Lack of interleukin-2 (IL-2) expression and selective expression of IL-10 mRNA in human renal cell carcinoma

Freshly isolated tumor-infiltrating lymphocytes (TIL) are often functionally deficient. Since one of the key functional parameters of an immune response is the local production of cytokines, we studied the expression of cytokine genes in freshly isolated renal cancer tissue. Using a PCR-assisted mRNA amplification assay, the constitutive expression of mRNA for 10 different cytokines was assessed in renal cancer tissue. We compared the cytokine mRNA expression in freshly isolated samples of renal carcinomas, renal cancer cell lines established from the tumor samples, peripheral blood mononuclear cells (PBMC) and non-tumor kidney tissue isolated from the same patients. IL-10 mRNA expression was detected only in tumor samples, while renal cancer lines, PBMC and non-tumorous kidney tissues were devoid of this cytokine. One-third of the tumor samples but none of the normal kidney samples also expressed G-CSF mRNA. IL-6, TNF-α and IFN-γ mRNA were expressed non-selectively in tumors, PBMC and normal renal tissue. Expression of IL-2, IL-3 and IL-4 mRNA was not detected in any of the tissues analyzed. Established renal cancer lines exhibited expression of IL-lα, IL-6, TNF-α and GM-CSF. Culture of tumor-derived T cells with anti-CD3 monoclonal antibody (MAb) resulted in expression of IL-2, IL-3 and IL-4 mRNA. In contrast, none of these cytokines was detected in culture with recombinant human IL-2 alone. Since IL-10 is known to suppress antigen presentation, these findings have important implications for the possible in vivo role of IL-10 as a suppressor of local anti-tumor response.     

E-selectin expression induced by pancreas-carcinoma-derived interleukin-1α results in enhanced adhesion of pancreas-carcinoma cells to endothelial cells

Cellular adhesion of sialyl-Lewis-a(SLe^a)-positive pancreas carcinoma to endothelial cells (EC) is augmented by activation of EC via up-regulated E-selectin expression on EC. Co-cultivation of pancreas-carcinoma cells, PCI-24, with human umbilical-vein endothelial cells (HUVEC) for 5 hr at the PCI-to-HUVEC ratio of 1:10 induced E-selectin expression on the endothelial-cell surface, augmenting SLe^a-positive pancreas-carcinoma cell attachment with HUVEC. Culture supernatants of 6 tested pancreas-carcinoma cell lines contained soluble, E-selectin-inducing factor(s). The E-selectin-inducing effect by the supernatants was blocked by the protein-kinase-C inhibitor, H7. Antibodies against SLe^a and E-selectin but not SLe^x or ICAM-I blocked the increased pancreas-carcinoma-to-endothelial attachment. Paraformaldehyde(PFA)-fixed PCI-24 cells also induced E-selectin on vascular endothelial cells upon direct contact with endothelial cells, indicating the presence of a membrane-bound form. The 6 pancreas-carcinoma lines all produced IL-1α mRNA and protein but not IL-1β or TNF-α protein and/or mRNA Absorption of IL-lα from the supernatants by IL-lα-specific antibody almost completely abolished E-selectin-inducing activity. Anti-IL-lα antibody also abolished the E-selectin-inducing activity of PFA-fixed PCI. IL-1α production by PCI cells was up-regulated by TNF-α. These observations suggest that substance(s) produced by pancreas-carcinoma cells, in this case, IL-1α, may contribute to pancreas-carcinoma-cell colonization in non-inflamed, distant locations in vivo, by activating vascular endothelial cells.     

Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer.

