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.     

Expression of high-affinity IL-4 receptors on human melanoma,ovarian and breast carcinoma cells

It has previously been shown that murine sarcoma cells express high-affinity IL-4 receptors (1L-4R) which are internalized after binding to the ligand (Puri et al., Cancer Res 1991; 51:3011-7). We have also reported that human renal cell carcinoma cells express high-affinity IL-4R. and IL-4 inhibits tumour growth in vitro (Obiri et al, J Clin Invest 1993; 91:88). In this study we investigated the expression and function of IL-4R on other human solid tumours. Human melanoma, ovarian carcinoma and breast carcinoma cell lines were assessed for the cell surface expression of IL-4R by radio-ligand receptor binding and for IL-4R gene expression by Northern blot analysis. Primary cultures of mesothelioma and neurofibrosarcoma cells were similarly investigated. Human melanoma, ovarian carcinoma and breast carcinoma cell lines expressed IL-4R on their cell surface with a dissociation constant (K_d) of 140-549 pM. These tumour lines expressed a single 4 kb species of mRNA for IL-4R. Similarly, primary cultures of mesothelioma and neurofibrosarcoma cells were positive for the IL-4R mRNA by Northern blot analysis. Fresh, non-cultured mesothelioma and neurofibrosarcoma tumour sections were also positive for the presence of IL-4R as determined by immunohistochemistry of frozen sections using anti-IL-4R antibody. In order to study possible functions of IL-4R, we evaluated the effects of 11.-4 on cell growth and its effect on MHC antigen expression in the presence or absence of interferon-gamma (IFN-γ)- In tissue culture, IL-4 reduced the growth of tumour cell lines and primary cell cultures studied. IL-4 had very title effect on MHC class I antigen expression on ovarian, breast and melanoma cell lines; however. MHC class I (HLA-DR) expression was enhanced on melanoma and breast carcinoma cells. IL-4 also enhanced the IFN-γ-induced class II expression on melanoma and breast carcinoma cells. Taken together, our observations indicate that IL-4R are expressed on a variety of human solid tumours and these receptors may be functional. IL-4 alone and in combination with IFN-γ may play a role in host immune response against cancers.     

Nicotinamide and 3-Aminobenzamide Reduce Interferon-γ -Induced Class II MHC (HLA-DR and -DP) Molecule Expression on Cultured Human Endothelial Cells and Fibroblasts

Abstract

We investigated the effects of nicotinamide and 3-aminobenzamide, known as inhibitors of poly(ADP-ribose) synthetase, on the expression of interferon-γ (IFN-γ) -induced class I and II major histocompatibility complex (MHC) molecules on the surface of cultured human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (HDF). Indirect immunofluorescent staining on HUVEC and HDF was performed using monoclonal antibodies against class I MHC (HLA-A, B, C) and class II MHC (HLA-DR, HLA-DP and HLA-DQ) molecules, and then the expression of these molecules was determined using a fluorescence flow cytometry. Human recombinant IFN-γ (100 U/ml) increased the expression of HLA-A, B, C molecules, and induced the expression of HLA-DR molecules and, to a lesser extent, of HLA-DP on both HUVEC and HDF. HLA-DQ molecules were not induced by IFN-γ on either cell type. Nicotinamide and 3-aminobenzamide in the concentration great-er than or equal to 1 mM reduced the IFN-γ -induced expression of HLA-DR and HLA-DP on both HUVEC and HDF, whereas neither agent in the concentration of up to 10 mM affected the IFN-γ -induced increase in HLA-A, B, C molecule expression. These data suggest that nicotinamide and 3-aminobenzamide suppress antigen presenting function of class II MHC positive endothelial cells and fibroblasts at the site of tissue inflammation.     

Differential inhibitory effects of indomethacin,dexamethasone, and interferon-gamma (IFN-γ) on IL-11 production by rheumatoid synovial cells

