目的 分析白细胞介素-36(IL-36)家族成员在糖尿病肾病(diabetic kidney disease, DKD)外周血中的水平，探讨重组人IL-36α对DKD患者单核细胞功能的调控作用。 方法 连续入组2型糖尿病患者41例、DKD患者36例、对照者20名，分离血浆和外周血单个核细胞，酶联免疫吸附法检测血浆IL-36α、IL-36β、IL-36γ、IL-36受体拮抗剂(IL-36Ra)水平，分选单核细胞，实时定量PCR检测单核细胞中IL-36受体亚基mRNA表达。使用重组IL-36α刺激单核细胞，检测培养上清中毒性分子和细胞因子水平，流式细胞术检测程序性死亡受体-1(PD-1)和细胞毒性T淋巴细胞相关蛋白4(CTLA-4)表达。单核细胞与Vero细胞共培养，评估单核细胞的杀伤功能。 结果 2型糖尿病组和DKD组血浆IL-36α、IL-36β水平显著高于对照组，DKD组血浆IL-36α水平亦高于2型糖尿病组(P<0.05)。IL-36γ和IL-36Ra水平在3组之间差异无统计学意义(P>0.05)。单核细胞中IL-36受体亚基mRNA表达在3组之间差异无统计学意义(P>0.05)。DKD组肿瘤坏死因子α(TNF-α)水平高于对照组和2型糖尿病组(P<0.05)，PD-1和CTLA-4水平低于对照组和2型糖尿病组(P<0.05)。DKD组单核细胞诱导Vero细胞死亡的比例显著高于对照组和2型糖尿病组(P<0.05)。重组人IL-36α刺激后，DKD患者单核细胞分泌颗粒酶B和TNF-α水平显著升高(P<0.05)，单核细胞诱导Vero细胞死亡的比例显著升高(P＝0.024)。 结论 DKD组单核细胞分泌颗粒酶B和IL-36α水平升高诱导单核细胞功能增强。 Objective To analyze the level of interleukin-36(IL-36) family cytokines in peripheral blood, and explore the regulatory role of recombinant human IL-36α in monocyte function in patients with diabetic kidney disease(DKD). Methods A total of 41 type 2 diabetes mellitus(T2DM) patients, 36 DKD patients, and 20 controls were consecutively enrolled. Plasma and peripheral blood mononuclear cells(PBMCs) were isolated. Enzyme-linked immunosorbent assay(ELISA) was used to measure plasma levels of IL-36α, IL-36β, IL-36γ, and IL-36 receptor antagonist(IL-36Ra). PBMCs were sorted, and real-time quantitative PCR was performed to detect the mRNA expression of IL-36 receptor subunits in monocytes. Monocytes were stimulated with recombinant IL-36α, and levels of cytotoxic molecules and cytokines in the culture supernatant were measured. Flow cytometry was used to assess the expressions of programmed death receptor-1(PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA-4). Co-culture of monocytes with Vero cells was performed to evaluate monocyte cytotoxicity. Results Plasma levels of IL-36α and IL-36β in the T2DM and DKD groups were significantly higher than those in the control group. The DKD group also showed higher plasma levels of IL-36α compared to the T2DM group(P<0.05). There were no significant differences in IL-36γ and IL-36Ra levels among the three groups(P>0.05). The mRNA expression of IL-36 receptor subunits in monocytes was comparable among the three groups(P>0.05). The DKD group had higher level of tumor necrosis factor-alpha(TNF-α) compared to the control and T2DM groups(P<0.05). The levels of PD-1 and CTLA-4 were lower in the DKD group than those in the control and T2DM groups(P<0.05). The proportion of monocyte-induced Vero cell death was significantly higher in the DKD group compared to the control and T2DM groups(P<0.05). After stimulation with recombinant human IL-36α, monocytes from DKD patients showed a significant increase in the secretion of granzyme B and TNF-α(P<0.05), as well as an increased proportion of monocyte-induced Vero cell death(P=0.024). Conclusion In DKD patients, elevated IL-36α and granzyme B levels in monocytes enhance monocyte function.
Expression of interleukin-36 and its regulatory effect on monocyte function in patients with diabetic kidney disease
Objective To analyze the level of interleukin-36(IL-36) family cytokines in peripheral blood, and explore the regulatory role of recombinant human IL-36α in monocyte function in patients with diabetic kidney disease(DKD). Methods A total of 41 type 2 diabetes mellitus(T2DM) patients, 36 DKD patients, and 20 controls were consecutively enrolled. Plasma and peripheral blood mononuclear cells(PBMCs) were isolated. Enzyme-linked immunosorbent assay(ELISA) was used to measure plasma levels of IL-36α, IL-36β, IL-36γ, and IL-36 receptor antagonist(IL-36Ra). PBMCs were sorted, and real-time quantitative PCR was performed to detect the mRNA expression of IL-36 receptor subunits in monocytes. Monocytes were stimulated with recombinant IL-36α, and levels of cytotoxic molecules and cytokines in the culture supernatant were measured. Flow cytometry was used to assess the expressions of programmed death receptor-1(PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA-4). Co-culture of monocytes with Vero cells was performed to evaluate monocyte cytotoxicity. Results Plasma levels of IL-36α and IL-36β in the T2DM and DKD groups were significantly higher than those in the control group. The DKD group also showed higher plasma levels of IL-36α compared to the T2DM group(P<0.05). There were no significant differences in IL-36γ and IL-36Ra levels among the three groups(P>0.05). The mRNA expression of IL-36 receptor subunits in monocytes was comparable among the three groups(P>0.05). The DKD group had higher level of tumor necrosis factor-alpha(TNF-α) compared to the control and T2DM groups(P<0.05). The levels of PD-1 and CTLA-4 were lower in the DKD group than those in the control and T2DM groups(P<0.05). The proportion of monocyte-induced Vero cell death was significantly higher in the DKD group compared to the control and T2DM groups(P<0.05). After stimulation with recombinant human IL-36α, monocytes from DKD patients showed a significant increase in the secretion of granzyme B and TNF-α(P<0.05), as well as an increased proportion of monocyte-induced Vero cell death(P=0.024). Conclusion In DKD patients, elevated IL-36α and granzyme B levels in monocytes enhance monocyte function.
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