Lack of BCL-2 confers interferon-alpha sensitivity to B-cell lymphomas

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder with pathological manifestations usually including splenomegaly and pancytopenia. Interferons (IFNs), specifically of the alpha subtypes, have shown a significant anti-tumor effect in HCL patients, with improvement of hematological parameters within the first few months of treatment. However, the therapeutic effect of IFN-alpha is still rather limited. The mechanisms responsible for the beneficial action of IFN-alpha in HCL patients are unclear. A continuous line of cells (Eskol) from a patient diagnosed with HCL was established and shown to have several properties of HCL. Even though, Eskol cells are very resistant to anti-proliferative activity of IFN-alpha, Daudi cells, another human B-cell-derived cell line, are very sensitive to anti-proliferative activity of IFN-alpha and are commonly used as a model cell to test anti-proliferative effect of IFN-alpha. To understand the molecular reason(s) behind the observed obvious differences to IFN sensitivity of above cells, we have analyzed the expression levels of BCL2, caspase-1, Laminin and PARP in these cells. We found that Daudi cells do not express BCL2 at all, and probably because of that, these cells have constantly cleaved, and probably activated form of caspase-1. However, when we over-expressed BCL2 in these cells, they lost processed form of caspase-1 and became resistant to anti-proliferative activity of IFN-alpha. These results let us to suggest that IFN-alpha sensitivity of B-cell lymphomas, once again, depends on the presence or absence of BCL2.     

2-5A synthetase activity induced by interferon alpha, beta, and gamma in human cell lines differing in their sensitivity to the anticellular and antiviral activities of these interferons

Two human cell lines (HEC-1, IF^r) which are resistant to the anticellular and/or antiviral action of HuIFN-α or -β have been tested with respect to the anticellular and antiviral activities of HuIFN-γ. HEC-1 cells were totally resistant to the antiviral activity of the IFN-γ, with either EMC or VSV as challenge virus. Per antiviral unit HuIFN-γ exerted a much stronger inhibitory effect on the growth of the interferon-sensitive cell line RSa than did IFN-α. IF^r cells were more resistant to the anticellular effect of IFN-γ than the wild-type cells (RSa), and HEC-1 cells were totally resistant to the anticellular effect of HuIFN-γ. 2-5A synthetase was present at a high basic level in untreated HEC-1 cells, as previously reported. This activity was not influenced by either HuIFN-β or -γ. Similar levels of 2-5A synthetase were induced by HuIFN-γ in IF^r and RSa cells; however, this induction was 10 times lower than that obtained for HuIFN-β. A 2′-phosphodiesterase activity was detected in control extracts from RSa and was not induced by either HuIFN-β or -γ. There was no correlation between the amplitude of 2-5A synthetase induction and extent of anticellular effect of either HuIFN-γ in different cell lines or of different interferons in a given cell line. Finally, HEC-1 represents the first example of a human cell line which is resistant to all three types of interferon (HuIFN-α, -β, and -γ).     

干扰素-α1b增强人γδT细胞对Daudi细胞系的细胞毒活性及相关机制

目的探讨干扰素-α1b(IFN-α1b)对人γδT细胞体外杀伤肿瘤细胞的促进作用及其相关机制。方法用IFN-α1b预处理的Daudi细胞作为靶细胞;用IFN-α1b预处理的γδT细胞作为效应细胞;LDH法检测γδT细胞对Daudi细胞的细胞毒活性;ELISA检测效靶细胞混合上清中γδT细胞分泌的颗粒酶B和γ干扰素水平;流式细胞计量技术检测IFN-α1b预处理Daudi细胞表面Fas受体和应激蛋白配体ULBP3/ULBP4的表达,以及γδT细胞表面活化分子CD69和CD25的变化。结果IFN-α1b分别预处理Daudi细胞和γδT细胞都能显著增强γδT细胞对Daudi细胞的细胞毒活性(P<0.0001);IFN-α1b预处理的γδT细胞分泌颗粒酶B和γ干扰素的能力显著提高(P<0.05);IFN-α1b能上调Daudi细胞表面Fas受体和应激配体ULBP4的表达水平,而应激配体ULBP3的表达没有变化;IFN-α1b还能上调γδT细胞表面CD69的表达水平,而CD25的表达没有变化。结论IFN-α1b能通过不同的途径促进人γδT细胞对肿瘤细胞系Daudi细胞的体外细胞毒活性,两者的联用能增强γδT细胞的肿瘤治疗疗效。     