IL-11, a member of the IL-6 type cytokines, has some biological activity related to the joint destruction in rheumatoid arthritis (RA), such as induction of osteoclast differentiation. However, its expression and regulation in rheumatoid inflamed joints has not been clarified. In the present study we examined the capacity of fresh rheumatoid synovial cells (fresh RSC) to produce IL-11, and the effect of indomethacin, dexamethasone and IFN-γ on IL-11 production. Fresh RSC obtained from eight patients with RA produced large amounts of IL-11, measured by ELISA, and showed strong expression of IL-11 mRNA, determined by Northern blotting. Indomethacin inhibited the production of IL-11 by about 55%. Prostaglandin E₂ (PGE₂) completely prevented the inhibition, suggesting that IL-11 production by fresh RSC was in part mediated by PGE₂. Dexamethasone inhibited the production of IL-11 by more than 80%. Interestingly, the inhibition was not abolished by PGE₂. IFN-γ inhibited the production of IL-11 from IL-1α-stimulated cultured rheumatoid synovial fibroblasts, although IFN-γ did not inhibit the production of IL-11 by fresh RSC. These results suggest that the production of IL-11 by rheumatoid synovia was differentially regulated by PGE₂ and IFN-γ, and that treatment with indomethacin or dexamethasone decreased the level of IL-11 at inflammatory joints in patients with RA.     

The Th1 and Th2 cytokines IFN-γ and IL-4 antagonize the inhibition of monocyte IL-1 receptor antagonist by glucocorticoids: involvement of IL-1

Monocytes express IL-1 and IL-1 receptor antagonist (IL-1Ra) in response to lipopolysaccharide (LPS). IL-1 self-induction contributes to the increase in IL-1 following LPS stimulation. LPS-stimulated IL-1 and IL-1Ra production are inhibited by glucocorticoids. In the present work we examined the regulation of IL-1Ra by Th1 cytokine IFN-γ, Th2 cytokine IL-4, glucocorticoids and IL-1 in human monocytes. We demonstrate that IL-1 contributes to LPS-induced IL-1Ra expression as shown by IL-1 blockade in LPS-stimulated monocytes using a specific anti-IL-1β antibody or recombinant IL-1Ra. Glucocorticoids inhibited IL-1β-stimulated IL-1Ra mRNA expression and protein production. Glucocorticoids inhibited both IL-1-mediated and non-mediated LPS stimulation of IL-1Ra expression. Both IFN-γ and IL-4 reversed the inhibitory effect of glucocorticoids on IL-1Ra expression and secretion. The effect of IFN-γ was blocked by pretreatment of monocytes with an anti-IL-1β blocking antibody, whereas the effect of IL-4 could not be blocked, demonstrating that IFN-γ acts through a mechanism dependent on endogenous IL-1 production, whereas IL-4 acts through an IL-1-independent one. Consistent with this finding, IFN-γ (but not IL-4) failed to reverse the inhibitory effect of glucocorticoids when stimulated by IL-1, and only IL-4 combined with IL-1 showed synergism resulting in an increase in IL-1Ra production. The differential regulation and involvement of IL-1 in the expression of IL-1Ra by IFN-γ, IL-4 and glucocorticoids sets the level of monocyte responsiveness during the Th1 or Th2 responses.     

Enhanced prostaglandin E2 production by monocytes in atopic dermatitis (AD) is not accompanied by enhanced production of IL-6, IL-10 or IL-12

AD is associated with a bias of the T helper cells to show increased IL-4 and reduced interferon-gamma (IFN-γ) production. The production of IFN-γ and IL-4 and the development of Th cells into either high IFN-γ or high IL-4 producers is strongly influenced by factors produced by antigen-presenting cells (APC), like IL-12 and prostaglandin E₂ (PGE₂). IL-12 selectively enhances IFN-γ production and favours the development of IFN-γ-producing Th cells, whereas PGE₂ selectively inhibits IFN-γ production by Th cells. The aim of this study was to test whether the increased IL-4/IFN-γ production ratio by Th cells in AD can be explained by an increased PGE₂/IL-12 production ratio by the APC. Monocytes were used as APC source. PGE₂ and IL-12 production by lipopolysaccharide (LPS)-stimulated monocytes from 12 AD patients and 12 non-atopic controls was determined using two complementary experimental systems, whole blood cultures and purified monocytes. In addition, we determined IL-6 production as a measure of monocyte activation, and IL-10 production because IL-12 production by monocytes is highly influenced by endogenously produced IL-10. The monocytes from AD patients showed normal production levels of IL-6 and IL-10, a two-fold, but non-significant decrease in IL-12 production, and a significantly (three-fold) higher PGE₂ production than those from non-atopic controls. Here we show for the first time that enhanced PGE₂ production by monocytes in AD is not accompanied by a general rise in cytokine production. We conclude that AD is indeed associated with an increased PGE₂/IL-12 production ratio by monocytes.     