Altered immune, inflammatory, and angiogenesis responses are observed in patients with head and neck squamous cell carcinoma (HNSCC), and many of these responses have been linked with aggressive malignant behavior and a decrease in prognosis. In this study, we examined the hypothesis that HNSCC cells produce cytokines that regulate immune, inflammatory, and angiogenesis responses. We identified important regulatory cytokines in supernatants of well-defined and freshly cultured HNSCC cell lines by ELISA and determined whether these cytokines are detected in tumor cell lines and tissue specimens by immunohistochemistry. The serum concentration of the cytokines and cytokine-dependent acute phase inflammatory responses (i.e., fibrinogen, C-reactive protein, and erythrocyte sedimentation rate) from patients with HNSCC was determined, and the potential relationship of serum cytokine levels to tumor volume was analyzed. Cytokines interleukin (IL)-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor were detected in similar concentration ranges in the supernatants of a panel of established University of Michigan squamous cell carcinoma (UM-SCC) cell lines and supernatants of freshly isolated primary HNSCC cultures. Evidence for the expression of IL-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and VEGF in HNSCC cells within tumor specimens in situ was obtained by immunohistochemistry. In a prospective comparison of the cytokine level and cytokine-inducible acute-phase proteins in serum, we report that cytokines IL-6, IL-8, and VEGF were detected at higher concentrations in the serum of patients with HNSCC compared with patients with laryngeal papilloma or age-matched control subjects (at P < 0.05). The serum concentrations of IL-8 and VEGF were found to be weakly correlated with large primary tumor volume (R2 = 0.2 and 0.4, respectively). Elevated IL-1- and IL-6-inducible acute-phase responses were also detected in cancer patients but not in patients with papilloma or control subjects (at P < 0.05). We therefore conclude that cytokines important in proinflammatory and proangiogenic responses are detectable in cell lines, tissue specimens, and serum from patients with HNSCC. These cytokines may increase the pathogenicity of HNSCC and prove useful as biomarkers or targets for therapy.     

A Comparison of the Characteristics of Cytokine Storm between Lichen Planus and Oral Squamous Cell Carcinoma

Introduction: Lichen planus (LP) is a relatively common chronic mucocutaneous disease that affects the skin and mucous membranes, including oral mucosa. The etiology of the disease is unknown. Some evidence suggests that the immune system and inflammation may play a role in the formation and progression of lichen planus. Some authorities believe that LP is a precancerous condition. The purpose of this study was to investigate the serum levels of the inflammatory cytokines CRP, IL-1, IL-6, and TNF- in patients with oral lichen planus and oral squamous cell carcinoma (OSCC), as well as to assess the relationship between these cytokine levels and clinical symptoms. Methods: A total of 75 subjects, with 25 in each group of oral lichen planus, healthy control, and oral squamous cell carcinoma, participated in this cross-sectional study. Serum levels of IL-1α, TNF-α, IL-6, and CRP were determined and compared. In comparison to the healthy control group, the lichen planus and oral squamous cell carcinoma groups had higher levels of CRP, IL-1α, IL-6, and TNF-α. Results: We discovered that the mean mRNA and protein levels of CRP, IL-1α, IL-6, and TNF-α were significantly higher in the blood and tissue of lichen planus and OSCC patients than in normal controls. Conclusion: Higher levels of CRP, IL-1α, IL-6, and TNF-α may be linked to OLP and oral carcinogenesis. More research with larger groups is required.     

Three cases of thyroid cancer, including a component of squamous cell carcinoma

A squamous cell carcinoma of the thyroid gland is rare and its prognosis is poor. Three cases of a rare thyroid cancer are reported, the first case being an adenosquamous cell carcinoma of a 75-year-old male and the second case being a pure squamous cell carcinoma of a 64-year-old male. These two cases were pathologically diagnosed on autopsy. The overall duration of these two cases was 6 months. The third case involved a 62-year-old male. His pathological diagnosis was a mixed squamous cell carcinoma and an undifferentiated carcinoma. Postoperatively 50 Gy of irradiation was performed. This patient is still alive and has shown no evidence of recurrence for 16 months.     

