Polymorphisms of Monocyte-Activating Cytokine/Chemokine Genes Affect the Susceptibility to, and Severity of Chronic ITP

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by the production of platelet antibodies, resulting in the destruction of platelets and inhibition of their production. A shift toward Th1 and Th17 cells together with impaired Tregs has been reported using quantitative RT-PCR and flow cytometry method. Further, recent reports have shown that CD16 (+) monocytes in ITP patients are polarized to secrete the Th1 cytokine IL-12, promoting Th1 responses. Enhanced phagocytosis of splenic macrophages was also observed in ITP patients. Thus, monocytes/macrophages are also involved in the pathogenesis of ITP. However, it remains unclear whether genetic factors of cytokines/chemokines activating monocytes increase the risk of chronic ITP. We investigated the impact of monocyte-related gene polymorphisms: TNF-α -857C/T, IFN-γ +874T/A, and MCP1+2518G/A on the susceptibility and clinical feature of chronic ITP (cITP) and Th1/Th2 ratio in peripheral blood of cITP patients,

Patients and methods: In this study, 126 Japanese cITP patients (92 females and 34 males with a median age of 47.7 [range: 2.4-82.3] years), as well as 202 race-matched healthy subjects were examined. The diagnosis of cITP was defined according to International Working Group criteria. Genotyping of the polymorphisms was determined by PCR based technique and direct sequencing. We evaluated Th1/Th2 ratio in peripheral blood of 15 normal donors and 25 ITP patients. Intracellular IL-4 (Th2 cytokine) and IFN-γ (Th1 cytokine) production was assessed in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin by flow cytometry.This study was approved by the Institutional Review Board of Gunma University Hospital.

Results: The platelet count ranged from 1×10⁹/L to 98×10⁹/L with a mean count of 32×10⁹/L at the initial diagnosis. Steroid treatment was given to 86 patients (58.1%), and splenectomy was performed in only 18 patients (12.2%). The genotype distributions of these three polymorphisms in healthy subjects were similar to those observed in previous studies of Japanese populations. Patients with cITP had a significantly higher frequency of the TNF-α -857 TT genotype (high producer type) and MCP1+2518 G allele (high-producer type) compared to healthy controls (14.0% vs 5.4%, p < 0.01; 67.6% vs. 60.6%, P < 0.01). However, no significant difference in the genotype frequencies of IFN-γ was observed between cITP patients and healthy controls. Thus, it was suggested that the TNF-α and MCP-1 polymorphisms shifted toward monocyte activation in cITP patients. We examined the association between these polymorphisms and the clinical characteristics of cITP patients. cITP patients with IFN-γ +874 non-AA genotype (high expression type) showed a lower minimum platelet count than those with an AA genotype (12.9 × 10⁹/L ± 10.7 ×10⁹/L vs 19.4×10⁹/L ± 18.9 ×10⁹/L, P < 0.05). We also examined the association between these polymorphisms and Th1/Th2 ratio in both normal donors and cITP patients. The median Th1/Th2 ratio in patients with cITP was significantly higher than that of healthy controls (31.4, range 0.6-98.8 vs. 17.8, range, 2.2-52.5, P < 0.001). Patients with the IFN-γ +874 non-AA genotype (high expression type) had a significantly higher Th1/Th2 ratio compared to those with the IFN-γ +874 AA genotype (low expression type) (71.5, range 29.4-92.8 vs. 27.5, range 0.6-98.8, P < 0.05). However, there was no significant association between the Th1/Th2 ratio and other polymorphism in cITP patients.

Conclusion: These findings suggested that the high expression type of monocyte-activating cytokine/chemokine genes increased the susceptibility to, and severity of chronic ITP