Functional expression of chemokine receptor CXCR4 on human epithelial cells

Chemokines and their receptors play an important role in the process of leucocyte recruitment at sites of inflammation. However, recent evidence suggests that these proteins can also regulate non-leucocyte cell functions such as angiogenesis, migration and proliferation. We have investigated the expression of the CXC chemokine receptor 4 (CXCR4) on primary cultures of type II alveolar epithelial cells, their transformed counterpart, the A549 cell line and also on other epithelial cell lines from various tissues. We found that all epithelial cell types tested express mRNA for CXCR4. Flow cytometric analysis and immunocytochemical staining shows that CXCR4 chemokine receptor is abundantly expressed on the surface of A549 epithelial cells. Furthermore, A549 cells responded to the CXCR4 ligand, stromal-derived factor-1alpha (SDF-1alpha) with a rapid and robust calcium mobilization and not to other CXC chemokines, suggesting that CXCR4 is functionally active and is able to couple to G-protein signalling mechanisms. A549 cells did not proliferate in response to either SDF-1alpha or interleukin-8 (IL-8) CXC chemokines. These findings may have important implications for epithelial physiology and pathology.     

Roles of Chemokine Receptor 4 （CXCR4） and Chemokine Ligand 12 （CXCL12） in Metastasis of Hepatocellular Carcinoma Cells

Chemokines are involved in human hepatocellular carcinoma （HCC） carcinogenesis. However, the exact mechanism of chemokines in HCC carcinogenesis remains unknown. Here we investigated the roles of chemokine receptor 4 （CXCR4） and chemokine ligand 12 （CXCL12） in the metastasis of HCC. We found that the expression levels of CXCR4 mRNA in HCC tissues, MHCC97 cells, and HUVEC cells were 2.52 ±1.13, 2.34 ±1.16 and 1.63 ±1.26, respectively and that the CXCR4 protein levels were 1.38 ± 0.13, 1.96± 0.32 and 1.86 ±0.21, respectively. In contrast, CXCR4 was not detected in normal hepatic tissues. In 78 HCC patients, we also found that the concentration of CXCL12 in cancerous ascitic fluid was 783-8,364 pg/ml and that CXCL12 mRNA level in HCC metastasis portal lymph nodes was 1.21 ± 0.87 but undetectable in normal hepatic tissues. Finally we discovered that recombinant human CXCL12 could induce MHCC97 cells and HUVEC cells to migrate with chemotactic indexes （CI） of 3.9 ±1.1 and 4.1± 1.6, respectively. Cancerous ascitic fluid could also induce the migration of MHCC97 cells with a CI of 1.9 ± 0.8. Thus, our data suggest that CXCR4 and CXCL12 may play an important role in the metastasis of HCC by promoting the migration of tumor cells.     

Lysophosphatidylcholine up-regulates CXCR4 chemokine receptor expression in human CD4 T cells

Oxidized low-density lipoprotein (OxLDL) is an inflammatory modulator in the atherosclerotic plaque. We examined the effect of lysophosphatidylcholine (lysoPC), a main phospholipid component of OxLDL, on inflammatory responses in human CD4 T cells. We found that lysoPC dose- and time-dependently increased expression of CXCR4, the chemokine receptor on CD4 T cells. This increase was inhibited by caffeic acid phenethyl ester or SN50, nuclear factor-kappaB inhibitors, and also by suppression of G2A expression, the specific receptor for lysoPC, using antisense oligonucleotide. lysoPC enhanced CD4 T cell chemotaxis in response to stromal cell-derived factor-1 (SDF-1), the exclusive ligand for CXCR4. lysoPC also enhanced SDF-1-stimulated production of inflammatory cytokines interleukin-2 and interferon-gamma by CD4 T cells activated by anti-CD3 immunoglobulin G. In conclusion, this study demonstrates that lysoPC directly modulates inflammatory responses in human CD4 T cells. The data suggest that the presence of lysoPC and SDF-1 in atherosclerotic lesions may trigger inflammatory responses mediated by CD4 T cells, which may play an important role in progression of atherosclerosis.     

Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma: pattern of expression and correlation with clinical outcome

Nasopharyngeal carcinoma (NPC) is a tumor derived from epithelial cells and Epstein-Barr virus infection has been reported to be a cause of this disease. Chemokine receptor CXCR4 was found to be involved in HIV infection and was highly expressed in human malignant breast tumors and the ligand for CXCR4, CXCL12 (SDF-1), exhibited high expression in organs in which breast cancer metastases are often found. The metastatic pattern of NPC is quite similar to that of malignant breast tumors. In this study, we investigated the expression of CXCR4 in nasopharyngeal carcinoma (NPC) tissues by immunohistostaining. We found different staining patterns, which included localization in the nucleus, membrane, cytoplasm or a combination of them. The staining intensity was also variable among samples. The metastatic rates in patients with high compared to low or absent expression was 38.6% versus 19.8%, respectively (P = 0.004). High expression of CXCR4 was associated with poor overall survival (OS = 67.05% versus 82.08%, P = 0.0225). These results suggest that CXCR4 may be involved in the progression of NPC and that a high level of CXCR4 expression could be used as a prognostic factor.     

CC chemokine receptor 5 and CXC chemokine receptor 6 expression by lung CD8+ cells correlates with chronic obstructive pulmonary disease severity

Chronic obstructive pulmonary disease (COPD) is a progressive disease associated with a cellular inflammatory response. CD8(+) T cells are implicated in COPD pathogenesis, and their numbers significantly correlate with the degree of airflow limitation. Dendritic cells (DCs) are important sentinel immune cells, but little is known about their role in initiating and maintaining the CD8 T-cell response in COPD. To investigate the mechanisms for CD8(+) T-cell recruitment to the lung, we used resected human lung tissue to analyze chemokine receptor expression by CD8(+) T cells and chemokine production by CD1a(+) DCs. Among 11 surveyed chemokine receptors, only CC chemokine receptor (CCR5), CXC chemokine receptor (CXCR) 3, and CXCR6 correlated with COPD severity as defined by criteria from the Global Initiative for Chronic Obstructive Lung Disease. The CD8(+) T cells displayed a Tc1, CD45RA(+) effector memory phenotype. CD1a(+) DCs produced the respective ligands for CCR5 and CXCR3, CCL3 and CXCL9, and levels correlated with disease severity. CD1a(+) DCs also constitutively expressed the CXCR6 ligand, CXCL16. In conclusion, we have identified major chemokine elements that potentially mediate CD8(+) T-cell infiltration during COPD progression and demonstrated that CD1a(+) mucosal-associated DCs may sustain CD8(+) T-cell recruitment/retention. Chemokine targeting may prove to be a viable treatment approach.     

趋化因子受体CCR1在人大肠癌组织中的表达及其与淋巴结转移的相关性

目的: 研究趋化因子受体CCR1在大肠癌中的表达,探讨其与淋巴转移的关系.方法: 采用SP免疫组织化学方法检测98例临床新鲜标本,其中包括15例癌旁正常大肠黏膜组织及83例大肠癌组织中CCR1的表达情况,计算大肠癌组织中的微淋巴管密度(LMVD)与微血管密度(MVD),并进行CCR1表达与大肠癌淋巴结转移的相关性分析.结果: CCR1在浸润程度深、分化程度低、有淋巴结转移及Dukes分期较晚的大肠癌组织中的阳性表达率高于浸润程度浅(P<0.05)、分化程度高(P<0.05)、无淋巴结转移(P<0.01)及Dukes分期较早(P<0.05)的大肠癌组织.CCR1表达阳性的大肠癌组织LMVD高于CCR1表达阴性的大肠癌组织LMVD(P<0.01).而CCR1表达阳性的大肠癌组织与CCR1表达阴性的大肠癌组织中,MVD无统计学差异.结论: 趋化因子受体CCR1在大肠癌组织中表达增高,并可能与大肠癌的淋巴结转移增加有关,具有一定的诊断价值.     

