A novel human glassy-cell carcinoma cell line producing IL-6 and IL-8 from uterine cervix

Summary A novel human cell line, TOM-2, was established from a rare uterine cervical cancer, glassy cell carcinoma (GCC). TOM-2 is the second established GCC cell line so far reported. The cells were intermediately or poorly differentiated with dysplastic nuclei and polygonal shape and secreted two tumor markers and cytokines, i.e., CA-125 and SCC, interleukin (IL)-1α, -6, and -8, and TNF-α. Growth of TOM-2 was so strongly dependent on population density that it was not possible to determine the plating efficiency. In mass culture, the following characteristics were observed: doubling time, 83 h; mode of chromosome number, 79; human papillomavirus type 18 DNA, detectable; tumorigenicity, easily transplantable into subcutis of nude mice; chemosensitivity in vitro, considerably sensitive to Cisplatin and 5-FU but not to 9 other antineoplastic agents. This novel cell line will be useful for developing new therapeutic strategies for the rare cancer, GCC.     

IL-8与胃癌关系的研究进展

IL-8是趋化性细胞因子,可以促进炎症细胞趋化和诱导细胞增殖,在胃癌组织中高表达。影响IL-8在胃癌组织中表达水平的因素包括幽门螺杆菌感染、NF-κB的干预、IL-8的基因多态性、表皮生长因子（EGF）的水平。IL-8主要参与胃癌的血管形成、促进金属蛋白酶的表达、影响E-cad和ICAM-1的水平、促进细胞有丝分裂、促使肿瘤转移等,对胃癌的发生、发展、预后均起重要的作用。选择性阻断IL-8及其受体具有抑癌作用,有望成为胃癌治疗的基础药物之一。     

Heparin potentiates in vivo neutrophil migration induced by IL-8

Chemokine IL-8 attracts neutrophils by a haptotactic gradient, made possible by its interaction with proteoglycans of the extracellular matrix. Heparan sulfate, but not heparin, potentiates the attraction exerted in vitro by IL-8. In the present study we first confirmed this in vitro phenomenon, observing that IL-8 activity was potentiated 100% by heparan sulfate, but not by heparin. Then, we evaluated the interference of heparan sulfate or heparin on in vivo neutrophil migration induced by IL-8. The activity of rat IL-8 (3.5 g/animal) preincubated with heparan sulfate (50 g/animal) or heparin (77 g/animal) was assayed on the rat dorsal air pouch. Contrary to in vitro experiments, heparin, but not heparan sulfate, potentiated the in vivo IL-8 activity two-fold. We investigated the relationship between this observation and that reported by others, that IL-8-induced migration depends on the presence of mast cells, which contain heparin-rich granules. We studied the neutrophil migration induced by IL-8 (3.5 g/animal) into the rat peritoneal cavity depleted of mast cells. Neutrophil migration was reduced by 32% when compared to that observed in normal animals. The response of depleted rats was reconstituted by preincubation of IL-8 with heparin (77 g/animal). These data suggest that heparin released from cytoplasmic granules may be the contribution of mast cells to IL-8-induced neutrophil migration.     

Paracrine interleukin-8 affects mesenchymal stem cells through the Akt pathway and enhances human umbilical vein endothelial cell proliferation and migration