肺癌患者血清中21种细胞因子的表达水平及其临床意义

目的:检测肺癌患者血清中21种细胞因子[干扰素诱导的T细胞趋化因子(IFN-inducible T cellαchemoattractant,ITAC)、GM-CSF、Fractalkine、IFN-γ、IL-10、巨噬细胞炎性蛋白-3α(macrophage inflammatory protein-3α,MIP-3α)]、IL-12(p70)、IL-13、IL-17A、IL-1β、IL-2、IL-4、IL-21 、IL-23、IL-5、IL-6、IL-7、IL-8、MIP-1α、MIP-1β和TNF-α的表达情况并分析其意义.方法:收集2015年3月至6月的山西省肿瘤医院40例肺癌患者;采用液相芯片技术检测30名健康人和40例初诊肺癌患者治疗前血清中21种细胞因子的表达水平,分析其与肺癌临床特征之间的关系及表达存在明显差异细胞因子间的相关性.结果:肺癌患者血清中11种细胞因子[(GM-CSF、Fractalkine、IFN-γ、MIP-3α、IL-12 (p70)、IL-1β、IL-2、IL-6、IL-7、IL-8、TNF-α]的表达水平明显升高(P＜0.05或P＜0.01),肺癌患者非转移组与转移组血清中21种细胞因子的表达水平比较无明显差异(P＞0.05),肺腺癌(adenocarcinoma,AC)组血清IFN-γ与MIP-1β水平明显高于鳞癌(squamous cell carcinoma,SCC)组(均P＜0.05),肺SCC组血清ITAC表达水平明显高于小细胞肺癌(small cell lung cancer,SCLC)组(P＜0.05).高表达的11种细胞因子在两组间表达的相关性不同.结论:肺癌患者血清中IL-6、IL-8等11种细胞因子表达升高,可能参与了肺癌发生发展,这些高表达的细胞因子或许可用于肺癌的辅助诊断,并为肺癌治疗提供新的靶标.     

IL-6 as an intracrine growth factor for renal carcinoma cell lines

Interleukin-6 (IL-6) is produced at high levels by renal cell carcinoma cell lines. The molecular mechanisms involved in its possible role as an autocrine growth factor were investigated. IL-6 and IL-6 receptor expression was investigated in 8 renal cell carcinoma (RCC) cell lines. The modulation of RCC cell line proliferation by an anti-IL-6 Ab, an IL-6 antisense oligonucleotide (ASON) directed against the second exon of IL-6 and cytokines inhibiting IL-6 production (IL-4 and IL-13) was investigated. All 8 RCC cell lines expressed IL-6 mRNA, produced IL-6 and expressed the soluble and membrane-bound gp130 chain of IL-6 receptor. The gp80 chain of IL-6 receptor was undetectable at the surface of the 8 RCC cell lines tested, while the soluble form of gp80 was detectable in the supernatant of one of these cell lines. The addition of a blocking IL-6 Ab did not inhibit the proliferation of any of the 8 RCC cell lines. In contrast, IL-6 ASON inhibited specifically IL-6 production and the proliferation of all RCC cell lines. Exogenous IL-6 failed to restore RCC cell line proliferation blocked by ASON, indicating that IL-6 acts through an intracrine loop in RCC cell lines. IL-13 and IL-4 inhibited the proliferation of 7 of the 8 cell lines without interfering with IL-6 or IL-6 receptor expression. IL-6 ASON inhibited the proliferation of the 8 RCC cell lines tested additively with IL-4 or IL-13. IL-6 is an intracrine growth factor in renal cell carcinoma cell lines.     

Effects of bacillus Calmette-Guérin and interferon-α-2B on human bladder cancer in vitro

The cytolytic and anti-proliferative effects of bacillus Calmette-Guérin (BCG) and/or interferon-α-2b (IFN-α-2b) on 5 human bladder carcinoma cell lines, RT4, RT112, MGH, SD and J82, were determined. The cell lines showed different sensitivities to BCG and IFN-α-2b. BCG had direct dose-dependent cytolytic and anti-proliferative effects on MGH, J82 and SD (grade 3 cell lines), whereas RT4 and RT112 (grades 1 and 2, respectively) were less sensitive. Surprisingly, higher concentrations of BCG enhanced cell growth of RT4. IFN-α-2b also had cytolytic and anti-proliferative effects on all 5 cell lines. Thus, the RT4 and RT112 cell lines that were not sensitive to BCG were highly sensitive to IFN-α-2b. A combination of BCG and IFN-α-2b had additive anti-proliferative effects on MGH, J82 and RT112. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production by these 5 cell lines was measured after stimulation with BCG and/or IFN-α-2b, by ELISA immunoassays. Production of IL-6 and TNF-α was significantly increased in MGH and J82 cell lines by the combination of BCG and IFNα-2b. The enhanced cytolytic and anti-proliferative effects of the combination of BCG and IFN-α-2b may be related to the induction of cytokines.Int. J. Cancer 71: 851-857, 1997. © 1997 Wiley-Liss Inc.     