CELL ADHESION MOLECULE EXPRESSION WITHIN HUMAN GLOMERULAR AND KIDNEY ORGAN CULTURE

A glomerular and kidney organ culture method was developed to study cytokine inducibility of adhesion molecules and MHC antigen expression. Glomerular cells showed constitutive expression of intercellular cell adhesion molecule-1 (ICAM-1) and MHC class I and II antigens, but not vascular cell adhesion molecule-1 (VCAM-1), E-selectin, or P-selectin. Expression of E-selectin on glomerular endothelial cells (ECs) was induced by interleukin-1 (IL-1), tumour necrosis factor (TNF), interferon-γ (IFN-γ) and granulocyte/macrophage-colony stimulating factor (GM-CSF); this induction was inhibited by transforming growth factor beta (TGFβ) and by IL-4. P-selectin expression was never seen within glomeruli. VCAM-1 was constitutively expressed by Bowman's capsule and proximal tubules and was weakly induced on glomerular ECs by TNF and IL-4 in combination. Glomerular endothelial ICAM-1 expression was increased by IL-1, TNF, IFN-γ, and GM-CSF, while TGFβ inhibited induction by TNF and IL-1. Expression of MHC class I and II antigens by glomerular ECs was constitutive; further upregulation of MHC class II by IFN-γ was observed. These studies suggest that leukocyte–endothelial cell interactions that occur within the kidney follow broadly similar principles as are proposed to occur elsewhere in the body but, in addition, there are subtle differences that reflect local conditions. © 1997 by John Wiley & Sons, Ltd.     

Enhanced monocyte Fc phagocytosis by a homologue of interleukin-10 encoded by human cytomegalovirus

Human cytomegalovirus (HCMV) expresses several homologues of human interleukin 10 (hIL-10) possessing immunomodulatory properties which may promote viral infection by modulating the function of myeloid cells. We examined the phenotype and phagocytic capability of human monocytes exposed to hIL-10, an HCMV-encoded hIL-10 homologue expressed during the productive phase of infection (cmvIL-10), and a differentially spliced form of cmvIL-10 expressed during latent and productive phases of infection, (LAcmvIL-10). hIL-10 and cmvIL-10 upregulated expression of Fcγ receptors, stimulated phagocytosis of IgG-opsonised erythrocytes and decreased MHC class II (HLA-DR) expression on purified monocytes within 24 h. In contrast, LAcmvIL-10 decreased HLA-DR expression at later times (48 h and 72 h) but did not increase Fcγ receptor expression. We conclude that cmvIL-10 promotes differentiation of monocytes towards a pro-phagocytic phenotype and that LAcmvIL-10 does not affect monocytes by the same mechanism as cmvIL-10. The significance of these properties to cytomegalovirus pathogenesis is discussed.     

IFN-γ Inhibits the Suppressive Effects of PGE2 on the Production of Tumor Necrosis Factor-α by Mouse Macrophages

Tumor necrosis factor-α (TNF-α) has become known as a central mediator of responses to endotoxin, rheumatoid diseases, and other forms of inflammation. Current investigations indicate that the production of TNF-α is controlled by other mediators, including interferon-γ (IFN-γ) and prostaglandin E₂ (PGE₂). In the present study, we investigated the regulatory effects of IFN-γ and/or PGE₂ on LPS-induced TNF-α production and mRNA expression in mouse peritoneal macrophages using the enzyme immunoassay and Northern blot analysis, respectively. In response to 10 ng/ml of LPS, TNF-α production reached a maximum at approximately 4 hrs, followed by rapid decline. At the molecular level, TNF-α mRNA accumulated rapidly after LPS exposure, reaching a peak by 3 hr, and declined more rapidly than did the production of TNF-α. Exposure of macrophages to 100 U/ml of IFN-γ caused an increase in both the TNF-α production and mRNA expression induced by LPS. Exogenous PGE₂ caused a dose dependent reduction in LPS-induced TNF-α mRNA accumulation as well as TNF-α production. Macrophages primed with IFN-γ showed the reduced responsiveness to the suppressive effect of PGE₂ on the production of TNF-α and the accumulation of TNF-α mRNA. These findings indicate that the suppressive effects induced by PGE₂ on the accumulation of TNF-α mRNA as well as the production of TNF-α can be reduced by the pretreatment of macrophages with IFN-γ. These studies demonstrate the role of IFN-γ as an immunomodulating compound that may effectively regulate TNF-α production by modulation of macrophage responsiveness to PGE₂.     