Interleukin-8 (IL-8) promotes cell homing and angiogenesis, but its effects on activating human bone marrow mesenchymal stem cells (BMSCs) and promoting angiogenesis are unclear. We used bioinformatics to predict these processes. In vitro, BMSCs were stimulated in a high-glucose (HG) environment with 50 or 100 μg/ml IL-8 was used as the IL-8 group. A total of 5 μmol/l Triciribine was added to the two IL-8 groups as the Akt inhibitor group. Cultured human umbilical vein endothelial cells (HUVECs) were cultured in BMSCs conditioned medium (CM). The changes in proliferation, apoptosis, migration ability and levels of VEGF and IL-6 in HUVECs were observed in each group. Seventy processes and 26 pathways were involved in vascular development, through which IL-8 affected BMSCs. Compared with the HG control group, HUVEC proliferation absorbance value (A value), Gap closure rate, and Transwell cell migration rate in the IL-8 50 and IL-8 100 CM groups were significantly increased (P<0.01, n=30). However, HUVEC apoptosis was significantly decreased (P<0.01, n=30). Akt and phospho-Akt (P-Akt) protein contents in lysates of BMSCs treated with IL-8, as well as VEGF and IL-6 protein contents in the supernatant of BMSCs treated with IL-8, were all highly expressed (P<0.01, n=15). These analyses confirmed that IL-8 promoted the expression of 41 core proteins in BMSCs through the PI3K Akt pathway, which could promote the proliferation and migration of vascular endothelial cells. Therefore, in an HG environment, IL-8 activated the Akt signaling pathway, promoted paracrine mechanisms of BMSCs, and improved the proliferation and migration of HUVECs.     

The involvement of IL-6 and IL-8 in acute invasive gastroenteritis of children

The involvement of the proinflammatory cytokines, interleukin 8 (IL-8) and 6 (IL-6), was studied during the first 72 h of acute invasive gastroenteritis. Study population included 33 infants and young children aged six months to six years and seven age-matched controls. As a group, patients with acute invasive gastroenteritis had an increased serum level of IL-8 and IL-6 as compared with healthy controls (p < 0.002 and p < 0.001, respectively). Subjects were then divided into two groups based on stool cultures (proven and non-proven bacterial cultures). Patients with bacterial-proven acute invasive gastroenteritis tended to have increased IL-8 serum concentrations (p < 0.07) as compared with those with non-proven bacterial etiologies and IL-6 levels were only detected in subjects with positive bacterial cultures (p < 0.05). When dividing each sub-group into early and late blood drawing with respect to disease onset, no statistical differences were found in each group but subjects with bacterial-proven etiologies had significant higher IL-6 levels as compared with non-proven etiologies at the two time points (p < 0.019 and p < 0.015, respectively).In conclusion, the proinflammatory cytokines, IL-6 and IL-8, are involved in acute invasive gastroenteritis. The difference in IL-6, and to a lesser degree IL-8, between proven and non-proven bacterial etiologies, needs further investigation.     

Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis

Neutrophils occur in tissues of the female reproductive tract (FRT) under non-infected conditions. These cells generally enter tissues under the influence of chemoattractants called chemokines. Primary epithelial cells (EC) from FRT were a potent source of chemokines, IL-8 being the chief neutrophil chemoattractant secreted. Blocking with neutralizing anti-IL-8 showed that IL-8 did not account for all of the chemoattraction observed. A mixture of 25 ng/mL rIL-8 and 1 ng/mL rGM-CSF mediated 2.7-fold more chemotaxis than that expected if the two agents were additive. We then found that GM-CSF was produced by EC in amounts that synergised strongly with IL-8 to enhance chemotaxis. Treatment of uterine EC conditioned medium with saturating doses of anti-IL-8 plus anti-GM-CSF antibodies produced an 84% inhibition of chemotaxis. These findings demonstrate that the majority of neutrophil chemoattractant activity produced by FRT EC results from the synergistic effects of IL-8 and GM-CSF.     

Expression and one-step ion-exchange purification of (AAR)IL-8 (human IL-8 receptor antagonist)

Interleukin-8 (IL-8) is C-X-C chemokine, which is produced by a variety of cells. IL-8 plays an important role in the inflammatory response and may be a therapeutic target for some inflammatory diseases. To develop an IL-8 receptor antagonist, (AAR)IL-8 (IL-8 receptor antagonist) was constructed and successfully expressed in Escherichia coli. (AAR)IL-8 could be easily purified by one-step SP-Sepharose fast flow column after the lysate of recombinant bacterial cells was heated at 70 degrees C for 10 min. The purity of (AAR)IL-8 is more than 95%. This purification process resulted in final purified yields of 4.29 mg (AAR)IL-8/g cell paste. In addition, the purified (AAR)IL-8 can significantly inhibit the chemotaxis that was induced by human IL-8 in vitro and in vivo. These results showed that this purification process is very simple and effective. It could be easily amplified at a larger scale. (AAR)IL-8 might find use as a new therapeutic IL-8 receptor antagonist for some acute and chronic inflammatory diseases.     

