The adhesion molecule and cytokine profile of multiple sclerosis lesions

The expression of the adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and their respective receptors on leukocytes, very late activation antigen-4 (VLA-4) and lymphocyte function–associated antigen-1 (LFA-1), together with a selection of proinflammatory and immunomodulatory cytokines (interleukin [IL]-1, IL-2, IL-4, IL-10, tumor necrosis factor-α [TNF-α], transforming growth factor-β [TGF-β], and interferon-γ [IFN-γ]) was examined by immunocytochemistry in multiple sclerosis (MS) lesions of different ages and compared with central nervous system (CNS) tissue from other neurological diseases, both inflammatory and noninflammatory, and normal CNS tissue. These molecules play key roles in lymphocytic infiltration and interactions during tissue inflammation and are in large part normally not expressed by CNS cells. High levels of expression of all the molecules tested were found in MS, particularly in chronic active lesions. Positivity for all molecules was also seen in other neurological diseases, even in noninflammatory conditions. There was some suggestion that the VCAM-1/VLA-4 adhesion pathway was expressed at higher levels in chronic MS lesions, while ICAM-1/LFA-1 was used more uniformly in lesions of all ages. Of the cytokines examined, there was increased expression of TNF-α and IL-4 in MS; this was found to be statistically significant when compared with noninflammatory neurological diseases. The expression of most adhesion molecules and some cytokines was negligible in normal CNS tissue although low-level reactivity for ICAM-1 TGF-β, IL-4, TNF-α, and IL-10 was detected, perhaps indicative of immunoregulatory mechanisms. Microglial cells and astrocytes were the major CNS cell types expressing cytokines. The results indicate a potential in the CNS for widespread induced expression of molecules involved in the inflammatory cascade. No adhesion or cytokine molecule or pattern of expression unusual for MS was apparent.     

Transforming growth factor–beta inhibition of cytokine-induced vascular cell adhesion molecule-1 expression in human astrocytes

Leukocyte transmigration across the blood-brain barrier (BBB) is a cardinal feature of central nervous system (CNS) inflammation. Astrocytes form an integral part, both structurally and functionally, of the BBB. Vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobulin gene superfamily, is involved in extravasation into inflamed tissues and activation of T-lymphocytes. In this study, we investigated the role of TGF-β, an immunosuppressive cytokine, in regulating cytokine-induced VCAM-1 expression in astrocytes. Human astroglioma cell lines and primary human fetal astrocytes were examined for VCAM-1 gene expression after treatment with proinflammatory cytokines (TNF-α, IL-1β, IFN-γ) in the absence or presence of TGF-β. Astroglioma cell lines as well as primary human fetal astrocytes expressed low levels of VCAM-1 constitutively, and the proinflammatory cytokines induced marked increases in VCAM-1 expression, particularly TNF-α and IL-1β. The inclusion of TGF-β1 or TGF-β2 with the proinflammatory cytokines inhibited VCAM-1 gene expression to varying degrees (33–93%) in all the astroglioma cell lines and primary fetal cells. These results indicate that TGF-β is an important regulator of cytokine induced VCAM-1 expression on astrocytes and may prove useful clinically in controlling CNS inflammation. GLIA 22:171–179, 1998. © 1998 Wiley-Liss, Inc.     

Interleukin-1β (IL-1β)-induced modulation of the hypothalamic IL-1β system,tumor necrosis factor-α, and transforming growth factor-β1 mRNAs in obese (fa/fa) and lean (Fa/Fa) Zucker rats: Implications to IL-1β feedback systems and cytokine–cytokine interactions

Interleukin-1β (IL-1β) induces anorexia, fever, sleep changes, and neuroendocrine alterations when administered into the brain. Here, we investigated the regulation of the IL-1β system (ligand, receptors, receptor accessory protein, and receptor antagonist), tumor necrosis factor-α (TNF-α), transforming growth factor (TGF)-β1, and TGF-α mRNAs in the hypothalamus of obese (fa/fa) and lean (Fa/Fa) Zucker rats in response to the intracerebroventricular microinfusion of IL-1β (8.0 ng/24 hr for 72 hr, a dose that yields estimated pathophysiological concentrations in the cerebrospinal fluid). IL-1β increased IL-1β, IL-1 receptor types I and II (IL-1RI and IL-1RII), IL-1 receptor accessory protein soluble form (IL-1R AcP II), IL-1 receptor antagonist (IL-1Ra), TNF-α, and TGF-β1 mRNAs in the hypothalamus from obese and lean rats. IL-1β–induced IL-1β system and ligand (IL-1β, TNF-α, and TGF-β1) mRNA profiles were highly intercorrelated in the same samples. Levels of membrane-bound IL-1R AcP and TGF-α mRNAs did not change. Heat-inactivated IL-1β had no effect. The data suggest 1) the operation of an IL-1β feedback system (IL-1β/IL-1RI/IL-1R Acp II/IL-1RII/IL-1Ra) and 2) potential cytokine–cytokine interactions with positive (IL-1β ←→ TNF-α) and negative (TGF-β1 → IL-1β/TNF-α) feedback. Dysregulation of the IL-1β feedback system and the TGF-β1/IL-1β-TNF-α balance may have implications for neurological disorders associated with high levels of IL-1β in the brain. J. Neurosci. Res. 49:541–550, 1997. © 1997 Wiley-Liss, Inc.     