IFN-λ在人食管癌细胞系中诱导的生物学活性的初步研究

目的 初步研究IFN-λ在7种人食管癌细胞系中诱导的生物学作用。方法 PCR检测IL-28α和IL-10β的基因及抗病毒分子基因的表达,流式细胞术检测主要组织相容性抗原的表达及细胞周期,二苯基溴化四氮唑蓝(MTT)检测细胞增殖。结果 各食管癌细胞表达IL-28α和IL-10β的基因;IFN-λ诱导或上调2′5′-寡腺苷酸合成酶（2′5′-OAS）和黏病毒抗性蛋白A(MxA)的基因表达;IFN-λ可上调Ⅰ类主要组织相容性抗原分子的表达;IFN-λ可通过调控细胞周期的方式抑制食管癌细胞生长增殖。结论 人食管癌细胞株表达IFN-λ受体复合体,IFN-λ具有抗病毒、抗增殖和免疫调节的生物学活性。     

细胞因子对HLA-B27启动子的调节作用及其在强直性脊柱炎发病机制中的意义

目的： 构建含HLA-B27启动子的HeLa稳定转染细胞株，观察7种细胞因子对HLA-B27启动子及其上游NF-κB及ISRE顺式作用元件活性的调节作用，探讨强直性脊柱炎（AS）等B27相关疾病的发生机制。方法:转染HeLa细胞，抗生素筛选单克隆构建含HLA-B27启动子的稳定转染细胞株。构建HeLa-B27稳定细胞株和HeLa-NF-κB稳定细胞株，在瞬时转染pISRE-luc的HeLa细胞中加入白细胞介素1α（IL-1α）、 白细胞介素1β（IL-1β）、 肿瘤坏死因子α（TNF-α）、干扰素α（IFN-α）、干扰素β（IFN-β）、 干扰素γ（IFN-γ）和转化生长因子β（TGF-β），观察7种细胞因子对B27启动子及其上游NF-κB和ISRE作用元件的调节作用。另外在HeLa-B27 稳定细胞株培养液中同时加入3种细胞因子单克隆抗体和相应细胞因子，观察其对启动子活性的调节作用。结果:TNF-α、IFN-α、IFN-β 和 IFN-γ 均能明显增强HeLa细胞B27启动子活性。细胞培养96 h 后，IFN-β为最强的启动子诱导剂（5.4倍，P<0.05）；细胞培养8 h 内，TNF-α、IL1-α 和 IL1-β,可诱导NF-κB的活性增加30倍左右（P<0.05），IFN-α 和IFN-β 可诱导ISRE的活性增加12倍左右（P<0.05），抗TNF-α 抗体对于I类IFN 增加的B27 启动子活性没有明显的抑制作用。结论：TNF-α和IFN 可通过结合于B27 启动子中各种转录因子结合元件调控HLA-B27启动子的转录活性，IFN-β 可能在强直性脊柱炎等B27 相关的脊柱关节病的发病机制中起着重要作用。     

Human and rhesus plasmacytoid dendritic cell and B-cell responses to Toll-like receptor stimulation

