Laboratory Assessment of Patients with Systemic Mastocytosis

加塔蛋白是谱系限制性转录因子，主要在发育期间调节基因表达的组织特异性模式。加塔-2似乎调节早期肥大细胞基因表达，并已显示在模型系统中正调节肥大细胞特性。加塔-2缺乏是人类受试者中的一种多变的疾病，具有显著可变的临床表现。与肥大细胞区室相关，许多加塔-2缺陷受试者具有髓系异常，包括骨髓细胞减少、血细胞减少和髓系肿瘤。鉴于加塔-2破坏对肥大细胞影响的现有数据仅限于非人类或体外研究，我们研究了杂合功能丧失型GATA 2突变对临床过敏、培养的原代肥大细胞生长、形态和GATA 2缺乏患者活化的影响。 回顾性研究显示，在加塔-2缺陷受试者中，与IgE介导的对食物、环境过敏原、药物和/或叮咬昆虫的速发型超敏反应一致的记录临床症状的总体患病率较低（21%; 6/28）。与这一发现相一致的是，没有加塔-2缺陷受试者被处方肾上腺素自动注射器，只有4名（14%）被处方第二代抗组胺药、白三烯调节剂或肥大细胞稳定剂。所有加塔-2缺陷受试者和27名健康志愿者（对照组）接受了一组常见环境过敏原和食物过敏原的皮肤点刺试验（SPT）。相对于加塔-2缺陷受试者，对照受试者表现出更高的致敏率和更多的多敏性。加塔-2缺陷受试者的SPT反应与血清中特异性IgE水平不一致，尽管总IgE水平较低（中位IgE为5.9 kU/L），但4例受试者可检测到抗原特异性IgE，其中3例对3种或4种过敏原致敏。加塔-2缺陷受试者对肥大细胞介质组胺的反应没有受损，如通过风团面积量化的，这表明观察到的SPT反应降低和临床过敏是肥大细胞内在的和抗原依赖的。 接下来，我们着手确定导致加塔-2缺陷型肥大细胞中抗原依赖性脱颗粒受损的机制，并将KIT和FcRI确定为候选分子;这些分子都有助于人类受试者中肥大细胞的活化和存活，并且已显示在许多系统中依赖于完整的加塔-2活性。流式细胞术分析显示，FcRI在加塔-2缺陷受试者的嗜碱性粒细胞（P <0.05）和浆细胞样树突状细胞（P <0.05）中的表达显著降低。 在皮肤活检标本中，加塔-2缺陷型肥大细胞的KIT表达同样减少，而肥大细胞数量与对照组的数量却矛盾地相当。尽管与对照组相比，加塔-2缺乏患者的骨髓肥大细胞在抽吸物中鉴定为CD 45 +KIT+ CD 34 SSC high细胞，KIT表达相当，但与特发性过敏反应受试者相比，这些细胞的数量实际上增加（P = 0.01），与惰性系统性肥大细胞增多症受试者的数量相当。与惰性系统性肥大细胞增多症一样，11例受试者中有3例骨髓肥大细胞计数增加与基础血清类胰蛋白酶（BST）水平升高（>11.4 ng/mL）相关。然而，检查骨髓中剩余的KIT+髓样细胞，发现表达减少。同样，在加塔-2缺陷患者的外周血中，发现需要KIT才能存活的CD 34+祖细胞明显减少。 进一步的研究表明，在成功培养的加塔-2缺陷型肥大细胞中，FcRI和KIT表达降低。成功培养的原代肥大细胞（来自5名加塔-2缺陷受试者和6对健康志愿者）用生物素化的IgE致敏，并使用链霉抗生物素蛋白交联FcRI。肥大细胞脱颗粒，作为衡量-氨基己糖苷酶释放，相对于控制值减少。干细胞因子（SCF）的加入增强了IgE介导的肥大细胞脱颗粒在对照组，但是，它并没有显着改变加塔-2缺乏肥大细胞脱颗粒。加塔-2缺陷型肥大细胞对IgE交联的反应以及加入SCF后，细胞内Ca 2+通量同样受损。这些数据与原发性加塔-2缺陷型肥大细胞对IgE和SCF的应答中的脱粒功能损害一致，这与我们已经确定的FcRI和KIT表达减少有关。 总之，加塔-2缺陷受试者存在IgE介导的肥大细胞活化受损和IgE介导的临床过敏性疾病减少的证据。这些发现是抗原依赖性的，与KIT和FcRI表达减少相关，并且不归因于肥大细胞中的数字问题或对肥大细胞介质组胺的反应受损。

GATA proteins are lineage-restricted transcription factors that regulate tissue-specific patterns of gene expression, principally during development. GATA-2 appears to regulate early mast cell gene expression and has been shown to positively regulate mast cell identity in model systems. GATA-2 deficiency is a protean disorder in human subjects with markedly variable clinical expressivity. Relevant to the mast cell compartment, many GATA-2deficient subjects have myeloid lineage abnormalities, including marrow hypocellularity, cytopenias, and myeloid neoplasms. Given that the data available on the effects of GATA-2 disruption in mast cells are limited to nonhuman or in vitro studies, we examined the effects of heterozygous loss-of-function GATA2 mutations on clinical allergy, cultured primary mast cell growth, morphology, and activation in patients with GATA2 deficiency. Retrospective investigation revealed that the overall prevalence of documented clinical symptoms consistent with IgE-mediated immediate hypersensitivity to foods, environmental allergens, medications, and/or stinging insects was low (21%; 6/28) in GATA-2deficient subjects. Consistent with this finding, none of the GATA-2deficient subjects had been prescribed epinephrine autoinjectors, and only 4 (14%) had been prescribed second-generation antihistamines, leukotriene modifiers, or mast cell stabilizers. All GATA-2deficient subjects and 27 unselected volunteers (control) underwent skin prick tests (SPTs) to a panel of common environmental allergens and food allergens. Control subjects demonstrated a greater rate of sensitization and a greater number were polysensitized relative to GATA-2deficient subjects. SPT