Nuclear expression of CXCR4 in tumor cells of non-small cell lung cancer is correlated with lymph node metastasis

The stromal-derived factor 1α (CXCL12)/chemokine receptor CXCR4 system plays an important role in the metastatic process of a variety of cancers, with CXCR4 frequently expressed by tumor cells homing to CXCL12-rich compartments. The current study evaluated a possible association of CXCR4 expression with lymph node metastasis in primary non–small cell lung cancer. CXCR4 expression levels were evaluated using immunohistology in 46 non–small cell lung cancer specimens of patients without or with lymph node involvement (N0 = 24, N1/N2/N3 = 22). Evaluation of immunostaining was performed semiquantitatively by visual assessment. Statistical analyses with multiple testing adjustments for confirmatory comparisons were performed to assess relevant parameters associated with lymph node metastases. In all samples of non–small cell lung cancer, tumor cells stained positively for cytoplasmic CXCR4. The intensity of the CXCR4 staining varied considerably between specimens: 2 (4%) tumors demonstrated weak cytoplasmic CXCR4, 22 (48%) intermediate, and 22 (48%) strong staining. Membranous staining was absent; however, nuclear staining of CXCR4 was observed in 5 non–small cell lung cancer samples. Statistical analyses of the association between presence of lymph node metastases and CXCR4 expression levels revealed that cytoplasmic CXCR4 expression was not associated with the presence of lymph node metastases. However, nuclear CXCR4 was significantly correlated with increasing lymph node stage (P = .008), linear-to-linear association. The association between aberrant expression of CXCR4 in the nucleus of non–small cell lung cancer and metastasis to lymph nodes points toward a potential tumor metastasis promoting function of nuclear CXCR4.     

Salmonella reduces tumor metastasis by downregulation C-X-C chemokine receptor type 4

Tumor metastasis is the main reason for the death of most cancer patients. C-X-C chemokine receptor type 4 (CXCR4) has been demonstrated to be overexpressed in numerous types of cancer. CXCR4 selectively binds with stromal cell-derived factor 1 (SDF1), also known as C-X-C family chemokine ligand 12 (CXCL12) (CXCL12/SDF-1), which induced tumor proliferation and metastasis. Recently, the use of conventional cancer treatments had some limitation; bacteria treatment for cancer becomes a trend that overcomes these limitations. Plenty of studies show that Salmonella has anti-tumor and anti-metastatic activity. The current study aimed to investigate Salmonella suppresses CXCR4 protein expression and tumor cell migration ability in B16F10 melanoma and LL2 lung carcinoma cells. Salmonella reduced CXCR4 protein expression through downregulating Protein Kinase-B (Akt)/Mammalian Target of Rapamycin (mTOR) signaling pathway. In cells transfected with constitutively active Akt plasmids, a reverse effect of Salmonella-induced inhibition of CXCR4 was observed. Tumor cells have chemotactic response to CXCL12 in migration assay, and we found that Salmonella reduced tumor chemotactic response after CXCL12 treatment. The C57BL/6 mice were intravenously injected with B16F10 and LL2 cells pre-incubated with or without Salmonella, the tumor size and lung weight of Salmonella group had obviously decreased, indicating anti-metastatic effect that confirmed the findings from the in vitro experiments.     

The murine chemokine receptor CXCR4 is tightly regulated during T cell development and activation

We have characterized the murine homolog of the HIV-co-receptor CXCR4 during T cell development and activation. Our data demonstrate that this chemokine receptor, although highly conserved between human and mouse, is differently expressed and regulated in both species. Mitogenic activation resulted in an increase of surface CXCR4 on murine T cells within 2 days, whereas the receptor was strongly down-regulated on human T cells during this period. Furthermore, intraperitoneal immunization of mice resulted in a strong increase of splenic and mesenteric cytotoxic T cells co-expressing CXCR4. It is interesting that, on thymocytes, expression of CXCR4 is restricted to CD4+CD8+ cells. Stromal cell-derived factor-1alpha, a natural ligand of CXCR4, induced chemotaxis of thymocytes and was found to counteract dexamethasone-induced apoptosis to a certain extent in these cells. Thus, our data show that expression of CXCR4 is tightly controlled on murine T cells and indicate that this highly conserved chemokine receptor might serve different functions in humans and mice.     