Interferon-α (IFN-α) produced at high levels by human plasmacytoid dendritic cells (pDCs) can specifically regulate B-cell activation to Toll-like receptor (TLR) 7/8 stimulation. To explore the influence of IFN-α and pDCs on B-cell functions in vivo, studies in non-human primates that closely resemble humans in terms of TLR expression on different subsets of immune cells are valuable. Here, we performed a side-by side comparison of the response pattern between human and rhesus macaque B cells and pDCs in vitro to well-defined TLR ligands and tested whether IFN-α enhanced B-cell function comparably. We found that both human and rhesus B cells proliferated while pDCs from both species produced high levels of IFN-α in response to ligands targeting TLR7/8 and TLR9. Both human and rhesus B-cell proliferation to TLR7/8 ligand and CpG class C was significantly increased in the presence of IFN-α. Although both human and rhesus B cells produced IgM upon stimulation, only human B cells acquired high expression of CD27 associated with plasmablast formation. Instead, rhesus B-cell differentiation and IgM levels correlated to down-regulation of CD20. These data suggest that the response pattern of human and rhesus B cells and pDCs to TLR7/8 and TLR9 is similar, although some differences in the cell surface phenotype of the differentiating cells exist. A more thorough understanding of potential similarities and differences between human and rhesus cells and their response to potential vaccine components will provide important information for translating non-human primate studies into human trials.     

IFN-α expression and antiviral effects are subtype and cell type specific in the cardiac response to viral infection

The interferon-β (IFN-β) response is critical for protection against viral myocarditis in several mouse models, and IFN-α or -β treatment is beneficial against human viral myocarditis. The IFN-β response in cardiac myocytes and cardiac fibroblasts forms an integrated network for organ protection; however, the different IFN-α subtypes have not been studied in cardiac cells. We developed a quantitative RT-PCR assay that distinguishes between 13 highly conserved IFN-α subtypes and found that reovirus T3D induces five IFN-α subtypes in primary cardiac myocyte and fibroblast cultures: IFN-α1, -α2, -α4, -α5, and -α8/6. Murine IFN-α1, -α2, -α4, or -α5 treatment induced IRF7 and ISG56 and inhibited reovirus T3D replication in both cell types. This first investigation of IFN-α subtypes in cardiac cells for any virus demonstrates that IFN-α is induced in cardiac cells, that it is both subtype and cell type specific, and that it is likely important in the antiviral cardiac response.     

干扰素α对Jurkat细胞中CCR5表达的影响

目的:通过研究干扰素(IFN-α)对Jurkat E6-1细胞内CCR5 mRNA表达及基因表达调控的影响,探讨采用干扰素治疗相关疾病的方法。方法:用不同浓度的IFN-α刺激处于对数生长期的CD4+T淋巴细胞系Jurkat E6-1细胞。培养48小时后,提取细胞的总RNA,逆转录成cDNA,进行RT-PCR和Real time-PCR扩增目的基因CCR5;利用脂质体转染荧光素酶报告系统检测Jurkat E6-1细胞内CCR5活性变化情况。结果:(1)IFN-α在浓度为100 U/ml时对CCR5 mRNA的表达有明显抑制作用;在浓度为1 000 U/ml时对CCR5 mRNA的表达表现为增强作用;在浓度为10 000 U/ml时对CCR5 mRNA的表达增强作用有所减弱。(2)当IFN-α的浓度为100 U/ml时荧光素酶的活性最低;当IFN-α的浓度为1 000 U/ml时荧光素酶的活性最高;当IFN-α的浓度为10 000 U/ml时荧光素酶的活性又有所减低。结论:IFN-α作为一类很好的免疫调节、抗病毒生物制剂,不同剂量在一定范围内会对细胞CCR5的表达有显著影响。     

Age-associated decline in IL-2 and IL-12 induction of LAK cell activity of human PBMC samples

Previously we reported that young and elderly natural killer (NK) cell activity against the standard NK sensitive K562 cell line can be augmented to the same degree by IL-2 and IFN-. We have extended these studies to include IL-12. Similar to IL-2 and IFN-, IL-12 can enhance NK cytotoxicity to the same degree in both young and elderly samples over a wide range of doses and incubation times when K562 cells are used as targets. However, in contrast to our findings with the NK system, we have observed that induction of lymphokine activated killer (LAK) cell activity, as defined by the ability of peripheral blood mononuclear cells (PBMC) samples to lyse the normally NK resistant Daudi cell line, was significantly decreased in the elderly samples compared to young samples. Comparable age-associated differences were observed in LAK activity after induction with IL-2, IL-12, and IFN- at varying doses and incubation times. We hypothesize an age-associated deficiency either in the mechanism of LAK induction or in target cell recognition.     