转4-1BBL 基因的小鼠肝癌细胞瘤苗刺激脾细胞产生细胞因子的研究

 目的 研究转4-1BBL基因的小鼠肝癌细胞瘤苗体外刺激同系小鼠脾细胞产生细胞因子（IL-2、TNF-α和GM-CSF）的能力。方法 以丝裂霉素C（MMC）处理高表达转m4-1 BBL基因的小鼠Hepa1-6肝癌细胞，制成肿瘤细胞瘤苗（TCV），体外与同系小鼠脾淋巴细胞共同培养后，观察其对脾细胞产生细胞因子（IL-2、TNF-α和GM—CSF）的影响。结果 TCV-4-1 BBL刺激后，脾细胞体外分泌细胞因子IL-2、TNF-α和GM-CSF的水平明显增高。结论 转4-1BBL基因的小鼠肝癌细胞瘤苗能刺激脾细胞产生细胞因子IL-2、TNF-α和GM-CSF。     

Expression of ADAMTS-1, ADAMTS-4, ADAMTS-5 and TIMP3 by hepatocellular carcinoma cell lines

Little is known about the expression or role of ADAMTS-1, -4 and -5 and their endogenous inhibitor TIMP3 in the liver in physiological and pathological conditions. Their expression was, therefore, investigated in the hepatocellular carcinoma cell lines HepG2 and HuH-7 using qRT-PCR and western blotting, and their cellular localisation by immunocytochemistry. Cytokine treatments were used to assess mRNA and protein modulation. ADAMTS-1, -4, -5 and TIMP3 mRNA and protein were detected in both HepG2 and HuH-7 cells. IL-1β and IL-6 treatments significantly modulated ADAMTS-1 mRNA expression and IL-1β treatment ADAMTS-4 mRNA expression in HepG2 cells. Modulations of mRNA by ≥ 5-fold did not translate to increased protein expression. This study showed that ADAMTS-1, -4, -5 and TIMP3 were expressed at differential levels in hepatocellular carcinoma cell lines. The pro-inflammatory cytokines IL-1β, TNF-α or IL-6 induced changes in mRNA expression, although these did not translate to the protein level.     

Surface membrane-expressed CD40 is present on tumor cells from squamous cell cancer of the head and neck in vitro and in vivo and regulates cell growth in tumor cell lines.

Because regional spread to lymph nodes without systemic spread is a relatively common event in squamous cell cancer of the head and neck (SCCHN), it is possible that lymphoid-related receptors or cytokines might directly impact the growth of these tumors. In the present study, we have shown by flow cytometry and Western blotting that the central lymphoid regulatory molecule, CD40, is expressed on the surface of all seven SCCHN tumor cell lines studied. Tumor cell lines also expressed epidermal growth factor (EGF) receptor, MHC class I, and CD95 (Fas) but did not uniformly express other important lymphoid regulatory molecules such as CD80, CD86, or interleukin (IL) 2 receptor components. CD40 ligation by trimeric CD40 ligand (CD40L) resulted in a 20-45% inhibition of tumor cell growth in three of seven cell lines tested. The cytokines IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-11, and IL-15 neither inhibited nor stimulated growth in any of the cell lines tested. EGF had pleiotropic effects on cell growth; it inhibited growth in two cell lines, stimulated growth in one cell line, and had no effect in four cell lines. When coligation by EGF and CD40L was studied, additive or supra-additive growth inhibition was seen in four cell lines. Three cell lines were unaffected by EGF, CD40, or coligation with both reagents. Examination of tumor tissues from 12 previously untreated patients representing a broad spectrum of patients presenting with SCCHN demonstrated CD40 expression in all 12 tumor specimens. This study supports the notion that CD40 is a regulatory molecule for the growth of SCCHN. The important role of CD40-CD40L interactions in the regulation of immune cells in the lymph node and the unique high-level expression of CD40L by these immune cells lend support to the hypothesis that this ligand/receptor pair is an important mediator of cell growth in SCCHN.     

