FcγRs在ITP中的表达及调控研究
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摘要
研究背景：
人Fcγ受体系统由活化性受体(FcγRⅠ,Ⅱa,Ⅲ)和抑制性受体(FcγRⅡb)两个家族组成,这两类作用相反的受体平衡一旦失调会导致许多自身免疫性疾病的发生。抗原-IgG免疫复合物(immune complex, IC)通过作用于免疫细胞上IgG恒定区Fcγ受体诱发多种免疫反应。根据RNA剪接异构体和蛋白结构不同,人类FcγR系统可分为FcγRⅠ、FcγRⅡ、FcγRⅢ三个亚群。FcγRⅠ又称CD64,为高亲和力的IgG受体,主要表达与单核细胞和巨噬细胞；FcγRⅢ即CD16,与单体IgG结合力较弱,但同IgG聚合体有中低度的亲和力,其主要表达于中性粒细胞、单核／巨噬细胞、自然杀伤细胞；FcγRⅡ又称CD32,按功能可进一步分为FcγRⅡa和FcγRⅡb两个亚型,为低到中亲和力的IgG受体,主要与聚合的IgG结合,表达于中性粒细胞、单核／巨噬细胞、树突状细胞(dendritic cells, DCs)、B细胞和血小板。按功能学分类,人FcγR系统可分为活化性FcγR和抑制性FcγR两个家族。FcγRⅡb为唯一抑制性的Fcγ受体,主要通过自身分子胞浆区偶联含有免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif, ITIM)传递抑制信号,在固有免疫和适应性免疫方面发挥重要负性调节作用；其余的FcγRⅠ、Ⅱa、Ⅲ均为活化性Fcγ受体,大都通过偶联含有免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activating motif, IT AM)的γ二聚体传递活化信号。Fcγ受体信号通路在多种自身免疫性疾病的发生及进展中发挥重要作用。多篇报道显示：在风湿性关节炎(rheumatoid arthritis, RA)、系统性红斑狼疮(systemic lupus erythematosus, SLE)、多发性硬化(multiple sclerosis, MS)自身免疫性溶血性贫血(autoimmune hemolytic anemia)等自身免疫性疾病中,往往可以发现活化性受体FcyRI的表达下调及抑制性受体FcyRIIb的表达上调,故活化性和抑制性Fcy受体失衡在自身免疫性疾病的发病过程中发挥重要作用。
原发性免疫性血小板减少症(primary immune thrombocytopenia, ITP),以往亦称为特发性血小板减少性紫癜(idiopathic thrombocytopenic purpura, ITP),是临床最为常见的出血性疾病,约占出血性疾病总数的30%。ITP临床表现为皮肤粘膜出血,月经过多,内脏出血,甚至颅内出血,严重影响人类健康。其发病机制主要为机体自身免疫耐受失衡,产生了针对血小板糖蛋白(glycoprotein, GP)特异性的自身抗体,大部分主要为针对GPⅡb/Ⅲa和／或GPⅠb/Ⅸ的IgG,该抗体与血小板膜自身GP抗原结合,通过作用于单核巨噬细胞Fcy受体(Fcγreceptor, FcyR)在网状内皮系统被巨噬细胞吞噬而过度清除。自身抗体的产生、激活、维持由表达FcγR的免疫细胞共同参与完成,是众多自身免疫性疾病的共同病理生理基础。除上述经典的抗体介导的血小板异常破坏途径外,多种细胞免疫机制异常,如Th1/Th2失衡、Th17亚群异常、细胞毒性T细胞(cytotoxic T lymphocyte, CTL)介导的血小板破坏、调节性T细胞数量或抑制功能异常等参与了ITP发病的异常病理生理过程,但上述异常的具体原因亟待明确。目前,对于ITP的治疗以糖皮质激素、脾切除、大剂量丙种球蛋白、免疫抑制剂等治疗为主,容易产生感染、骨髓抑制等副作用,约有1/3的患者对上述治疗无效,成为难治性ITP,常常迁延不愈,甚至危及生命。因此,深入了解ITP的发病机制,探讨阻断ITP特异发病途径并寻找新的药物作用靶点对展新的ITP的治疗策略有重要意义
ITP是最经典的一种Fcy受体介导的自身免疫性出血性疾病。做为FcγRs系统中唯一的抑制性受体,FcγRⅡb在ITP的免疫负性调节中发挥重要作用。Ashi最近报道显示：对清除幽门螺杆菌疗法有反应的ITP患者中,可以发现单核细胞抑制性受体FcγRⅡb的表达上调及活化性受体FcγRⅡa、FcyRⅠ的表达下调,而清除幽门螺杆菌疗法无反应的ITP患者中并不伴有该种变化,这提示幽门螺杆菌清除后可以通过恢复FcγRs受体平衡来缓解ITP病情。动物实验亦证实：过表达FcγRⅡa的转基因小鼠可显著加重抗体介导的血小板减少。此外,FcγRⅡa、Ⅱb亦参与抗体、补体介导的炎症反应：Shushakova研究显示C5a可直接上调肺泡巨噬细胞FcγRⅡb的表达。小鼠模型中,可溶性抗原特异的IgG可成功治疗ITP。大剂量丙种球蛋白静脉滴注(intravenous immunoglobulin, IVIg)已成功用于人类包括ITP在内的多种自身免疫性疾病中,且疗效机制研究发现IVI可上调抑制性FcγyRⅡb活性并下调激活性FcγRⅡa活性。口服大剂量地塞米松(high dose dexamethasone, HD-DXM)冲击疗法是当前皮质激素治疗ITP的研究热点,其疗效逐渐得到了广泛认可,尤其是治疗初治ITP患者。最近ITP国际工作组已将HD-DXM作为初诊ITP的标准一线疗法,但其疗效的具体机制尚不明确。初步研究证实：它可抑制抗体生成及减轻抗原抗体反应、改善毛细血管通透性、刺激骨髓造血及血小板向外周血的释放、恢复Th1/Th2平衡、增加Tregs数量等。迄今为止,尚未有系统的关于ITP患者与健康对照外周单核及B细胞上FcγRs表达及HD-DXM对ITP患者Fcγ受体系统影响的报道。
目的：
