of c-fos in cells from the human mast cell line HMC-1. This indicates that mast cells express functional receptors for IL-13. IL-13 and IL-4 decreased the proliferation rate of HMC-1 cells. However, IL-13 was less potent than IL-4. Human mast cells constitutively express the adhesion molecule ICAM-1 (CD54) and the receptor for stem cell factor (Kit) (CD117). The expression of CD54 was increased after treatment with IL-13 or IL-4, whereas the expression of Kit was decreased. Also in this action IL-4 was more potent than IL-13. By culturing mononuclear cells from cord blood in the presence of stem cell factor there is a differentiation of tryptase-positive mast cells in the cultures. This process was inhibited when IL-4 was present. In contrast, IL-13 did not affect the expression of tryptase during differentiation of stem cell factor dependent cord blood-derived mast cells. Taken together, these findings indicate that IL-13 has regulatory effects on human mast cells. The effect overlaps with but is also different from that of IL-4.     

Tryptase as severity marker in drug provocation tests

BACKGROUND: In the absence of objective symptoms, it is difficult to assess an adverse reaction during drug provocation testing. We evaluated the value of serum tryptase levels to distinguish between positive, negative and, even more important, so-called 'hysterical' reactions (conversion symptoms). The latter are occasionally observed in drug provocation tests when the patient experiences ambiguous subjective symptoms. METHODS: In a prospective single-center study, 303 patients underwent 785 drug provocation tests. Blood was taken for tryptase measurement on each test day before and after drug challenge, and the changes in serum tryptase levels in patients with no reactions were compared with those who experienced immediate reactions to a drug. RESULTS: Thirty-four of 785 drug provocations were clinically judged as being positive. Despite objective symptoms, median serum tryptase values in the afternoon were even lower than baseline levels. However, this decrease was not statistically significant. In the 751 patients suffering no objective reactions, the median values of post-testing tryptase values were statistically significantly decreased as compared with pretesting values. CONCLUSIONS: The measurement of serum tryptase levels does not appear to be helpful to differentiate mild allergic or nonallergic reactions from 'hysterical' ones. The milder decrease in the group with objective drug reactions might indicate slight mast cell activation in some patients. More severe clinical drug reactions led to stronger mast cell degranulation. Mild reactions did not increase the tryptase levels consistently.     

肥大细胞类胰蛋白酶与支气管哮喘

支气管哮喘 (以下简称哮喘 )一直被认为与I型变态反应密切相关 ,而肥大细胞是介导I型变态反应的重要成分之一。肥大细胞的活化信号主要来自变应原与IgE的桥联 ,但神经肽及其它因素也能使肥大细胞活化。活化肥大细胞脱颗粒 ,释放出颗粒中预先合成和新合成的各种介质 ,如类胰蛋白酶、类糜蛋白酶、组胺、肝素等。这些介质作用于靶分子、靶细胞和靶组织 ,引起哮喘在病理、病理生理等方面的变化。目前普遍认为哮喘是一种复杂的慢性呼吸道炎症 ,其主要特征为对特异性和非特异性刺激物诱发的气道高反应性、炎症细胞浸润及气道重塑[1,2 ,2 1] 。虽…     

Tryptase阳性细胞在移植后不同时间段排斥肾中的分布

目的　了解Tryptase阳性肥大细胞在肾移植后不同时间段的排斥肾组织中的分布规律。方法　采用抗Tryptase单抗免疫组化染色法与甲苯胺蓝特殊染色方法 ,对移植后 8d～ 7a手术切除的排斥肾进行了染色。结果　在移植后的各个时间段的排斥肾中均见到较正常肾明显偏多的肥大细胞 ,且随着移植时间的延长呈增长的趋势。其中移植后 7a以上组的肥大细胞明显高于 1～ 2a组和 5～ 6a组 (P <0 .0 1)。甲苯胺蓝染色结果与Tryptase免疫组化结果相一致 ,但Tryptase免疫组化方法更为敏感。结论　肥大细胞可能参与了肾移植的排斥反应 ,此反应可能导致肾组织的纤维化变性