Plasmatic cytokine network in patients with laryngeal carcinoma after surgical treatment

Alterations in host immunity, inflammation, angiogenesis and metabolism are all prominent clinical features in patients with laryngeal squamous cell carcinoma (LSCC). Although the origin of the signals and mechanisms underlying these responses are not well understood, their local and systematic nature suggest that squamous cell carcinoma-produced cytokines with proinflammatory and immunoregulatory activity may contribute to the pathogenesis of LSCC. In order to gain a better insight into the roles and relationships of the cytokines, we investigated serum IL-6, IL-10 and IL-12 concentrations in LSCC patients under baseline conditions and after surgery. In comparison with controls, all the patients had higher plasma IL-10 concentrations before surgical treatment (T0), while plasma IL-6 and IL-12 concentrations were higher in 22 (84.6%) and 24 patients (92.3%). The differences in plasma IL-6, IL-10 and IL-12 concentrations at T0 and T1 were statistically significant (p<0.001, p<0.0046 and p<0.011). Our finding suggest that plasma cytokines are overexpressed in LSCC patients. There was an independent increase in plasma IL-6 levels before and after surgical treatment. Furthermore, the up- and down-regulation of plasma IL-10 and IL-12 suggest a regulatory relationship between them.     

Differentiation-dependent expression and mitogenic action of interleukin-6 in human colon carcinoma cells: relevance for tumour progression

Interleukin (IL)-6 mRNA expression in general is low in normal, adenomatous and cancerous human colon mucosa, except in rather undifferentiated lesions, in which IL-6 is overexpressed. Cytokeratin (CK) 8-positive carcinoma cells were identified by double immunostaining as almost exclusive source of IL-6. Likewise, in five (sub)clones of primary culture COGA-1 and COGA-13 human colon carcinoma cells, and in three established cell lines (Caco-2/AQ, Caco-2/15 and HT-29), efficient translation of IL-6 mRNA into protein was observed only in the least differentiated COGA-13 cells. Notably, IL-1beta (5 ng/ml) enhanced IL-6 release in COGA-13 cultures by three orders of magnitude. Although all cell clones studied expressed the IL-6 receptor (IL-6R), rhIL-6 (1-100 ng/ml) had a significant effect on cellular proliferation only in highly differentiated Caco-2 cells. Our data imply that IL-6, when released from rather undifferentiated carcinoma cells, particularly in response to IL-1beta, can advance tumour progression through paracrine growth stimulation of normal or highly differentiated colon tumour cells with intact STAT-3-mediated IL-6 signalling.     

Establishment of 2 human thyroid-carcinoma cell-lines (8305c, 8505c) bearing p53 gene-mutations

New cell lines, designated 8305C and 8505C, were established from undifferentiated thyroid carcinomas of a 67 year-old-female patient and a 78-year-old-female patient, respectively. Pathologically both these primary undifferentiated carcinoma tissues contained residual well differentiated components, suggesting well differentiated to undifferentiated carcinoma progression. Cell kinetic analysis indicate that the cell population doubling time is 43 h for 8305C and 36 h for 8505C. The saturation density at confluency is 5.7 x 10(4) cells/cm2 for 8305C and 1.1 x 10(5) cells/cm2 for 8505C. To identify genetic changes that may have occurred in these two cell lines, tumor suppressor genes p53, Rb, APC and MCC were analyzed. Sequence analysis confirmed a C:G to T:A transition at the first base of p53 gene codon 273 in 8305C and a C:G to G:C transversion at the first base of p53 codon 248 in 8505C. Polymerase chain reaction-loss of heterozygosity assays confirmed allelic deletion of p53 gene from the 8505C cell line. Loss of heterozygosity of other tumor suppressor genes were not observed. Given that p53 mutations associate with undifferentiated carcinoma but not with well differentiated carcinoma during multistep carcinogenesis of the thyroid, these cell lines should prove useful for research into the role of p53 gene mutations in malignant transformation.     