检测初诊活动性ITP患者及健康对照外周血FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表面分子表达及FcγRⅡ的亚型Ⅱa及Ⅱb mRNA及蛋白表达；研究HD-DXM治疗后ITP患者FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表达变化、FcγRIIa/IIb mRNA及蛋白表达变化；检测ITP患者治疗前后单核／巨噬细胞对致敏自身血小板吞噬能力变化及Th1/Th2细胞因子平衡变化；体外检测ITP患者单核细胞在DXM存在情况下培养时FcγRⅡa/Ⅱb mRNA表达变化,探讨HD-DXM影响FcγRs系统变化的可能机制。
材料和方法：
(1)外周血FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表面分子表达及FcγRⅡ的亚型Ⅱa及Ⅱb mRNA及蛋白表达检测部分,抽取23例初诊活动性ITP患者治疗前、治疗后2周及20例健康对照的外周血,分离血浆及单个核细胞。
(2)流式细胞仪染色法测定外周血FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表面分子表达；免疫磁珠阳性分选外周血CD14+单核细胞；实时定量聚合酶链反应(real-time reverse-transcription polymerase chain reaction, real-time RT-PCR)检测单核细胞FcγRⅡa、Ⅱb的mRNA表达；免疫沉淀(immunoprecipitation, IP)和western blotting检测FcγRⅡa、Ⅱb的蛋白表达；酶联免疫吸附实验(enzyme-linked immunoabsorbent assay, ELISA)检测ITP治疗前后及正常对照血浆中IFN-γ、IL-4的水平。
(3)体外实验部分抽取14例治疗前ITP患者外周血做细胞培养。免疫磁珠阳性分选外周血CD14+单核细胞,加入不同剂量的DXM培养16h, real-time RT-PCR检测FcγRⅡa、Ⅱb的mRNA表达变化；
(4)抽取10例健康对照、10例ITP患者治疗前及治疗2周后外周血,贴壁法分离外周血单核细胞并刺激,与CMFDA(5-chloromethylfluorescein diacetate)探针标记过的致敏自身血小板在37°C或4°C共培养,流式细胞仪检测血小板被自身单核／巨噬细胞吞噬情况,应用MFI37℃/MFI4℃做为吞噬指数以判定吞噬能力。
结果：
(1)流式测定的FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ表面分子表达高低以平均荧光强度表示(mean fluorescence intensity, MFI),结果显示：①初诊ITP患者单核细胞表面分子FcγRⅠ表达(504.6±58.6)显著高于正常对照(309.8±52.9；P<0.001)；经HD-DXM治疗后2周,FcγRⅠ表达较治疗前显著降低(317.5±69.4；P<0.001)：FcγRⅠ在治疗后与正常对照组无显著差异(P>0.05)。②单核细胞表面分子FcγRⅡ(Ⅱa+Ⅱb)、Ⅲ在ITP组治疗前(FcγRⅡ82.6±160; FcγRⅢ9.9±4.6)、治疗后(FcγRⅡ75.4±18.9; FcγRⅢ8.9±3.0)及正常对照组(FcγRⅡ83.2±16.3; FcγRⅢ9.3±3.2)之间无显著差异(FcγRⅡP=0.947; FcγRⅢP=0.694; ANOVA).③B细胞表面分子FcγRⅡ(Ⅱa+Ⅱb)在ITP组治疗前(82.3±13.0)治疗后(82.7±11.8)及正常对照组(83.2±12.1)之间差异无显著性(P=0.954；ANOVA).
(2)单核细胞FcγRⅡa和FcγRⅡb mRNA和蛋白表达：①real-time RT-PCR结果：我们应用FcγRⅡa和FcγRⅡb mRNA的比率变化表示HD-DXM对FcγRⅡa和FcγRⅡb mRNA表达的影响。初诊ITP患者FcγRⅡa/Ⅱb mRNA的比率值为5.88±5.12,与正常对照组存在显著差异(0.31±0.29；P<0.001)；接受HD-DXM治疗后FcγRⅡa/Ⅱb mRNA的比率值(0.61±0.86)较治疗前显著降低(P<0.001)。②western blotting结果：应用相对光密度值高低来代表FcγRⅡa和FcγRⅡb蛋白表达量。初诊ITP患者单核细胞FcγRⅡa表达量接受HD-DXM治疗后显著降低(2.05±0.81 vs 1.07±0.96, P< 0.001, Student(?) test),并伴有FcγRⅡb表达的显著升高(0.13±0.31 vs 1.23±1.25, P< 0.001, Whitney U test)
(3)ITP患者HD-DXM治疗后单核／巨噬细胞FcγR介导的吞噬致敏血小板能力的改变。应用CMFDA做探针标记血小板,初诊ITP患者吞噬指数为(MFI ratio:3.5±0.7),显著高于正常对照(2.2±0.5；P<0.05)。HD-DXM治疗后吞噬指数降为(MFI ratio:2.6±1.0),较治疗前有显著差异(P<0.05)。
(4)ITP患者治疗前后血浆IFN-γ、IL-4水平变化。初诊ITP患者治疗前Thl系列细胞因子IFN-γ较正常对照显著增高(62.7±13.0 vs 37.4±4.8 pg/ml, P< 0.001)和IL-4的显著降低(8.9±2.7 vs 18.3±4.7 pg/ml, P< 0.001). HD-DXM治疗后,IFN-γ水平较治疗前显著降低并伴有IL-4的显著升高(IFN-γ:40.3±11.0 pg/ml, P< 0.001; IL-4:18.5±4.2 pg/ml, P< 0.001).