Interferon-α as angiogenesis inhibitor: Learning from tumor models

Interferon-α (IFN-α), a cytokine with marked therapeutic activity in transplantable tumor models, has been identified as powerful angiogenesis inhibitor. The effects of IFN-α on the vasculature have been mainly attributed to inhibition of basic fibroblast growth factor production by tumor cells or downregulation of IL-8 and vascular endothelial growth factor gene expression. Moreover, IFN-α has direct effects on endothelial cells (EC), including impairment of their proliferation and migration. The gene expression profile induced by IFN-α in EC has recently been defined, and it was found that several genes encoding negative regulators of angiogenesis are upmodulated, thus providing a potential amplification mechanism for this biological activity. The anti-angiogenic effects of IFN-α appear to be associated with increased hypoxia and ischemic necrosis in subcutaneous xenograft models, whereas in transgenic mouse models, IFN-α may simultaneously interfere with both blood vessels and tumor cell proliferation, leading to regression of tumors without necrosis. The consequences of IFN-α therapy on the invasive and metastatic behavior of tumor cells are currently unknown. Finally, as effective anti-angiogenic therapy with IFN-α demands sustained localized production of this cytokine, innovative strategies of targeted delivery of the IFN-α gene into tumors are discussed.     

Growth and major histocompatibility antigen expression regulation by IL-4, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) on human renal cell carcinoma

We have recently shown that human renal cell carcinoma (RCC) tumour lines express high-affinity IL-4 receptors. Binding of IL-4 to RCC cells induced a growth inhibition in the range of 20 68%. To enhance the growth inhibitory effect of IL-4. we have tested the effects of two additional cytokines capable of directly affecting tumour cell growth. IFN-γ caused a significant inhibition of RCC tumour cell growth (up to 70%) in a dose-dependent manner, whereas the effect of TNF-α was more limited (0 20% inhibition). The addition of IL-4 to IFN-γ on RCC cells sensitive to lL-4 induced a greater inhibition of cell growth than that seen with each cytokine alone. IL-4 and IFN-γ rendered RCC cells more responsive to the inhibitory effect mediated by TNF-α, The combination of TNF-Q with IL-4 and IFN-γ induced an optimal growth inhibition (up lo 90 98%) of RCC cells. In addition to a direct anti-proliferative effect, we have demonstrated that these cytokines can also enhance the expression of MHC antigens on the surface of RCC tumour cell lines which may render the cells more immunogenic, All RCC lines tested expressed class 1 antigens, but not class II antigens. IFN-γ induced class II expression and up-regulated the expression of class I antigens on RCC cells. Class II antigen expression was detectable following 48 h incubation, and greater after 72 h with IFN-7. lL-4 minimally affected class I expression, whereas TNF-(v up-regulated class I antigen expression. IL-4 or TNF-α did not induce class II expression. The combination of The three cytokines slightly augmented the up-regulation of class I and class II antigens observed with IFN-γ alone. These observations confirm the direct interaction of IL-4, IFN-γ and TNF-a with RCC tumour cells. both at the level of growth regulation and MHC antigen expression, and suggest a therapeutic potential of the combination of the three cytokines for renal ceil carcinoma.     

Patients with systemic lupus erythematosus (SLE) have a circulating inducer of interferon-alpha (IFN-α) production acting on leucocytes resembling immature dendritic cells

Patients with active SLE often have an ongoing production of IFN-α. We therefore searched for an endogenous IFN-α-inducing factor (IIF) in SLE patients and found that their sera frequently induced production of IFN-α in cultures of peripheral blood mononuclear cells (PBMC) from healthy blood donors, especially when the PBMC were costimulated with the cytokines IFN-α2b and granulocyte-macrophage colony-stimulating factor (GM-CSF). The phenotype of the IFN-α-producing cells (IPC) as determined by flow cytometry corresponded to that of the natural IPC, resembling immature dendritic cells. The IIF activity in SLE sera was sometimes as high as that of a virus and was present especially in patients with active disease and with measurable IFN-α levels in serum. The IIF had an apparent molecular weight of 300–1000 kD and appeared to consist of both immunoglobulin and DNA, possibly being immune complexes. This endogenous IFN-α inducer may be of pathogenic significance, since a reported occasional adverse effect of IFN-α therapy in patients with non-autoimmune disorders is development of anti-dsDNA antibodies and SLE.     