IL-8 increases integrin expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Over-expression of IL-8 has been detected in many human tumors. However, the effects of IL-8 in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IL-8 increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and AP-1 pathways after IL-8 treatment were demonstrated, and IL-8-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and AP-1 cascades. Taken together, our results indicated that IL-8 enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the PI3K/Akt/AP-1 signal transduction pathway.     

Med1 controls CD8 T cell maintenance through IL-7R-mediated cell survival signalling

Under steady-state conditions, the pool size of peripheral CD8⁺ T cells is maintained through turnover and survival. Beyond TCR and IL-7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8⁺ T cell proportion in spleens but not in thymi of mice with T cell-specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild-type (WT) and Med1-deficient CD8⁺ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1-deficient CD8⁺ T cells compared with WT counterparts. Finally, Med1-deficient CD8⁺ T cells exhibited a decreased expression of interleukin-7 receptor α (IL-7Rα), down-regulation of phosphorylated-STAT5 (pSTAT5) and Bim up-regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8⁺ T cells through IL-7Rα/STAT5 pathway-mediated cell survival.     

双歧杆菌对肠上皮细胞生长和白介素-8分泌功能的影响

目的探讨双歧杆菌对人肠上皮细胞株HT29生长及其IL-8分泌水平的影响。方法HT29细胞在96孔板上生长24h后分为正常细胞对照组、高剂量双歧杆菌共培养组（细菌终浓度为1×10^10CFU／m1）、低剂量双歧杆菌共培养组（细菌终浓度为1×10^6CFU／ml）、轮状病毒感染对照组,分别加入不同剂量双歧杆菌和感染轮状病毒共培养,继续培养24h,光镜下观察细胞生长状态,MTT比色法检测细胞活性情况,ELISA检测细胞培养上清中IL-8表达水平。结果光镜下观察到双歧杆菌与HT29细胞共培养后细胞形态无明显改变,共培养24h后MTT检测双歧杆菌对HT29细胞增殖和调亡无明显影响,但轮状病毒感染对照组细胞病变脱落,活细胞数量明显减少。共培养6h,其余3组细胞培养上清中IL-8分泌较正常细胞对照组增加（P〈0．05）,高剂量双歧杆菌组增加较低剂量双歧杆菌组差异有显著性（P〈0．05）,但两个剂量组均明显低于轮状病毒感染阳性对照组的IL-8分泌增加水平（P〈0．05）;感染后24h,细胞培养上清中IL-8分泌水平高于正常细胞对照组（P〈0．05）,但高、低剂量双歧杆菌组之间差异无显著性（P〉0．05）,两个剂量组IL-8分泌增加水平均明显低于轮状病毒感染阳性对照组（P〈0．01）。结论两歧双歧杆菌共培养不影响HT29细胞的生长,双歧杆菌能够促进HT29细胞分泌细胞因子IL-8,但明显低于致病微生物刺激引起的细胞因子分泌水平改变,这种促进作用无时间-剂量依赖关系,提示双歧杆菌与肠道内致病微生物对肠道免疫功能的影响不同,双歧杆菌促进肠上皮细胞分泌IL-8可能与其参与的肠道黏膜免疫系统发育成熟相关。     

Epidermal growth factor receptor transactivation is implicated in IL-6-induced proliferation and ERK1/2 activation in non-transformed prostate epithelial cells

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.     