(5)DXM对ITP患者体外培养单核细胞FcγRⅡa、Ⅱb mRNA表达的影响。体外培养ITP患者的单核细胞在加入DXM后出现FcγRⅡa、Ⅱb mRNA表达的同时上调,这种效应在DXM为1μM时最明显。在DXM为0.5μM和1μM时,FcγRⅡb mRNA表达上调的幅度要高于FcγRⅡa的幅度(P<0.05)。
结论：
(1)初诊活动性ITP患者外周血单核细胞呈现活化性FcγRⅠ、FcγRⅡa的表达升高,及抑制性FcγRⅡb mRNA及蛋白表达降低；
(2)ITP患者经HD-DXM治疗后2周,单核细胞出现活化性FcγRL、FcγRIIa的表达降低,及抑制性FcγRⅡb mRNA及蛋白表达升高,FcγRⅡa/Ⅱb mRNA比率恢复正常；
(3)ITP患者治疗后伴随着单核细胞活化性和抑制性FcγRs受体平衡恢复,单核／巨噬细胞FcγR介导的自体致敏血小板吞噬能力降低；
(4)ITP患者呈现Th1细胞极化状态,经HD-DXM治疗后,Th1/Th2失衡状态得到纠正；
(5)DXM在体外可同时上调FcγRⅡa、Ⅱb mRNA表达,且FcγRⅡb上调幅度更高；
意义：本研究揭示HD-DXM治疗可以通过上调抑制性FcγRⅡb恢复单核细胞FcγRs系统平衡,从而缓解ITP患者症状,这提示了HD-DXM在ITP中发挥疗效的一种新的可能机制。
Background
The human Fcγreceptor (FcyR) system is composed of two opposing families, the activating FcγRsⅠ,Ⅱa,Ⅲand the inhibitory FcγRⅡb, the balance of which determines the magnitude of the inflammatory response. FcγRⅡb is the only FcyR that has an inhibitory function, and is expressed by a variety of immune cells, including B cells, monocytes, macrophages, dendritic cells (DCs), mast cells and basophils. FcγRⅡb can decrease antibody production by raising the activation threshold of B cells if cross-linked to the B cell receptor (BCR). It inhibits the function of activating FcyRs, such as phagocytosis and pro-inflammatory cytokine release by monocytes and DCs. The disturbed balance of the activating and inhibitory FcyRs has been found in many autoimmne diseases such as system lupus erythematosus (SLE) and rheumatoid arthritis (RA). Therapeutic response in RA and SLE was often associated with the restoration of the balance between activating and inhibitory FcγRs.
Primary immune thrombocytopenia (ITP), which is also named as idiopathic thrombocytopenia previously, is one of the most common hemorrhagic disorders, and accounts for about 30 percent of all bleeding disorders. ITP is often manifested by skin and mucosal hemorrhage, menorrhagia, visceral bleeding, and even in some case shown as intracranial hemorrhage, having a serious effect on human health. ITP is an immune-mediated bleeding disorder in which platelets are opsonized by autoantibodies directed against platelet surface membrane glycoproteins (GPⅡb/Ⅲa, Ib/IX), and prematurely cleared by FcyR-bearing macrohpages in the reticuloendothelial system. The production, activation and maintenance of autoantibodies are completed by a variety of immune cells which express FcγRs. Previous studies have shown that leukocyte IgG-FcγR, such as FcγRⅡandⅢ, played an important role in the phagocytosis of autoantibody coated platelets. McKenzie et al. demonstrated that human FcγRIIa was necessary for antibody-mediated platelet clearance by using a murine model transgenic for human FcyRⅡa and lacking murine FcγRⅠand FcγRⅢ. Samuelsson et al. showed that decreased expression of murine inhibitory FcyRⅡb was associated with increased platelet destruction. In a multivariate logistic regression analysis of data from 60 children with ITP, the presence of the FcyRⅡbT232 variant predicted a chronic disease course. Recently, it was reported that Helicobacter pylori (H. pylori) eradication could induce a significant platelet count increase in a subset of H. pylori infected ITP patients, and that effect was shown to be mediated by shifting monocyte Fcγreceptor balance toward the inhibitory FcyRⅡb. The pathophysiology of ITP is heterogeneous and complex. Besides the classical mechanism of GP-specific autoantibody-mediated platelet destruction, several cellular defects about immune modulation, such as the disturbed balance of T helper 1 (Th1)/Th2, the decreased number and dysfunction of regulatory T cells, the elevated number of Th17 cells, and cytotoxicity T lymphocytes (CTLs)-mediated platelet destruction, have been described. The precise reason for these abnormalities remains being elucidated.
The therapeutic regimens for ITP include glucorticosteroids (GCs), intravenous immunoglobulin (IVIG), splenectomy, thrombopoietin receptor agonists, and other immunosuppressive drugs. IVIG could induce a beneficial response in ITP patients via up-regulating FcγRⅡb on monocytes/macrophages, and the same results were showed in mouse models of ITP. High-dose dexamethasone (HD-DXM) have been widely recognized as the first-line therapy for patients who need to be managed. However, the effects of HD-DXM on FcγR regulation remain unelucidated in ITP patients. In the present study, the difference in the expression of FcγRs in ITP patients compared with normal controls, and the reguation of FcγRs by HD-DXM were investigated.
Objective
To compare the surface molecule expression of FcγRⅠ/CD64,Ⅱ(Ⅱa+Ⅱb)/CD32,Ⅲ/CD16 on peripheral blood monocytes and the expression of FcγRⅡ/CD32 on B cells, as well as the mRNA and protein expression of FcγRIIa and FcyRⅡb on mnoocytes in newly diagnosed ITP patients and healthy controls; To investigate the effect of HD-DXM on FcγRⅠ,Ⅱ(Ⅱa,Ⅱb,Ⅱa+Ⅱb, respectively),Ⅲregulation and the phagocytic ability change of monocytes/macrophages in ITP patients; To determine the Th1/Th2 cytokine change in plasma of ITP patients after HD-DXM administration; To investigate the effect of DXM on FcγRⅡa,Ⅱb mRNA expression in in vitro cultured monocytes from newly diagnosed ITP patients.
Materials and Methods
(1) For surface molecule expression of FcγRⅠ,Ⅱ,Ⅲon monocytes and the expression of FcγRⅡ/CD32 on B cells,23 newly diagnosed ITP patients and 20 healthy controls were enrolled. In vitro monocyte culture was performed in another 14 newly-diagnosed ITP patients before HD-DXM treatment, and samples from 10 of them were also used for assays for the phagocytic capacity of monocyte-derived macrophages.
(2) The surface molecule expression of FcγRⅠ/CD64,Ⅱ(Ⅱa+Ⅱb)/CD32,Ⅲ/CD16 on peripheral blood monocytes and the expression of FcγRⅡ/CD32 on B cells were determined by flow cytometry. CD14+monocytes from peripheral blood mononuclear cells (PBMCs) were positively selected by immunomagnetic beads. mRNA expression of FcγRⅡa,Ⅱb was quantified on sorted monocytes by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). Immunoprecipitation (IP) and western blotting were used to determine the protein expression of FcγRⅡa,Ⅱb. Plasma levels of IFN-γand IL-4 were determined by enzyme-linked immunoabsorbent assay (ELISA).