responses in GATA-2deficient subjects were discordant with specific IgE levels in serum, where despite low total IgE levels (median IgE, 5.9 kU/L), 4 subjects had detectable antigen-specific IgE, 3 of whom were polysensitized to 3 or 4 allergens. GATA-2deficient subjects did not have impaired responses to the mast cell mediator histamine, as quantified by wheal area, suggesting the reduced SPT responses and clinical allergy observed were both mast cell intrinsic and antigen dependent. We next set out to identify mechanisms leading to impairment of antigen-dependent degranulation in GATA-2deficient mast cells and identified KIT and FcRI as candidates; these molecules both contribute to mast cell activation and survival in human subjects and have been shown to depend on intact GATA-2 activity in a number of systems. Flow cytometric analysis revealed that FcRI expression was significantly reduced in basophils (P < .05) and plasmacytoid dendritic cells (P < .05) from GATA-2deficient subjects. KIT expression was likewise reduced in GATA-2deficient mast cells in skin biopsy specimens, whereas mast cell numbers were paradoxically comparable with those of control subjects. Although bone marrow mast cells identified in aspirates as CD45+KIT+CD34SSChigh cells from patients with GATA-2 deficiency had comparable KIT expression when compared with those from control subjects with disease, these cells were actually increased (P = .01) in number compared with those in subjects with idiopathic anaphylaxis and comparable in number to subjects with indolent systemic mastocytosis. Like indolent systemic mastocytosis, increased bone marrow mast cell counts were associated with increased basal serum tryptase (BST) levels (>11.4 ng/mL) in 3 of 11 subjects. However, examining the remaining KIT+ myeloid cells in bone marrow, expression was found to be reduced. Likewise, significantly fewer CD34+ progenitor cells, which require KIT for survival, were identified in the periphery of GATA-2deficient patients. Further studies revealed that FcRI and KIT expression were reduced in successfully cultured GATA-2deficient mast cells. Successfully cultured primary mast cells (from 5 GATA-2deficient subjects and 6 paired healthy volunteers) were sensitized with biotinylated IgE, and FcRI was cross-linked using streptavidin. Mast cell degranulation, as measured by -hexosaminidase release, was reduced relative to control values. Stem cell factor (SCF) addition enhanced IgE-mediated mast cell degranulation in control subjects; however, it did not significantly alter GATA-2deficient mast cell degranulation. Intracellular Ca2+ flux was likewise impaired in GATA-2deficient mast cells in response to IgE cross-linking, as well as after the addition of SCF. These data are consistent with functional impairment in degranulation of primary GATA-2deficient mast cells in response to both IgE and SCF, which was associated with the reduced FcRI and KIT expression we have identified. In summary, GATA-2deficient subjects have evidence of impaired IgE-mediated mast cell activation and reduced IgE-mediated clinical allergic disease. These findings are antigen dependent and associated with reduced KIT and FcRI expression and are not attributable to a numeric problem in mast cells or an impaired response to the mast cell mediator histamine.