Expression And Regulation Of Glucocorticoid Receptor Isoforms In Patients With Immune Thrombocytopenia And Its Correlation With Glucocorticoid Resistance

Objective: Alternative splicing of glucocorticoid receptor (GR) precursor RNA,which produces isoforms of GR, may cause resistance to glucocorticoids (GC) inpatients with adult immune thrombocytopenia (ITP). In order to demonstrate thishypothesis, we examined the expression of glucocorticoid receptor mRNA (GRα, GRβ,GRγ, GRp), as well as GRα and GRβ proteins.Methods: We examined the expression of GR mRNA (GRα, GRβ, GRγ, GRp) inmononuclear cells of peripheral blood of62newly diagnosed ITP patients and38healthy volunteers by real time PCR. Among the ITP patients,39accepted GC therapy,who were further categorized into GC sensitive group（GCS）and GC resistant group(GCR) according to GC response. Expression of GRα, GRβ, GRγ and GRp wereanalyzed in paired groups. The correlation between these GR isoforms and plateletcount was analyzed. GRα and GRβ protein of PBMC in20GCS patients,8GCRpatients and20healthy volunteers were tested by western blot.Results: The data did not follow a normal distribution and was expressed asmedian and interquartile range. The expression of GRα mRNA，GRβ mRNA and thesum of GR isoforms were significantly increased in ITP patients than in normal subjects(P<0.05); there was no significant difference in mRNA expression of GRγ and GRp.Moreover, no significant difference in mRNA expression was observed in GRα, GRβ,GRγ and GRp isoforms between GCS and GCR patients. However, the expression ofGRα mRNA, tended to be higher in GCS group1.11(0.30-2.69) than in GCR group 0.20(0.05-1.41). The expression of GRγ mRNA tended to be higher in GCR group0.27（0.00-3.23）than in GCS group0.20(0.05-1.41). The expression of GRβ, GRγ andGRp mRNA were very low. A positive correlation was observed between3paired-group, that is, between GRα and GRp mRNA; GRβ and GRp mRNA; GRγ andGRp mRNA. There was no significant difference between GR isoforms and plateletcount. GRα protein level was significantly higher in GCS group than in GCR group(P<0.05), while GRβ protein could not be detected.Conclusion: Our study suggested that the sum of GR isoforms, GRα mRNA andGRβ mRNA were increased in adult ITP patients before treatment. GC resistance wassignificantly related with the expression of GRα while it was mildly associated with theexpression of GRγ. The expression of GRβ mRNA was so low that it was not detectedat protein level, which points out that GRβ mRNA may be not associated with GCresistance. Our data presented here should therefore be considered preliminary andfurther confirmatory studies are warranted on an expanded patient based scale. Objective: To investigate the expression of heat shock protein90(HSP90),nuclear factor-κB (NF-κB) and activator protein-1(AP-1) in patients with primaryimmune thrombocytopenia (ITP) and its correlation with glucocorticoid resistance.Methods: HSP90mRNA was tested by real time PCR in44newly diagnosedITP patients and32healthy volunteers. Then patients with ITP were divided intoseveral subgroups according to glucocorticoid response, gender, age, stage and severityof disease，and difference between each pair of subgroups were analyzed. Plateletassociated IgG (PAIgG) was measured by a competitive enzyme immunoassay (ELISA).We tested and compared P65/NF-κB protein and C-Jun/AP-1protein from nuclear cellsof20GC sensitive patients,7GC resistance patients and20healthy volunteers bywestern blot.Results: No significant difference was found in expression of HSP90mRNA andHSP90/GRα between ITP patients and healthy volunteers（P＞0.05）, whereas thelevel of HSP90expression and HSP90/GRα tended to be lower in ITP patients. Thelevel of HSP90expression was significantly higher in acute ITP patients than chronicITP patients（P<0.05）. There was no significant difference between GC resistant andGC sensitive patients; between female and male; between younger and older age groupsand finally between mild and severe patients. However, the latter in each group pairswere more prone to become GC resistance whereas the expression of HSP90mRNAtended to be lower in subsequent subgroup. The ratio of HSP90and GRα tended to behigher in GC resistance patients. No significant correlation between HSP90mRNA andplatelet count or megakaryocytes or PAIgG was found in ITP patients. Expression ofp65protein was significantly higher in GCR group than in GCS group (P<0.05), whileno significant difference of C-Jun protein was noted.Conclusion：Our study suggested that HSP90mRNA correlated with the diseasestage in ITP. HSP90mRNA was decreased in ITP patients. The ratio of HSP90and GRαmay be associated with GC resistance. Increased expression of p65/NF-κB protein plays an important role in development of GC resistance in ITP. However, C-Jun/AP-1protein may be not related with GC resistance in ITP. Backgroud and Objective: Dysfunction of cytokine-mediated cellular immunityhas been considered to play an important role in the pathophysiology of adult immunethrombocytopenia (ITP). CD4+T lymphocytes are important part of T lymphocytes.They are divided into four subtypes, T helper1(Th1),T helper2(Th2), T-reg cells(regulatory T cells）and T helper17(Th17). So our study aimed to investigate the roleof CD4+T lymphocytes in the pathegenesis of ITP by testing the cytokines of thesecells.Methods: We measured the concentration of IL-2, IFN-γ (Th1), IL-4, IL-10(Th2), TGF-β1(T-reg) and IL-17(Th17) in the plasma from52patients with adult ITPand30age-and sex-matched normal controls by using enzyme-linked immunosorbentassay (ELISA). The results were analyzed by gender, age, disease status, diseasestages and disease severity. Moreover，correlation were computed between thesecytokines, the platelet count, megakaryocytes and the platelet associated IgG (PAIgG)in ITP.Results: The expressions of both IL-4and IL-10were significantly increased inITP than in normal controls (P<0.05). However, the expressions of IFN-γ, IL-2, TGF-β1and IL-17were significantly decreaced in ITP (P<0.05). Interestingly only IL-17wassignificantly higher in chronic ITP compared to acute ITP (P<0.05), and there was nosignificant difference between cytokines expression among the other subgroups.Furthermore, a positive correlation was observed between PAIgG and IFN-γ (r=0.48,P=0.02), while no significant correlations between other cytokines and platelet count,megakaryocytes or PAIgG in ITP was recorded.Conclusion： Our findings indicated that ITP pathogenesis is closely associatedwith the imbalance of cytokines, presenting as an increased Th2cytokines anddecreased Th1, T-reg and Th17cytokines. The concentration of IFN-γ positivelycorrelated with PAIgG in ITP group. Thus, adjusting the imbalance of CD4+T lymphocytes and their cytokines may improve patient’s condition and finally mayrepresent a possible new targeted drug therapy for ITP.