(3) Monocytes from additional 14 newly-diagnosed ITP patients before treatment were adjusted to 1 * 106/ml in RPMI-1640 culture medium supplemented with 10%heat-inactivated human pooled AB serum at a denstity of 2 * 106 cells/well in a 6-well culture plate and incubated in humidified air in 5% CO2 at 37℃in the presence of different concentration of DXM. the monocyte-derived macrophages were collected for real-time PCR analysis of FcγRⅡa/Ⅱb mRNA expression.
(4) In vitro phagocytosis of IgG-opsonized platelets by macrophages was carried out in another 10 newly-diagnosed ITP patients. Platelets were labeled with 5-chloromethylfluorescein diacetate (CMFDA) and oposonized with 5μg murine IgG2a anti-human major histocompatibility complex (MHC) class I monoclonal antibody (W6/32). Monocytes were activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), incubated with opsonized CMFDA-labeled platelets, and Intracellular FL1 GM-Green platelet fluorescence in the nucleated events was determined by flow cytometry. The phagocytic index was calculated as the MFI obtained at 37℃divided by the MFI at 0℃.
Results
(1)①Quantification of FcγRⅠexpression on monocytes showed a significantly higher mean fluorescence intensity (MFI) in ITP patients (504.6±58.6) compared with normal controls (309.8±52.9, P< 0.001), and after 4-day HD-DXM therapy, FcγRⅠexpression showed a significant decrease (317.5±69.4, P< 0.001) compared with the levels before treatment.②〧FcγRⅡ(Ⅱa+Ⅱ) expression on monocytes from ITP patients showed a slight increase after HD-DXM therapy compared with the levels before treatment (82.6±16.0 vs.74.5±8.9), but this increase did not get statistically significant difference. Also there was no difference between ITP group and control individuals (83.2±16.3, P=0.947, ANOVA). After 4-day HD-DXM administration, monocytes from 17 patients (73.9%) showed increased FcyRII expression, and 6 patients (26.1%) had decreased FcγRⅡexpression. There were no significant changes in FcγRⅢexpression on monocytes detected by FACS with anti-FcγRⅢmAb after HD-DXM administration, and no difference was found among patients before, after therapy and the normal control group (9.9±4.6, 8.9±3.0, and 9.3±3.2, respectively; P= 0.694, ANOVA).③The FcγR expression on B cells was also determined by flow cytometry in ITP patients and controls. As shown in Fig.2A, B, the expression of FcγRⅡon B cells in ITP patients prior to HD-DXM administration was similar to normal controls (82.0±13.0 vs.83.2±12.1), and HD-DXM treatment had no effect on B cell FcγRⅡexpression (82.7±11.8,p=0.954, ANOVA).
(2)①Results showed that the ratio of FcγRⅡa/Ⅱb mRNA expression on monocytes was significantly higher in untreated ITP patients than normal controls (5.88±5.12 vs.0.31±0.29, P< 0.001). After HD-DXM therapy, the ratio of FcyRIIa/IIb mRNA decreased significantly (0.61±0.86, P< 0.001) in comparison with the level before treatment.②The patients after therapy had a marked downregulation of FcyRIIa protein expression on monocytes compared with the levels before treatment (2.05±0.81 vs.1.07±0.96, P< 0.001, Student's t test). At the same time, the decreased FcγRⅡa protein levels were associated the upregulated FcyRIIb levels. The relative densitometry of monocyte FcyRIIb in ITP group was 0.13±0.31, which increased significantly after HD-DXM therapy (1.23±1.25, P< 0.001, Mann-Whitney U test).
(3) Monocytes from untreated ITP patients demonstrated higher phagocytic capacity compared with healthy controls (MFI ratio:3.5±0.7 vs.2.2±0.5, P< 0.05). After HD-DXM therapy, phagocytic capacity of monocytes decreased significantly (2.6±1.0, P< 0.05).
(4) significantly higher levels of plasma IFN-γand lower IL-4 were found in untreated patients compared with healthy controls (IFN-γ:62.7±13.0 vs.37.4±4.8 pg/mL, P< 0.001; and IL-4:8.9±2.7 vs.18.3±4.7 pg/mL, P< 0.001, respectively). Plasma IFN-y levels in untreated patients decreased significantly after HD-DXM administration (40.3±11.0 pg/mL, P< 0.001), while post-treatment levels of IL-4 increased significantly compared with the levels before treatment (18.5±4.2pg/mL,P< 0.001).
(5) DXM could increase the mRNA expression of both FcyγRⅡa and FcγRⅡb on monocytes, with FcγRⅡb at higher amplitudes. At the concentration of 0.5 and 1μM, effect of DXM on the regulation of FcyRⅡa and FcγRⅡb (with FcγRⅡb at higher amplitudes) got statistical significance.
Conclusions
(1) Expression of FcγRⅠand FcγRⅡa on monocytes in newly diagonosed ITP patients was significantly higher compared with healthy controls. On the contrary, mRNA and protein expression of the inhibitory FcγRⅡb were significantly lower in ITP patients compared with healthy controls.
(2) It was demonstrated that HD-DXM therapy could downregulate FcγRⅠ, FcγRⅡa expression, while upregulate FcγRⅡb expression, on monocytes. The FcγRⅡandⅢexpression showed no change after HD-DXM therapy. mRNA ratio of FcγRⅡa/Ⅱb in ITP patients were comparative to that of the ratio in healthy controls
(3) Accompanied by the restored FcγRⅡa/Ⅱb balance, the FcyR-mediated phagocytic capacity of monocyte-derived macrophages in ITP patients decreased after HD-DXM therapy.
(4) DXM could increase the mRNA expression of both FcγRⅡa and FcγRⅡb in in vitro cultured monocytes, with FcyRIIb at higher amplitudes.
In summary, decreased FcγRⅡb expression and increased FcγRⅡa, and FcγRⅠexpression in untreated ITP patients suggested the possible role of the disturbed FcyR balance in the pathogenesis of ITP. HD-DXM may reduce inflammation in ITP by restoring the balance of activating and inhibitory FcγRs, raising the threshold for monocyte activation and decreasing the phagocytic capacity of monocytes.