In vitro sensitivity of human metapneumovirus to type I interferons

Human metapneumovirus (hMPV) has been recognized as an important respiratory pathogen. Due to its relatively recent discovery, only limited information is available on the relationship between hMPV and type I interferons (IFN). This study was designed to determine whether in vitro hMPV is sensitive to the antiviral activity of IFN-β, leukocyte IFN-α, and several IFN-α subtypes in a human Hep-2 cell line. The results showed that 50% inhibitory concentration values against hMPV for the various type I IFN preparations were significantly higher than those against the IFN-sensitive vesicular stomatitis virus, and some IFN-α subtypes appeared to be more active against hMPV than others, with IFN-α subtypes 5, 6, 8, and 10 being the most potent, and IFN-α2, 17, and 21 the least potent. The results show that hMPV grown in Hep-2 is partially resistant to the antiviral activity of type I IFNs. Additional studies are required to understand whether and to what extent the relatively low sensitivity of hMPV to IFNs influences the clinical outcomes of infected individuals.     

Interferon-α induces unabated production of short-lived plasma cells in pre-autoimmune lupus-prone (NZB×NZW)F1 mice but not in BALB/c mice

IFN-α is known to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanisms remain unclear. We previously showed that within weeks, exposure to IFN-α in vivo induces lupus in pre-autoimmune lupus-prone NZB×NZW F1 (NZB/W) but not in BALB/c mice. In the current study, we show that in vivo expression of IFN-α induces sustained B-cell proliferation in both BALB/c and NZB/W mice. In NZB/W but not BALB/c mice, B-cell proliferation was accompanied by a rapid and unabated production of autoantibody-secreting cells (ASCs) in secondary lymphoid organs, suggesting that a B-cell checkpoint is altered in the autoimmune background. The majority (>95%) of ASCs elicited in IFN-α-treated NZB/W mice were short-lived and occurred without the induction of long-lived plasma cells. A short course of cyclophosphamide caused a sharp drop in IFN-α-elicited short-lived plasma cells, but the levels recovered within days following termination of treatment. Thus, our work provides new insights into effectiveness and limitations of the current SLE therapies.     

IFN-α对人胃癌细胞株BGC-823生长的抑制作用

目的：观察干扰素-α(IFN-α)对人胃癌细胞株BGC-823生长和浸润转移的抑制作用及其作用机理。方法:在体外培养体系中检测IFN-α对BGC-823细胞株生长增殖能力的影响，MTT法检测细胞活力，流式细胞仪检测细胞周期。同时采用免疫组化法观察了IFN-α对肿瘤细胞E-cadherin、MMP-2表达的调节作用，电镜下观察肿瘤细胞间连接的超微结构的变化。结果:IFN-α对人胃癌细胞株BGC-823的生长有明显抑制作用，并表现出剂量依赖性，IFN-α浓度≥106 U/L时可有效抑制细胞增殖，抑制率≥12.2%，细胞周期在G1-S期之间发生阻滞；在IFN-α诱导下细胞E-cadherin表达水平升高而MMP-2表达下降，超微结构出现了细胞粘附连接数量增多、结构相对紧密等变化 。结论：IFN-α可通过影响细胞周期来抑制人胃癌细胞株BGC-823的生长；IFN-α能调节E-cadherin、MMP-2的表达，密切细胞间的连接，具有限制胃癌细胞侵袭转移的潜能。     

