基质金属蛋白酶14通过促进CD100脱落增强急性B淋巴细胞白血病患者单核细胞功能

Matrix metalloproteinase 14 enhances monocyte function through promoting CD100 shedding in patients with B-cell acute lymphoblastic leukemia

目的 检测不同活性形式的CD100在急性B淋巴细胞白血病(B-ALL)患者中的表达,观察基质金属蛋白酶14(MMP14)对CD100的表达调控及对B-ALL患者单核细胞功能的影响。方法 入组44例B-ALL患者和22例对照者,采集外周血并分离血浆和外周血单个核细胞(PBMCs),酶联免疫吸附试验(ELISA)检测血浆MMP14和可溶型CD100(sCD100)水平,流式细胞术检测CD14+单核细胞中膜结合型CD100(m CD100)水平。使用磁珠分离法分选CD14+单核细胞,使用脂多糖刺激培养;反转录实时定量PCR法检测肿瘤坏死因子相关凋亡诱导配体(TRAIL)和Fas配体(FasL)mRNA相对表达量;ELISA法检测颗粒酶A、颗粒酶B、干扰素-γ(IFN-γ)、白细胞介素-1β(IL-1β)、IL-6水平;比较B-ALL患者和对照者单核细胞功能差异。使用重组人MMP14刺激B-ALL患者纯化的单核细胞,加入抗CD72抗体,通过检测TRAIL和FasL mRNA相对表达量以及颗粒酶A、颗粒酶B、IFN-γ、IL-1β、IL-6水平变化评估MMP14对单核细胞功能影响。结果 B-ALL患者MMP14水平低于对照者(P<0.05),sCD100水平亦显著低于对照者(P<0.05);B-ALL患者单核细胞中mCD100阳性的比例高于对照者(P<0.05),mCD100平均荧光强度(MFI)亦高于对照者(P<0.0001);B-ALL患者单核细胞中TRAIL和FasL mRNA相对表达量均低于对照者(P<0.05),B-ALL患者单核细胞分泌颗粒酶A、颗粒酶B、IFN-γ、IL-1β、IL-6均低于对照者(P<0.05)。重组MMP14刺激培养可诱导B-ALL患者单核细胞培养上清中s CD100水平升高(P<0.005),而mCD100细胞比例和mCD100 MFI则降低(P<0.05)。重组MMP14刺激可促进B-ALL患者单核细胞中TRAIL和FasL m RNA表达(P<0.05),增加颗粒酶A和颗粒酶B分泌水平(P<0.05),但对IFN-γ、IL-1β和IL-6分泌无明显影响(P>0.05)。阻断CD72不影响MMP14介导B-ALL患者单核细胞分泌颗粒酶A、颗粒酶B水平升高(P>0.05)。结论 B-ALL患者中存在sCD100和mCD100表达失衡,单核细胞功能不全。MMP14可通过诱导mCD100脱落增强B-ALL患者单核细胞功能。

CD100 is a member of semaphorin family, and plays an important role in infectious diseases and cancers. This study was designed to investigate the expressions of soluble and membrane-bound CD100 in patients with B-cell acute lymphoblastic leukemia(B-ALL), and to assess the influence of matrix metalloproteinase 14(MMP14) on CD100 expression and CD14+monocyte function in B-ALL patients. Total of 44 B-ALL patients and22 controls were enrolled in this study. The peripheral blood was collected, and plasma and peripheral blood mononuclear cells(PBMCs) were isolated. PlasmaMMP14 and soluble CD100(s CD100) were measured byenzyme-linked immunosorbent assay(ELISA);membrane-bound CD100(m CD100) expression on CD14+monocytes was measured by flow cytometry. CD14+monocytes were purified by magnetic activated cellsorting, and then cultured with lipopolysaccharide stimulation. m RNA relative levels of tumor necrosis factor-relatedapoptosis-inducing ligand(TRAIL) and Fas ligand(Fas L) were semi-quantified by reverse transcriptional real-timePCR. The expression of granzyme A, granzyme B, interferon-γ(IFN-γ), interleukin-1β(IL-1β), and IL-6 weremeasured by ELISA. The functional difference of CD14+monocytes was compared between B-ALL patients andcontrols. The influence of MMP14 on monocyte function was assessed by measuring TRAIL and Fas L m RNA levelsas well as granzyme A, granzyme B, IFN-γ, IL-1β, and IL-6 levels. Data showed that MMP14 and s CD100 levelswere lower in B-ALL patients compared with controls(P<0.05), while the ratio of m CD100-positive cells inmonocytes was higher(P<0.05), and the mean fluorescence intensity of m CD100 on monocytes was elevated in B-ALL patients compared with controls(P<0.000 1). Compared with controls, the m RNA relative levels of TRAIL andFas L in monocytes from B-ALL patients were down-regulated(P<0.05), and the levels of Granzyme A, granzyme B,IFN-γ, IL-1β and IL-6 also reduced in monocytes of B-ALL patients(P<0.05). Recombinant MMP14 stimulationelevated s CD100 secretion(P=0.0004), reduced m CD100 percentage and m CD100 MFI(P<0.05), promoted TRAILand Fas L m RNA expressions(P<0.05), enhanced granzyme A and granzyme B secretion(P<0.05), but did notstrongly affected the levels of IFN-γ, IL-1β or IL-6(P>0.05). CD72 blockade did not affect MMP14-inducedelevation of granzyme A and granzyme B secretions by monocytes in B-ALL patients(P>0.05). In conclusion, thereis monocyte dysfunction and an imbalance between s CD100 and m CD100 expression in B-ALL patients. AndMMP14 can enhance monocyte function via inducing mCD100 shedding in B-ALL patients.