人Fcγ受体系统由活化性受体(FcγRⅠ,Ⅱa,Ⅲ)和抑制性受体(FcγRⅡb)两个家族组成,这两类作用相反的受体平衡一旦失调会导致许多自身免疫性疾病的发生。抗原-IgG免疫复合物(immune complex, IC)通过作用于免疫细胞上IgG恒定区Fcγ受体诱发多种免疫反应。根据RNA剪接异构体和蛋白结构不同,人类FcγR系统可分为FcγRⅠ、FcγRⅡ、FcγRⅢ三个亚群。FcγRⅠ又称CD64,为高亲和力的IgG受体,主要表达与单核细胞和巨噬细胞；FcγRⅢ即CD16,与单体IgG结合力较弱,但同IgG聚合体有中低度的亲和力,其主要表达于中性粒细胞、单核／巨噬细胞、自然杀伤细胞；FcγRⅡ又称CD32,按功能可进一步分为FcγRⅡa和FcγRⅡb两个亚型,为低到中亲和力的IgG受体,主要与聚合的IgG结合,表达于中性粒细胞、单核／巨噬细胞、树突状细胞(dendritic cells, DCs)、B细胞和血小板。按功能学分类,人FcγR系统可分为活化性FcγR和抑制性FcγR两个家族。FcγRⅡb为唯一抑制性的Fcγ受体,主要通过自身分子胞浆区偶联含有免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif, ITIM)传递抑制信号,在固有免疫和适应性免疫方面发挥重要负性调节作用；其余的FcγRⅠ、Ⅱa、Ⅲ均为活化性Fcγ受体,大都通过偶联含有免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activating motif, IT AM)的γ二聚体传递活化信号。Fcγ受体信号通路在多种自身免疫性疾病的发生及进展中发挥重要作用。多篇报道显示：在风湿性关节炎(rheumatoid arthritis, RA)、系统性红斑狼疮(systemic lupus erythematosus, SLE)、多发性硬化(multiple sclerosis, MS)自身免疫性溶血性贫血(autoimmune hemolytic anemia)等自身免疫性疾病中,往往可以发现活化性受体FcyRI的表达下调及抑制性受体FcyRIIb的表达上调,故活化性和抑制性Fcy受体失衡在自身免疫性疾病的发病过程中发挥重要作用。
原发性免疫性血小板减少症(primary immune thrombocytopenia, ITP),以往亦称为特发性血小板减少性紫癜(idiopathic thrombocytopenic purpura, ITP),是临床最为常见的出血性疾病,约占出血性疾病总数的30%。ITP临床表现为皮肤粘膜出血,月经过多,内脏出血,甚至颅内出血,严重影响人类健康。其发病机制主要为机体自身免疫耐受失衡,产生了针对血小板糖蛋白(glycoprotein, GP)特异性的自身抗体,大部分主要为针对GPⅡb/Ⅲa和／或GPⅠb/Ⅸ的IgG,该抗体与血小板膜自身GP抗原结合,通过作用于单核巨噬细胞Fcy受体(Fcγreceptor, FcyR)在网状内皮系统被巨噬细胞吞噬而过度清除。自身抗体的产生、激活、维持由表达FcγR的免疫细胞共同参与完成,是众多自身免疫性疾病的共同病理生理基础。除上述经典的抗体介导的血小板异常破坏途径外,多种细胞免疫机制异常,如Th1/Th2失衡、Th17亚群异常、细胞毒性T细胞(cytotoxic T lymphocyte, CTL)介导的血小板破坏、调节性T细胞数量或抑制功能异常等参与了ITP发病的异常病理生理过程,但上述异常的具体原因亟待明确。目前,对于ITP的治疗以糖皮质激素、脾切除、大剂量丙种球蛋白、免疫抑制剂等治疗为主,容易产生感染、骨髓抑制等副作用,约有1/3的患者对上述治疗无效,成为难治性ITP,常常迁延不愈,甚至危及生命。因此,深入了解ITP的发病机制,探讨阻断ITP特异发病途径并寻找新的药物作用靶点对展新的ITP的治疗策略有重要意义
ITP是最经典的一种Fcy受体介导的自身免疫性出血性疾病。做为FcγRs系统中唯一的抑制性受体,FcγRⅡb在ITP的免疫负性调节中发挥重要作用。Ashi最近报道显示：对清除幽门螺杆菌疗法有反应的ITP患者中,可以发现单核细胞抑制性受体FcγRⅡb的表达上调及活化性受体FcγRⅡa、FcyRⅠ的表达下调,而清除幽门螺杆菌疗法无反应的ITP患者中并不伴有该种变化,这提示幽门螺杆菌清除后可以通过恢复FcγRs受体平衡来缓解ITP病情。动物实验亦证实：过表达FcγRⅡa的转基因小鼠可显著加重抗体介导的血小板减少。此外,FcγRⅡa、Ⅱb亦参与抗体、补体介导的炎症反应：Shushakova研究显示C5a可直接上调肺泡巨噬细胞FcγRⅡb的表达。小鼠模型中,可溶性抗原特异的IgG可成功治疗ITP。大剂量丙种球蛋白静脉滴注(intravenous immunoglobulin, IVIg)已成功用于人类包括ITP在内的多种自身免疫性疾病中,且疗效机制研究发现IVI可上调抑制性FcγyRⅡb活性并下调激活性FcγRⅡa活性。口服大剂量地塞米松(high dose dexamethasone, HD-DXM)冲击疗法是当前皮质激素治疗ITP的研究热点,其疗效逐渐得到了广泛认可,尤其是治疗初治ITP患者。最近ITP国际工作组已将HD-DXM作为初诊ITP的标准一线疗法,但其疗效的具体机制尚不明确。初步研究证实：它可抑制抗体生成及减轻抗原抗体反应、改善毛细血管通透性、刺激骨髓造血及血小板向外周血的释放、恢复Th1/Th2平衡、增加Tregs数量等。迄今为止,尚未有系统的关于ITP患者与健康对照外周单核及B细胞上FcγRs表达及HD-DXM对ITP患者Fcγ受体系统影响的报道。
目的：