干扰素-α联合高三尖杉酯碱下调K562细胞端粒酶逆转录酶及端粒酶活性诱导细胞凋亡

目的：研究干扰素-α(IFN-α)联合高三尖杉酯碱(HHT)对K562细胞端粒酶逆转录酶及端粒酶的作用，并探讨其与细胞凋亡的关系。方法:IFN-α、HHT以及两药联合作用K562细胞后用MTT法检测细胞增殖，吉姆萨-瑞氏染色观察细胞形态，PI-Annexin VFITC双染色、流式细胞仪检测细胞凋亡,Krupp改良的荧光素标记的端粒重复扩增方法(TRAP)检测K562细胞端粒酶活性，RT-PCR方法检测hTERT mRNA表达变化。结果:5×106U·L-1 IFN-α分别联合5-40 μg·L-1 HHT诱导K562细胞凋亡从而抑制其生长，单用HHT时IC50为27.35 μg·L-1，联用5×106 U·L-1 IFN-α后IC50降为18.72 μg·L-1；IFN-α与HHT联用明显下调hTERT mRNA表达并抑制K562细胞的端粒酶活性。结论：IFN-α联合HHT可以诱导细胞凋亡，这可能与下调细胞hTERT mRNA的表达水平，抑制端粒酶活性有关。两药联合作用强于单独用药，提示临床IFN-α联合HHT治疗慢性髓细胞白血病（CML）可能比单用HHT效果更好。     

SC58125 通过抑制IκBα的降解调节TNF-α 诱导的人结肠癌HT29细胞的凋亡

目的： 了解SC58125与TNF-α是否具有协同诱导HT29细胞凋亡的作用，同时探讨其可能的分子机制。方法： 用MTT、Hoechst 33342染色及琼脂糖凝胶电泳，观察SC58125/TNF-α对HT29结肠癌细胞增殖与凋亡的作用；用EMSA及Western blotting检测转录因子NF-κB的结合活性、IκBα以及磷酸化IκBα的表达。结果： SC58125与TNF-α在抑制HT29细胞增殖及诱导其凋亡方面具有明显的协同作用；表现为细胞核浓缩、核碎裂及“DNA梯带”形成；凋亡过程中伴随caspase活性的激活。经TNF-α刺激后的HT29细胞，IκBα，磷酸化IκBα迅速降解，NF-κB的结合活性大大提高，而加入SC58125后，IκBα降解及NF-κB的结合活性明显受抑制。结论： SC58125与TNF-α在诱导HT29细胞凋亡方面具有明显的协同作用，这可能与激活caspase-3 及抑制IκBα降解有关。     

Inhibitory effect of PGE(2) on the killing of Paracoccidioides brasiliensis by human monocytes can be reversed by cellular activation with cytokines

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a deep mycosis endemic in Latin America. Studies to elucidate the host-parasite relationship in this mycosis have demonstrated that non-activated phagocytes fail to kill the etiologic agent. Investigations of human monocytes have shown that the lack of fungicidal activity is partially associated with the capacity of a high-virulence strain to induce PGE(2) release by these cells. This eicosanoid inhibits production of TNF-α, the cytokine involved in cell activation for release of H(2)O(2), the fungicidal metabolite. Cell priming with IFN-γ was shown to partially reverse this inhibitory effect. In this study, we asked whether monocyte challenge with a low-virulence strain of this fungus would also result in PGE(2) release and consequently inhibition of antifungal activities. We also assessed whether PGE(2,) besides inhibiting production of TNF-α, a monocyte-activating cytokine, also affects IL-10. The latter, in contrast to TNF-α is a monocyte-suppressing cytokine. Finally, we evaluated whether priming cells with other cytokines, namely TNF-α and GM-CSF, could be more effective than IFN-γ in reversing the PGE(2) inhibitory effect. The results revealed that the less virulent P. brasiliensis strain also induces human monocytes to release PGE(2). However, the inhibitory effect of PGE(2) was less pronounced when cells were challenged with this strain than with the more virulent one. It was also demonstrated that PGE(2), while inhibits TNF-α production, tends to increase IL-10 levels. Priming with GM-CSF or TNF-α was more effective than IFN-γ in compensating for the inhibitory PGE(2) effect, since these cytokines induce cells to produce higher H(2)O(2) and TNF-α levels.