Targeting T cell responses by selective chemokine receptor expression

Immune responses require the orchestrated migration of T cells throughout the body. Conventional CD4+ and CD8+ alphabeta T cells undergo clonal expansion in the secondary lymphoid tissues, during which they are programmed to migrate into specific non-lymphoid tissues and other lymphoid effector sites such as B cell follicles. By contrast, T cell populations expressing receptors with limited diversity (i.e. gammadelta T cells and NK T cells) appear to be preprogrammed to localize in non-lymphoid tissues where they monitor tissue integrity or serve regulatory functions. By promoting chemotaxis and integrin activation, chemokines and their receptors (in conjunction with surface adhesion molecules) control these T cell homing events. Thus, expression of chemokine receptors defines T cells with tropism for particular tissues and/or microenvironments, and identifies T cell subsets with distinct functional properties.     

CCR5 and CXCR4 chemokine receptor expression and beta-chemokine production during early T cell repopulation induced by highly active anti-retroviral therapy.

Expression of chemokine receptors and beta-chemokine production by peripheral blood mononuclear cells (PBMC) were determined in HIV-1-infected individuals before and after highly active anti-retroviral therapy (HAART) and their relationship to viral load, T cell phenotype and the expression of immunological activation markers was examined. We found that the expression of CCR5 is up-regulated in HIV-1-infected individuals while CXCR4 appears down-regulated on both CD4 and CD8 T cells compared with normal controls. These alterations are associated with the high levels of viral load. In addition, a relationship was observed between the degree of immune activation and chemokine receptor expression on T cells. However, after 3 months of combined anti-retroviral regimen, expression of CXCR4 significantly increased while CCR5 decreased when compared with pretherapy determinations. This was seen in strict association with a dramatic decrease of viral load and an increase of both CD45RA+/CD62L+ (naive) and CD45RA-/CD62L+ or CD45RA+/CD62L- (memory) T cells accompanied by a significant decrease of the expression of immune activation markers such as HLA-DR and CD38. At enrolment, both spontaneous and lectin-induced RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta production by PBMC were higher in HIV-1-infected individuals compared with normal controls, although differences for MIP-1beta were not statistically significant. However, RANTES and MIP-1alpha production decreased during HAART at levels closer to that determined with normal controls, while MIP-1beta production was less consistently modified. These data indicate that the expression of chemokine receptors CCR5 and CXCR4 and the production of beta-chemokines are altered in HIV-infected individuals, and suggest that their early modifications during HAART reflect both the peripheral redistribution of naive/memory T cell compartments and the decrease in levels of T cell activation. Such modifications in the expression of host determinants of viral tropism and the production of anti-viral molecules may play a role in the emergence of virus variants when a failure of HAART occurs.     

Tumor-associated macrophages promote the metastasis of ovarian carcinoma cells by enhancing CXCL16/CXCR6 expression

This study investigated the underlying mechanism by which C-X-C motif chemokine ligand 16 (CXCL16)/C-X-C motif chemokine receptor 6 (CXCR6) signaling is activated by tumor-associated macrophages and assists in regulating the metastasis of ovarian carcinoma. Specimens of ovarian carcinoma tissue and adjacent tissue were collected from 20 ovarian carcinoma patients. Human THP-1 cells were induced to differentiate into macrophages, which were then co-cultured with SKOV3 cells and low concentrations of tumor necrosis factor-α (TNF-α) to simulate the inflammatory microenvironment of ovarian carcinoma. Additionally, small interfering RNA (siRNA) targeting CXCR6 was transfected into SKOV3 cells; after which, the levels of nuclear factor kappa B p65 (NF-κB p65) protein and phosphorylated PI3K and Akt were measured. The migration and invasion abilities of the SKOV3 cells were also tested. The levels of TNF-α, interluekin-6 (IL-6), NF-κB p65, CXCL16, and CXCR6 expression in the ovarian carcinoma tissues were higher than those in the precancerous tissues. CXCR6 expression was positively correlated with TNF-α, IL-6, and CXCL16 expression. Co-culture of SKOV3 cells with macrophages significantly promoted CXCL16, CXCR6, NF-κB, and p65 expression by the SKOV3 cells, increased their levels of phosphorylated PI3K and Akt, and increased the migration and invasion abilities of SKOV3 cells. Silencing of CXCR6 or blocking the PI3K/Akt signal pathway markedly attenuated the expression of NF-κB p65 and phosphorylation of PI3K and Akt, as well as the migration and invasion abilities of SKOV3 cells. These findings demonstrate that macrophages can promote the migration and invasion of ovarian carcinoma cells by affecting the CXCL16/CXCR6 pathway.