检测初诊活动性ITP患者及健康对照外周血FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表面分子表达及FcγRⅡ的亚型Ⅱa及Ⅱb mRNA及蛋白表达；研究HD-DXM治疗后ITP患者FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表达变化、FcγRIIa/IIb mRNA及蛋白表达变化；检测ITP患者治疗前后单核／巨噬细胞对致敏自身血小板吞噬能力变化及Th1/Th2细胞因子平衡变化；体外检测ITP患者单核细胞在DXM存在情况下培养时FcγRⅡa/Ⅱb mRNA表达变化,探讨HD-DXM影响FcγRs系统变化的可能机制。
材料和方法：
(1)外周血FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表面分子表达及FcγRⅡ的亚型Ⅱa及Ⅱb mRNA及蛋白表达检测部分,抽取23例初诊活动性ITP患者治疗前、治疗后2周及20例健康对照的外周血,分离血浆及单个核细胞。
(2)流式细胞仪染色法测定外周血FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ的表面分子表达；免疫磁珠阳性分选外周血CD14+单核细胞；实时定量聚合酶链反应(real-time reverse-transcription polymerase chain reaction, real-time RT-PCR)检测单核细胞FcγRⅡa、Ⅱb的mRNA表达；免疫沉淀(immunoprecipitation, IP)和western blotting检测FcγRⅡa、Ⅱb的蛋白表达；酶联免疫吸附实验(enzyme-linked immunoabsorbent assay, ELISA)检测ITP治疗前后及正常对照血浆中IFN-γ、IL-4的水平。
(3)体外实验部分抽取14例治疗前ITP患者外周血做细胞培养。免疫磁珠阳性分选外周血CD14+单核细胞,加入不同剂量的DXM培养16h, real-time RT-PCR检测FcγRⅡa、Ⅱb的mRNA表达变化；
(4)抽取10例健康对照、10例ITP患者治疗前及治疗2周后外周血,贴壁法分离外周血单核细胞并刺激,与CMFDA(5-chloromethylfluorescein diacetate)探针标记过的致敏自身血小板在37°C或4°C共培养,流式细胞仪检测血小板被自身单核／巨噬细胞吞噬情况,应用MFI37℃/MFI4℃做为吞噬指数以判定吞噬能力。
结果：
(1)流式测定的FcγRⅠ、Ⅱ(Ⅱa+Ⅱb)、Ⅲ表面分子表达高低以平均荧光强度表示(mean fluorescence intensity, MFI),结果显示：①初诊ITP患者单核细胞表面分子FcγRⅠ表达(504.6±58.6)显著高于正常对照(309.8±52.9；P<0.001)；经HD-DXM治疗后2周,FcγRⅠ表达较治疗前显著降低(317.5±69.4；P<0.001)：FcγRⅠ在治疗后与正常对照组无显著差异(P>0.05)。②单核细胞表面分子FcγRⅡ(Ⅱa+Ⅱb)、Ⅲ在ITP组治疗前(FcγRⅡ82.6±160; FcγRⅢ9.9±4.6)、治疗后(FcγRⅡ75.4±18.9; FcγRⅢ8.9±3.0)及正常对照组(FcγRⅡ83.2±16.3; FcγRⅢ9.3±3.2)之间无显著差异(FcγRⅡP=0.947; FcγRⅢP=0.694; ANOVA).③B细胞表面分子FcγRⅡ(Ⅱa+Ⅱb)在ITP组治疗前(82.3±13.0)治疗后(82.7±11.8)及正常对照组(83.2±12.1)之间差异无显著性(P=0.954；ANOVA).
(2)单核细胞FcγRⅡa和FcγRⅡb mRNA和蛋白表达：①real-time RT-PCR结果：我们应用FcγRⅡa和FcγRⅡb mRNA的比率变化表示HD-DXM对FcγRⅡa和FcγRⅡb mRNA表达的影响。初诊ITP患者FcγRⅡa/Ⅱb mRNA的比率值为5.88±5.12,与正常对照组存在显著差异(0.31±0.29；P<0.001)；接受HD-DXM治疗后FcγRⅡa/Ⅱb mRNA的比率值(0.61±0.86)较治疗前显著降低(P<0.001)。②western blotting结果：应用相对光密度值高低来代表FcγRⅡa和FcγRⅡb蛋白表达量。初诊ITP患者单核细胞FcγRⅡa表达量接受HD-DXM治疗后显著降低(2.05±0.81 vs 1.07±0.96, P< 0.001, Student(?) test),并伴有FcγRⅡb表达的显著升高(0.13±0.31 vs 1.23±1.25, P< 0.001, Whitney U test)
(3)ITP患者HD-DXM治疗后单核／巨噬细胞FcγR介导的吞噬致敏血小板能力的改变。应用CMFDA做探针标记血小板,初诊ITP患者吞噬指数为(MFI ratio:3.5±0.7),显著高于正常对照(2.2±0.5；P<0.05)。HD-DXM治疗后吞噬指数降为(MFI ratio:2.6±1.0),较治疗前有显著差异(P<0.05)。
(4)ITP患者治疗前后血浆IFN-γ、IL-4水平变化。初诊ITP患者治疗前Thl系列细胞因子IFN-γ较正常对照显著增高(62.7±13.0 vs 37.4±4.8 pg/ml, P< 0.001)和IL-4的显著降低(8.9±2.7 vs 18.3±4.7 pg/ml, P< 0.001). HD-DXM治疗后,IFN-γ水平较治疗前显著降低并伴有IL-4的显著升高(IFN-γ:40.3±11.0 pg/ml, P< 0.001; IL-4:18.5±4.2 pg/ml, P< 0.001).