IL-1β and IFN-γ induce the regenerative epidermal phenotype of psoriasis in the transwell skin organ culture system. IFN-γ up-regulates the expression of keratin 17 and keratinocyte transglutaminase via endogenous IL-1 production

Skin biopsies from healthy human skin and non-lesional skin from patients with psoriasis were cultured for 24h and stimulated with interleukin-1β(IL-1β) and interferon-γ (IFN-γ) in a skin organ culture model and the induction of the psoriasiform regenerative epidermal phenotype was analysed using immunostaining. In the presence of IL-1β, the psoriasiform regenerative epidermal phenotype was clearly induced. This involved strong up-regulation of the expression of keratin 16, keratin 17, and keratinocyte transglutaminase (TGk) in the suprabasal layers, strong up-regulation and a shift of the expression of keratin 5 and integrin β₁from the basal to suprabasal keratinocytes, and induction of the expression of ICAM-1 and HLA-DR on basal keratinocytes. The effects of IL-1β in the organ cultures of normal skin could be completely neutralized by anti-IL-1 polyclonal antibodies. The effects of IFN-γ in healthy and non-lesional psoriatic skin were qualitatively similar to those of IL-1β. The IFN-γ-induced epidermal expression of keratin 17 and TGk could be completely blocked by culturing the biopsies in the presence of IL-1ra or anti-IL-1 antibodies, while the induction of HLA-DR and ICAM-1 was not inhibited. The induction of the psoriasiform regenerative epidermal phenotype by IFN-γ is partially mediated via endogenous epidermal IL-1. Copyright © 1999 John Wiley & Sons, Ltd.     

干扰素和肿瘤坏死因子对人脐静脉内皮细胞表达HLA-Ⅱ类抗原的影响

采用cell-ELISA法对IFN-α、γ及rTNFα单独或协同影响人脐静脉内皮细胞（HUVEC）表达HLA-Ⅱ类抗原作了定量观察。结果表明,IFNγ直接刺激 HUVEC可依浓度和时间依赖的方式提高其 HLA-Ⅱ类抗原表达量,而rTNFα、IFNα单独处理HUVEC无效。当rTNFα和IFNγ同时诱导时,对HUVEC表达HLA-DR、DP、DQ均表现抑制作用。但是,当先用IFNγ诱导HUVEC 24h后,再加入rTNFα则发现DR、DP、DQ的表达升高,起促进作用。     

Comparison of the effects of interleukin-1α, interleukin-lβ and interferon-γ-inducing factor on the production of interferon-γ by natural killer

Interferon-γ inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-γ (IFN-γ) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1γ. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1α, IL-1β and IGIF on NK cell production of IFN-γ. All three IL-1 types enhanced NK cell production of IFN-γ when induced by IL-2 or IL-12, although at high concentrations (>10 ng/ml), IGIF was five- to tenfold more potent than IL-1α or IL-1β. This effect correlated with enhanced levels of mRNA for IFN-γ when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-γ was not increased by IL-1α or IL-1β. The ability of IGIF to enhance IFN-γ production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-γ and demonstrate that the effect of IGIF on NK cell production of IFN-γ is similar to that of IL-1α and IL-1β but distinct from that of IL-12.     

An immunological model connecting the pathogenesis of stress,depression and carcinoma

Recently there has been considerable conjecture in the literature concerning a possible relationship between stress, depression and bereavement, and carcinoma. We shall propose a causal model in which the relationship between stress, depression and carcinoma is clarified. This relationship is grounded on dysregulation of the inflammatory cytokines in stress and depression. Stress is associated with increased expresson of interleukin-1 beta (IL-1p) and tumor necrosis factor-alpha (TNF-α), and reduced expression of IL-2, interferongamma (IFN-γ), major histocompatability complex (MHC) class II molecules and natural killer cell activity (NKA). Depression is associated with elevated IFN-γ and IL-1β, downregulated IL-2, and reduced NKA. Most organ-related carcinomas are associated with elevated TNF-α, which inhibits the activity of protein tyrosine phosphatase (PTPase), the enzyme that initiates activation of the MHC class I pathway. Sustained elevation of TNF-α inhibits the activity of PTPase which results in diminished expression of the MHC class I antigen on the cell surface and thus, malignant cells escape immune surveillance. Therefore, stress and depression can foster tumor progression by means of inhibiting the expression of MHC class I and II molecules and through the reduction of NKA.     