Src-inducible association of CrkL with procaspase-8 promotes cell migration

Procaspase-8, the zymogen form of the apoptosis-initiator caspase-8, undergoes phosphorylation following integrin-mediated cell attachment to an extracellular matrix substrate. Concordant with cell attachment to fibronectin, a population of procaspase-8 becomes associated with a peripheral insoluble compartment that includes focal complexes and lamellar microfilaments. Phosphorylation of procaspase-8 both impairs its maturation to the proapoptotic form and can promote cell migration. Here we show that the cytoskeletal adaptor protein CrkL promotes caspase-8 recruitment to the peripheral spreading edge of cells, and that the catalytic domain of caspase-8 directly interacts with the SH2 domain of CrkL. We show that the interaction is abolished by shRNA-mediated silencing of Src, in Src-deficient MEFs, and by pharmacologic inhibitors of the kinase. The results provide insight into how tyrosine kinases may act to coordinate the suppression caspase-8 mediated apoptosis, while promoting cell invasion.     

LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium.

We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kappaB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kappaB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC.     

Silencing CCL8 inhibited the proliferation and migration of PDGF-BB-stimulated human aortic smooth muscle cells

ABSTRACT

C-C motif Chemokine ligand 8 (CCL8) has been found in diseases’ pathogenesis. But its molecular mechanism in atherosclerosis (AS) remains to be elucidated. Human aortic smooth muscle cells (HASMCs) were stimulated by PDGF-BB to establish cell model. α-SMA in PDGF-BB-stimulated HASMCs was measured by immunofluorescence staining. Relative gene expressions in PDGF-BB-stimulated HASMCs were detected by quantitative real-time polymerase chain reaction and western blot. HASMCs proliferation, migration, and cell cycle were assessed by cell counting kit-8, wound-healing assay, and flow cytometry. HASMCs viability was increased after PDGF-BB stimulation, with α-SMA downregulation yet CCL8 upregulation. Silencing CCL8 inhibited PDGF-BB-stimulated HASMCs proliferation and migration, and increased cells percentage in G1 phases but decreased those in S phase. Also, silencing CCL8 decreased OPN and cyclinD1 expressions and AKT and ERK1/2 phosphorylation while increased those of α-SMA and Sm22α. However, upregulating CCL8 led to opposite effects, suggesting CCL8 could be an atherosclerosis therapeutic target.     

MiR-152 reduces human umbilical vein endothelial cell proliferation and migration by targeting ADAM17

As a cleavage enzyme of precursor TNF-α, the high expression level of ADAM17 in endothelial cells is an important factor in atherosclerosis. In this study, we demonstrate that ADAM17 is the target of miR-152. We found that miR-152 could reduce TNF precursor cleavage and inhibit cell proliferation and migration by targeting ADAM17 in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression pattern of miR-152 and corresponding target ADAM17 was opposite in HUVECs under hypoxic conditions. The levels of circulating miR-152 in AS patient sera were lower than those detected in the sera of normal individuals. Our results indicate that miR-152 may be involved in the development of human atherosclerosis and could be used as diagnostic biomarker or therapeutic target in atherosclerosis.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

Expression profile of IL-8 and growth factors in breast cancer cells and adipose-derived stem cells (ASCs) isolated from breast carcinoma

Adipose-derived stem cells (ASCs) are regarded as a major player of breast cancer microenvironment. By production of various growth factors and expression of regulatory molecules, it is postulated that ASCs protect breast cancer cells from the host immune responses. In this study, the expressions of insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), CXCL8 (IL-8) in breast cancer cells and adipose-derived stem cells isolated from breast tissue of women with breast cancer were investigated. The results were analyzed comparatively in normal ASCs isolated from healthy normal women. In case of breast cancer tissues, results were analyzed between high stage and low stage patients. The expressions of extracted mRNAs were determined using real-time quantitative RT-PCR. As a result, in breast cancer tissues, IGF-1 and IL-8 mRNAs had 28.6 and 56-fold more expressions in high stage compared to low stage patients. In ASCs, relative quantifications (RQ) of VEGF, IL-8, HGF and IGF-1 was about 2-fold higher in patients than controls. Data of this study conclude that presence of resident ASCs within the scaffold of breast tissue may support breast tumor growth and progression through the expressions of tumor promoting factors.