(5)DXM对ITP患者体外培养单核细胞FcγRⅡa、Ⅱb mRNA表达的影响。体外培养ITP患者的单核细胞在加入DXM后出现FcγRⅡa、Ⅱb mRNA表达的同时上调,这种效应在DXM为1μM时最明显。在DXM为0.5μM和1μM时,FcγRⅡb mRNA表达上调的幅度要高于FcγRⅡa的幅度(P<0.05)。
结论：
(1)初诊活动性ITP患者外周血单核细胞呈现活化性FcγRⅠ、FcγRⅡa的表达升高,及抑制性FcγRⅡb mRNA及蛋白表达降低；
(2)ITP患者经HD-DXM治疗后2周,单核细胞出现活化性FcγRL、FcγRIIa的表达降低,及抑制性FcγRⅡb mRNA及蛋白表达升高,FcγRⅡa/Ⅱb mRNA比率恢复正常；
(3)ITP患者治疗后伴随着单核细胞活化性和抑制性FcγRs受体平衡恢复,单核／巨噬细胞FcγR介导的自体致敏血小板吞噬能力降低；
(4)ITP患者呈现Th1细胞极化状态,经HD-DXM治疗后,Th1/Th2失衡状态得到纠正；
(5)DXM在体外可同时上调FcγRⅡa、Ⅱb mRNA表达,且FcγRⅡb上调幅度更高；
意义：本研究揭示HD-DXM治疗可以通过上调抑制性FcγRⅡb恢复单核细胞FcγRs系统平衡,从而缓解ITP患者症状,这提示了HD-DXM在ITP中发挥疗效的一种新的可能机制。
Background
The human Fcγreceptor (FcyR) system is composed of two opposing families, the activating FcγRsⅠ,Ⅱa,Ⅲand the inhibitory FcγRⅡb, the balance of which determines the magnitude of the inflammatory response. FcγRⅡb is the only FcyR that has an inhibitory function, and is expressed by a variety of immune cells, including B cells, monocytes, macrophages, dendritic cells (DCs), mast cells and basophils. FcγRⅡb can decrease antibody production by raising the activation threshold of B cells if cross-linked to the B cell receptor (BCR). It inhibits the function of activating FcyRs, such as phagocytosis and pro-inflammatory cytokine release by monocytes and DCs. The disturbed balance of the activating and inhibitory FcyRs has been found in many autoimmne diseases such as system lupus erythematosus (SLE) and rheumatoid arthritis (RA). Therapeutic response in RA and SLE was often associated with the restoration of the balance between activating and inhibitory FcγRs.
Primary immune thrombocytopenia (ITP), which is also named as idiopathic thrombocytopenia previously, is one of the most common hemorrhagic disorders, and accounts for about 30 percent of all bleeding disorders. ITP is often manifested by skin and mucosal hemorrhage, menorrhagia, visceral bleeding, and even in some case shown as intracranial hemorrhage, having a serious effect on human health. ITP is an immune-mediated bleeding disorder in which platelets are opsonized by autoantibodies directed against platelet surface membrane glycoproteins (GPⅡb/Ⅲa, Ib/IX), and prematurely cleared by FcyR-bearing macrohpages in the reticuloendothelial system. The production, activation and maintenance of autoantibodies are completed by a variety of immune cells which express FcγRs. Previous studies have shown that leukocyte IgG-FcγR, such as FcγRⅡandⅢ, played an important role in the phagocytosis of autoantibody coated platelets. McKenzie et al. demonstrated that human FcγRIIa was necessary for antibody-mediated platelet clearance by using a murine model transgenic for human FcyRⅡa and lacking murine FcγRⅠand FcγRⅢ. Samuelsson et al. showed that decreased expression of murine inhibitory FcyRⅡb was associated with increased platelet destruction. In a multivariate logistic regression analysis of data from 60 children with ITP, the presence of the FcyRⅡbT232 variant predicted a chronic disease course. Recently, it was reported that Helicobacter pylori (H. pylori) eradication could induce a significant platelet count increase in a subset of H. pylori infected ITP patients, and that effect was shown to be mediated by shifting monocyte Fcγreceptor balance toward the inhibitory FcyRⅡb. The pathophysiology of ITP is heterogeneous and complex. Besides the classical mechanism of GP-specific autoantibody-mediated platelet destruction, several cellular defects about immune modulation, such as the disturbed balance of T helper 1 (Th1)/Th2, the decreased number and dysfunction of regulatory T cells, the elevated number of Th17 cells, and cytotoxicity T lymphocytes (CTLs)-mediated platelet destruction, have been described. The precise reason for these abnormalities remains being elucidated.
The therapeutic regimens for ITP include glucorticosteroids (GCs), intravenous immunoglobulin (IVIG), splenectomy, thrombopoietin receptor agonists, and other immunosuppressive drugs. IVIG could induce a beneficial response in ITP patients via up-regulating FcγRⅡb on monocytes/macrophages, and the same results were showed in mouse models of ITP. High-dose dexamethasone (HD-DXM) have been widely recognized as the first-line therapy for patients who need to be managed. However, the effects of HD-DXM on FcγR regulation remain unelucidated in ITP patients. In the present study, the difference in the expression of FcγRs in ITP patients compared with normal controls, and the reguation of FcγRs by HD-DXM were investigated.
Objective
To compare the surface molecule expression of FcγRⅠ/CD64,Ⅱ(Ⅱa+Ⅱb)/CD32,Ⅲ/CD16 on peripheral blood monocytes and the expression of FcγRⅡ/CD32 on B cells, as well as the mRNA and protein expression of FcγRIIa and FcyRⅡb on mnoocytes in newly diagnosed ITP patients and healthy controls; To investigate the effect of HD-DXM on FcγRⅠ,Ⅱ(Ⅱa,Ⅱb,Ⅱa+Ⅱb, respectively),Ⅲregulation and the phagocytic ability change of monocytes/macrophages in ITP patients; To determine the Th1/Th2 cytokine change in plasma of ITP patients after HD-DXM administration; To investigate the effect of DXM on FcγRⅡa,Ⅱb mRNA expression in in vitro cultured monocytes from newly diagnosed ITP patients.
Materials and Methods
(1) For surface molecule expression of FcγRⅠ,Ⅱ,Ⅲon monocytes and the expression of FcγRⅡ/CD32 on B cells,23 newly diagnosed ITP patients and 20 healthy controls were enrolled. In vitro monocyte culture was performed in another 14 newly-diagnosed ITP patients before HD-DXM treatment, and samples from 10 of them were also used for assays for the phagocytic capacity of monocyte-derived macrophages.
(2) The surface molecule expression of FcγRⅠ/CD64,Ⅱ(Ⅱa+Ⅱb)/CD32,Ⅲ/CD16 on peripheral blood monocytes and the expression of FcγRⅡ/CD32 on B cells were determined by flow cytometry. CD14+monocytes from peripheral blood mononuclear cells (PBMCs) were positively selected by immunomagnetic beads. mRNA expression of FcγRⅡa,Ⅱb was quantified on sorted monocytes by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). Immunoprecipitation (IP) and western blotting were used to determine the protein expression of FcγRⅡa,Ⅱb. Plasma levels of IFN-γand IL-4 were determined by enzyme-linked immunoabsorbent assay (ELISA).