Cytokine production of lung cancer cell lines: Correlation between their production and the inflammatory/immunological responses both in vivo and in vitro

Cytokines produced by tumor cells may have various effects on antitumor immune responses and tumor growth. In the present study, the cytokine production of 31 lung cancer cell lines was evaluated, while any correlation with the histological type, the induction of tumor-specific cytotoxic T lymphocytes (CTL) in vitro, and angiogenesis and the infiltration of inflammatory cells in tumor tissues were also examined. Production of interleukin (IL)-1alpha, IL-1beta, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)-beta and vascular endothelial growth factor (VEGF) in the culture supernatant was measured using enzyme-linked immunosorbent assay. Each cytokine was produced in a substantial number of the tumor cell lines. In particular, IL-6, IL-8, TGF-beta and VEGF were produced in 18 (55%), 29 (94%), 31 (100%) and 28 (90%) of 31 cell lines, respectively. However, neither IL-4 nor TNF-alpha was produced at all by any tumor cell line. TGF-beta production was significantly higher in adenocarcinoma than in squamous cell carcinoma (P = 0.03). Immunohistochemical staining revealed the magnitude of macrophage infiltration, and angiogenesis in surgically resected tumor tissue specimens correlated well with GM-CSF and IL-8 production from the corresponding cell lines. Among six lung cancer cell lines, CTL were induced in the three lung cancer cell lines that produced a lower amount of TGF-beta (<100 pg/mL). These findings suggested that TGF-beta produced by tumor cells could inhibit the induction of CTL in vitro. The present results suggest that the production of various cytokines from tumor cells might exert various paracrine effects both in vivo and in vitro.     

The feasibility of monitoring NF-kappaB associated cytokines: TNF-alpha, IL-1alpha, IL-6, and IL-8 in whole saliva for the malignant transformation of oral lichen planus

Previous investigations have demonstrated that immune activation and chronic inflammation may be one of the causes of oncogenesis. A previous study from our lab has shown significant increases of NF-kappaB dependent cytokines, TNF-alpha, IL-1alpha, IL-6, and IL-8 in different oral fluids from oral lichen planus (OLP) patients. The aim of this analysis was to explore the potential of detecting these cytokines in whole unstimulated saliva (WUS) in monitoring the malignant transformation of OLP. Thirteen patients with OLP (with epithelial dysplasia), 13 cases with oral squamous cell carcinoma (OSCC), and 13 age-sex matched controls were enrolled in the study. The WUS samples were collected and the level of TNF-alpha, IL-1alpha, IL-6, and IL-8 in WUS was determined by ELISA. In moderate and severe dysplasia, the level of each cytokine was significantly higher than in control. In moderate dysplasia, TNF-alpha and IL-1alpha were significantly increased at a level without difference from OSCC, but IL-6 and IL-8 was detected at a concentration significantly lower than OSCC. In severe dysplasia, the level of TNF-alpha was also not significantly different from that of OSCC, and the level of IL-1alpha, IL-6, and IL-8 was still significantly lower than that of OSCC. The level of four cytokines between smokers and non-smokers in each group did not show a significant difference. These results indicate that the change of NF-kappaB dependent cytokines in WUS may in part reflect the malignant transformation of OLP and the analysis of these cytokines and may provide a useful, non-invasive surrogate endpoint for monitoring malignant transformation as well as the therapeutic response of OLP. This is the first in vivo study utilizing saliva to confirm preclinical data that NF-kappaB is upregulated in oral carcinogenesis.