猪IL-10在软骨细胞中的表达

为了构建猪IL 10真核表达载体并在猪软骨细胞中表达。将目的基因酶切连入真核表达载体pcDNA3 1,以FuGENE 6介导转入猪软骨细胞中表达 ,RT PCR检测IL 10在软骨细胞中mRNA表达水平 ,ELISA检测细胞培养上清中IL 10蛋白含量。观察其对效应细胞PBMC分泌IFN γ和软骨细胞MHCII类分子表达的影响。结果显示 ,成功构建了猪IL 10真核表达载体 ,并转入软骨细胞 ,检测到细胞内IL 10mRNA转录 ,细胞培养 2 4、 4 8和 72h后 ,经ELISA检测上清中IL 10蛋白含量分别为 196 1 9、 2 92 5 9和 2 95 3 4pg/ml,能明显抑制经PHA刺激的PBMC分泌IFN γ (抑制率达 6 8 1% )和软骨细胞MHCII类分子的表达。猪IL 10真核表达载体的成功构建并在软骨细胞中表达 ,发挥其免疫调节功能 ,为研究IL 10诱导软骨细胞体内同种异体移植耐受奠定了基础。     

IL-18在实验性暴发型肝衰竭发病机制中的作用

为探讨IL 18在暴发型肝衰竭发生中的表达变化及对其他细胞因子的调控作用。采用D 氨基半乳糖 (D Gal) 90 0mg/kg与脂多糖 (LPS ) 10 μg/kg诱导BALB/c小鼠暴发型肝衰竭 ,检测不同时间点血清转氨酶 (ALT、AST )和肝组织病理、DNA梯形条带 ,评估肝损伤情况 ;用半定量RT PCR和相应的分析软件分析不同时间点肝组织中IL 18mRNA、TNF αmRNA和IFN γmRNA表达及ELISA方法检测血浆IL 18、TNF α和IFN γ的蛋白表达。结果 :D Gal/LPS给予后 4h血清转氨酶明显升高 ,7h小鼠开始死亡 ,10h死亡率达 80 %。肝组织病理学检查发现 ,5h肝窦扩张、炎性细胞浸润、枯否细胞增生 ;7h肝细胞大量凋亡、坏死或肝组织出现大量出血性坏死 ;5h电镜示肝细胞核仁碎裂、线粒体肿胀或空泡变性 ;7h核仁边聚 ,呈半月型 ,表现为典型的凋亡形态学变化 ,线粒体大部分空泡变性。DNA电泳显示 5h始出现梯形条带。正常小鼠肝组织IL 18mRNA有少量表达 ,TNF αmRNA、IFN γmRNA微量表达。给药后 ,三者的mRNA分别在 1h、 2h、 3h达高峰 ,血浆中TNF α、IFN γ水平与其mRNA变化显著正相关 (rTNF α=0 4 3,P =0 0 1;rIFN γ=0 6 9,P <0 0 0 1) ,而血浆IL 18与其mRNA表达无明显相关 (r= 0 12 ,P =0 2 5 )。本实验诱导的暴发型肝衰竭模型中 ,肝细?     

大鼠胰岛β细胞损伤中IL-18的作用研究

新生Wistar大鼠离体胰岛与细胞因子孵育后 ,观测胰岛素释放和一氧化氮 (NO )生成的变化 ,并用逆转录 聚合酶链反应 (RT PCR )观察IL 18受体信号链 (IL 18Rβ )mRNA的表达水平。结果表明 :(1) 0 6 2 5～ 10nmol/L基因重组小鼠 (rm )IL 18孵育胰岛 2 4h后 ,对累积的和葡萄糖刺激的胰岛素释放以及NO生成均无显著效应 ;(2 ) 2 0 0U/ml基因重组大鼠 (rr)γ干扰素 (IFN γ )或 2 5 0U/ml基因重组人 (rh )肿瘤坏死因子 α (TNF α)单独存在对胰岛素释放和NO生成均无明显效应 ,也不能促使 10nmol/LrmIL 18对胰岛素释放和NO生成产生影响 ;(3) 2 0 0U/mlrrIFN γ +2 5 0U/mlrhTNF α或 15pg/mlrhIL 1β均明显促进NO生成和抑制胰岛素释放 ,而 10nmol/LrmIL 18则不影响IFN γ +TNF α或IL 1β的上述效应 ;(4 )即使经IL 1β和 (或 )IFN γ +TNF α或IL 12孵育后 ,大鼠胰岛素瘤 (RIN )细胞或离体大鼠胰岛仍未见IL 18RβmRNA的表达。提示IL 18在细胞因子所致胰岛β细胞损伤中不发挥直接作用 ,原因是IL 18受体在胰岛 β细胞中不表达。