(3) Monocytes from additional 14 newly-diagnosed ITP patients before treatment were adjusted to 1 * 106/ml in RPMI-1640 culture medium supplemented with 10%heat-inactivated human pooled AB serum at a denstity of 2 * 106 cells/well in a 6-well culture plate and incubated in humidified air in 5% CO2 at 37℃in the presence of different concentration of DXM. the monocyte-derived macrophages were collected for real-time PCR analysis of FcγRⅡa/Ⅱb mRNA expression.
(4) In vitro phagocytosis of IgG-opsonized platelets by macrophages was carried out in another 10 newly-diagnosed ITP patients. Platelets were labeled with 5-chloromethylfluorescein diacetate (CMFDA) and oposonized with 5μg murine IgG2a anti-human major histocompatibility complex (MHC) class I monoclonal antibody (W6/32). Monocytes were activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), incubated with opsonized CMFDA-labeled platelets, and Intracellular FL1 GM-Green platelet fluorescence in the nucleated events was determined by flow cytometry. The phagocytic index was calculated as the MFI obtained at 37℃divided by the MFI at 0℃.
Results
(1)①Quantification of FcγRⅠexpression on monocytes showed a significantly higher mean fluorescence intensity (MFI) in ITP patients (504.6±58.6) compared with normal controls (309.8±52.9, P< 0.001), and after 4-day HD-DXM therapy, FcγRⅠexpression showed a significant decrease (317.5±69.4, P< 0.001) compared with the levels before treatment.②〧FcγRⅡ(Ⅱa+Ⅱ) expression on monocytes from ITP patients showed a slight increase after HD-DXM therapy compared with the levels before treatment (82.6±16.0 vs.74.5±8.9), but this increase did not get statistically significant difference. Also there was no difference between ITP group and control individuals (83.2±16.3, P=0.947, ANOVA). After 4-day HD-DXM administration, monocytes from 17 patients (73.9%) showed increased FcyRII expression, and 6 patients (26.1%) had decreased FcγRⅡexpression. There were no significant changes in FcγRⅢexpression on monocytes detected by FACS with anti-FcγRⅢmAb after HD-DXM administration, and no difference was found among patients before, after therapy and the normal control group (9.9±4.6, 8.9±3.0, and 9.3±3.2, respectively; P= 0.694, ANOVA).③The FcγR expression on B cells was also determined by flow cytometry in ITP patients and controls. As shown in Fig.2A, B, the expression of FcγRⅡon B cells in ITP patients prior to HD-DXM administration was similar to normal controls (82.0±13.0 vs.83.2±12.1), and HD-DXM treatment had no effect on B cell FcγRⅡexpression (82.7±11.8,p=0.954, ANOVA).
(2)①Results showed that the ratio of FcγRⅡa/Ⅱb mRNA expression on monocytes was significantly higher in untreated ITP patients than normal controls (5.88±5.12 vs.0.31±0.29, P< 0.001). After HD-DXM therapy, the ratio of FcyRIIa/IIb mRNA decreased significantly (0.61±0.86, P< 0.001) in comparison with the level before treatment.②The patients after therapy had a marked downregulation of FcyRIIa protein expression on monocytes compared with the levels before treatment (2.05±0.81 vs.1.07±0.96, P< 0.001, Student's t test). At the same time, the decreased FcγRⅡa protein levels were associated the upregulated FcyRIIb levels. The relative densitometry of monocyte FcyRIIb in ITP group was 0.13±0.31, which increased significantly after HD-DXM therapy (1.23±1.25, P< 0.001, Mann-Whitney U test).
(3) Monocytes from untreated ITP patients demonstrated higher phagocytic capacity compared with healthy controls (MFI ratio:3.5±0.7 vs.2.2±0.5, P< 0.05). After HD-DXM therapy, phagocytic capacity of monocytes decreased significantly (2.6±1.0, P< 0.05).
(4) significantly higher levels of plasma IFN-γand lower IL-4 were found in untreated patients compared with healthy controls (IFN-γ:62.7±13.0 vs.37.4±4.8 pg/mL, P< 0.001; and IL-4:8.9±2.7 vs.18.3±4.7 pg/mL, P< 0.001, respectively). Plasma IFN-y levels in untreated patients decreased significantly after HD-DXM administration (40.3±11.0 pg/mL, P< 0.001), while post-treatment levels of IL-4 increased significantly compared with the levels before treatment (18.5±4.2pg/mL,P< 0.001).
(5) DXM could increase the mRNA expression of both FcyγRⅡa and FcγRⅡb on monocytes, with FcγRⅡb at higher amplitudes. At the concentration of 0.5 and 1μM, effect of DXM on the regulation of FcyRⅡa and FcγRⅡb (with FcγRⅡb at higher amplitudes) got statistical significance.
Conclusions
(1) Expression of FcγRⅠand FcγRⅡa on monocytes in newly diagonosed ITP patients was significantly higher compared with healthy controls. On the contrary, mRNA and protein expression of the inhibitory FcγRⅡb were significantly lower in ITP patients compared with healthy controls.
(2) It was demonstrated that HD-DXM therapy could downregulate FcγRⅠ, FcγRⅡa expression, while upregulate FcγRⅡb expression, on monocytes. The FcγRⅡandⅢexpression showed no change after HD-DXM therapy. mRNA ratio of FcγRⅡa/Ⅱb in ITP patients were comparative to that of the ratio in healthy controls
(3) Accompanied by the restored FcγRⅡa/Ⅱb balance, the FcyR-mediated phagocytic capacity of monocyte-derived macrophages in ITP patients decreased after HD-DXM therapy.
(4) DXM could increase the mRNA expression of both FcγRⅡa and FcγRⅡb in in vitro cultured monocytes, with FcyRIIb at higher amplitudes.
In summary, decreased FcγRⅡb expression and increased FcγRⅡa, and FcγRⅠexpression in untreated ITP patients suggested the possible role of the disturbed FcyR balance in the pathogenesis of ITP. HD-DXM may reduce inflammation in ITP by restoring the balance of activating and inhibitory FcγRs, raising the threshold for monocyte activation and decreasing the phagocytic capacity of monocytes.
引文
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