Liu Yu-Qing, Poon Ronnie-T, Hughes Jeremy, Li Qin-Yu, Yu Wan-Ching, Fan Sheung-Tat
Centre for the Study of Liver Disease and Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China.
World J Gastroenterol. 2005 Jan 14;11(2):164-70. doi: 10.3748/wjg.v11.i2.164.
Despite the presence of lymphocyte infiltration, human hepatocellular carcinoma (HCC) is typically a rapidly progressive disease. The mechanism of regulation of lymphocyte migration is poorly understood. In this study, we investigated various factors regulating T cell migration in HCC patients. We examined serum CXC chemokine levels in HCC patients and demonstrated the production of CXC chemokines by HCC cell lines. We determined the effect of both HCC patient serum and tumor cell conditioned supernatant upon lymphocyte expression of chemokine receptor CXCR3 as well as lymphocyte migration. Lastly, we examined the chemotactic responses of lymphocytes derived from HCC patients.
The serum chemokines IP-10 (CXCL10) and Mig (CXCL9) levels were measured by cytometric bead array (CBA) and the tumor tissue IP-10 concentration was measured by ELISA. The surface expression of CXCR3 on lymphocytes was determined by flow cytometry. The migratory function of lymphocytes to the corresponding chemokines was assessed using an in vitro chemotactic assay. Phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis.
Increased levels of IP-10 and Mig were detected in HCC patient serum and culture supernatants of HCC cell lines. The IP-10 concentration in the tumor was significantly higher than that in the non-involved adjacent liver tissues. HCC cell lines secreted functional chemokines that induced a CXCR3-specific chemotactic response of lymphocytes. Furthermore, tumor-cell-derived chemokines induced initial rapid phosphorylation of lymphocyte ERK followed by later inhibition of ERK phosphorylation. The culture of normal lymphocytes with HCC cell line supernatants or medium containing serum from HCC patients resulted in a significant reduction in the proportion of lymphocytes exhibiting surface expression of CXCR3. The reduction in T cell expression of CXCR3 resulted in reduced migration toward the ligand IP-10, and both CD4+ and CD8+ T cells from HCC patients exhibited diminished chemotactic responses to IP-10 in vitro compared to T cells from healthy control subjects.
This study demonstrates functional desensitization of the chemokine receptor CXCR3 in lymphocytes from HCC patients by CXCR3 ligands secreted by tumor cells. This may cause lymphocyte dysfunction and subsequently impaired immune defense against the tumor.
尽管存在淋巴细胞浸润，但人类肝细胞癌（HCC）通常是一种快速进展的疾病。淋巴细胞迁移的调控机制尚不清楚。在本研究中，我们调查了调节HCC患者T细胞迁移的各种因素。我们检测了HCC患者血清中CXC趋化因子水平，并证明了HCC细胞系可产生CXC趋化因子。我们确定了HCC患者血清和肿瘤细胞条件上清液对趋化因子受体CXCR3在淋巴细胞上的表达以及淋巴细胞迁移的影响。最后，我们检测了来自HCC患者的淋巴细胞的趋化反应。
通过细胞计数珠阵列（CBA）测量血清趋化因子IP-10（CXCL10）和Mig（CXCL9）水平，通过ELISA测量肿瘤组织中IP-10浓度。通过流式细胞术确定淋巴细胞上CXCR3的表面表达。使用体外趋化试验评估淋巴细胞对相应趋化因子的迁移功能。通过蛋白质印迹分析确定细胞外信号调节激酶（ERK）的磷酸化。
在HCC患者血清和HCC细胞系培养上清液中检测到IP-10和Mig水平升高。肿瘤中IP-10浓度明显高于未受累的邻近肝组织。HCC细胞系分泌功能性趋化因子，可诱导淋巴细胞产生CXCR3特异性趋化反应。此外，肿瘤细胞衍生的趋化因子诱导淋巴细胞ERK最初快速磷酸化，随后ERK磷酸化受到抑制。用HCC细胞系上清液或含有HCC患者血清的培养基培养正常淋巴细胞，导致表现出CXCR3表面表达的淋巴细胞比例显著降低。CXCR3在T细胞上表达的降低导致向配体IP-10的迁移减少，与健康对照受试者的T细胞相比，HCC患者的CD4+和CD8+T细胞在体外对IP-10的趋化反应均减弱。
本研究证明肿瘤细胞分泌的CXCR3配体使HCC患者淋巴细胞中的趋化因子受体CXCR3发生功能性脱敏。这可能导致淋巴细胞功能障碍，进而损害对肿瘤的免疫防御。
