The Expression And Regulation Of Fc?Rs In Primary Immune Thrombocytopenia

BackgroundThe human Fc?receptor (FcyR) system is composed of two opposing families, the activating Fc?Rs?,?a,?and the inhibitory Fc?R?b, the balance of which determines the magnitude of the inflammatory response. Fc?R?b 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. Fc?R?b 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 Fc?Rs.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 (GP?b/?a, 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 Fc?Rs. Previous studies have shown that leukocyte IgG-Fc?R, such as Fc?R?and?, played an important role in the phagocytosis of autoantibody coated platelets. McKenzie et al. demonstrated that human Fc?RIIa was necessary for antibody-mediated platelet clearance by using a murine model transgenic for human FcyR?a and lacking murine Fc?R?and Fc?R?. Samuelsson et al. showed that decreased expression of murine inhibitory FcyR?b was associated with increased platelet destruction. In a multivariate logistic regression analysis of data from 60 children with ITP, the presence of the FcyR?bT232 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 Fc?receptor balance toward the inhibitory FcyR?b. 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 Fc?R?b 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 Fc?R regulation remain unelucidated in ITP patients. In the present study, the difference in the expression of Fc?Rs in ITP patients compared with normal controls, and the reguation of Fc?Rs by HD-DXM were investigated.ObjectiveTo compare the surface molecule expression of Fc?R?/CD64,?(?a+?b)/CD32,?/CD16 on peripheral blood monocytes and the expression of Fc?R?/CD32 on B cells, as well as the mRNA and protein expression of Fc?RIIa and FcyR?b on mnoocytes in newly diagnosed ITP patients and healthy controls; To investigate the effect of HD-DXM on Fc?R?,?(?a,?b,?a+?b, respectively),?regulation 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 Fc?R?a,?b mRNA expression in in vitro cultured monocytes from newly diagnosed ITP patients.Materials and Methods(1) For surface molecule expression of Fc?R?,?,?on monocytes and the expression of Fc?R?/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 Fc?R?/CD64,?(?a+?b)/CD32,?/CD16 on peripheral blood monocytes and the expression of Fc?R?/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 Fc?R?a,?b 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 Fc?R?a,?b. Plasma levels of IFN-?and 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 Fc?R?a/?b 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 5?g 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)?Quantification of Fc?R?expression 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, Fc?R?expression showed a significant decrease (317.5±69.4, P< 0.001) compared with the levels before treatment.??Fc?R?(?a+?) 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 Fc?R?expression. There were no significant changes in Fc?R?expression on monocytes detected by FACS with anti-Fc?R?mAb 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).?The Fc?R expression on B cells was also determined by flow cytometry in ITP patients and controls. As shown in Fig.2A, B, the expression of Fc?R?on 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 Fc?R?expression (82.7±11.8,p=0.954, ANOVA).(2)?Results showed that the ratio of Fc?R?a/?b 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.?The 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 Fc?R?a 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-?and lower IL-4 were found in untreated patients compared with healthy controls (IFN-?: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 Fcy?R?a and Fc?R?b on monocytes, with Fc?R?b at higher amplitudes. At the concentration of 0.5 and 1?M, effect of DXM on the regulation of FcyR?a and Fc?R?b (with Fc?R?b at higher amplitudes) got statistical significance.Conclusions (1) Expression of Fc?R?and Fc?R?a on monocytes in newly diagonosed ITP patients was significantly higher compared with healthy controls. On the contrary, mRNA and protein expression of the inhibitory Fc?R?b were significantly lower in ITP patients compared with healthy controls.(2) It was demonstrated that HD-DXM therapy could downregulate Fc?R?, Fc?R?a expression, while upregulate Fc?R?b expression, on monocytes. The Fc?R?and?expression showed no change after HD-DXM therapy. mRNA ratio of Fc?R?a/?b in ITP patients were comparative to that of the ratio in healthy controls(3) Accompanied by the restored Fc?R?a/?b 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 Fc?R?a and Fc?R?b in in vitro cultured monocytes, with FcyRIIb at higher amplitudes.In summary, decreased Fc?R?b expression and increased Fc?R?a, and Fc?R?expression 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 Fc?Rs, raising the threshold for monocyte activation and decreasing the phagocytic capacity of monocytes.