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF-α) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1β or TNF-α, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1β or TNF-α, respectively. D54-MG cells require a minimal 1 h exposure to IL-1β in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-α. Simultaneous treatment of D54-MG cells with IL-1β and interferon-gamma (IFN-γ) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-α and IFN-γ. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1β (5.3-fold increase) and TNF-α (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1β and IFN-γ. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-γ, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.     

TNF-α and TGF-β act synergistically to kill Schwann cells

Interactions between cytokines and Schwann cells (SC) are important in development, repair, and disorders of the peripheral nervous system (PNS). Tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) are two prominent cytokines which may be involved in these processes and their gene products are upregulated in some experimental neuropathies. This study focuses on thein vitro effects of these cytokines, both singly and in combination, on cultured SC. Expression of both Type I and Type II TNF-α receptors was demonstrated on the SC surface by immunocytochemistry. Treatment of SC with a combination of TNF-α plus TGF-β causes significant detachment and cell death while treatment with each cytokine alone is not significantly cytotoxic. When compared with control cultures, SC treated with the combination of cytokines exhibit an increase in the number of cells with condensed nuclei and evidence of DNA fragmentation, characteristics consistent with cells undergoing programmed cell death. Thus, TNF-α plus TGF-β induce SC loss of adhesion which is predominantly due to cell death. Apoptotic mechanisms are likely to contribute to some extent to this cell death. These findings provide in vitro evidence to support the hypothesis that cytokines can directly damage SC in PNS disorders. J. Neurosci. Res. 53:747–756, 1998. © 1998 Wiley-Liss, Inc.     

Differential localization and expression of α and β isoenzymes of protein kinase c in the rat retina

Expression and cellular localization of three isoenzymes of Ca²⁺-dependent protein kinase C (PKCα, PKCβ, and PKCγ) in the adult rat retina were revealed by immunohistochemistry and in situ hybridization histochemistry with isoenzyme-specific antibodies and cRNA probes. Immunoreactivities and mRNA signals for PKCα were conspicuous in rod bipolar cells. A subgroup of amacrine cells expressed PKCα. The cells in the ganglion cell layer also displayed PKCα gene products. Positive immunoreactivities for PKCβ were localized as stripe patterns in the inner plexiform layer, corresponding to the stratification levels of axon terminals of cone bipolar cells. The somata of cone bipolar cells expressed PKCβ. Amacrine cells and retinal ganglion cells also displayed PKCβ gene products. The results obtained by immunohistochemistry were confirmed with colocalization of mRNA signals for PKCα and PKCβ on the somata. The cell membranes showed stronger immunoreactivities than did the cytoplasms for both PKCα and PKCβ. Neither immunoreactivities nor mRNA signals for PKCγ were detected in all retinal regions. The differential roles of Ca²⁺-dependent PKC isoenzymes could be revealed in physiological defined retinal neurons. J. Neurosci. Res. 54:655–663, 1998. © 1998 Wiley-Liss, Inc.     

Correlation of matrix metalloproteinases-2 and -9 with proinflammatory cytokines in Guillain-Barré syndrome

The role of matrix metalloproteinases (MMPs) and cytokines in the pathogenesis of Guillain-Barré syndrome (GBS) largely remains unknown. We studied the role of MMP-2, MMP-9, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in disease progression and recovery of patients with GBS. Sixty-five patients with GBS and 68 healthy controls were enrolled in the study. Serum levels of MMP-2, MMP-9, TNF-α, and IL-1β were analyzed by ELISA, and activities of MMPs were measured by zymography. Expression of MMP-9, TNF-α, and IL-1β was higher in the progressive phase and lower in the recovery phase of GBS than in controls. A positive correlation of MMP-2 with IL-1β and MMP-9 with TNF-α and IL-1β was observed with progressive-phase GBS. The study shows that up-regulation of MMP-9 along with proinflammatory cytokines (TNF-α and IL-1β) in the early course appears to be associated with immune-mediated disease progression resulting from inflammation in the peripheral nervous system, whereas, during the later phase, down-regulation of MMP-9 and proinflammatory cytokines is implicated in recovery from the disease.     

Multiple sclerosis: myelin basic protein induced mRNA expression of proinflammatory cytokines in mononuclear cells is suppressed by interferon-β 1b in vitro

Beta-interferon (IFN-β) is a promising treatment in multiple sclerosis (MS), reducing the exacerbation rate and MRI lesion burden, as well as the disease progression in relapsing-remitting MS. IFN-β was originally defined by its antiviral effects, but the interest has recently been focused on its immunomodulatory properties. Myelin basic protein (MBP) is one of several autoantigens considered to be the target for autoaggressive immune responses, which eventually might lead to the development of MS. To study in-vitro effects of IFN-β1b on MBP induced cytokine expression, mRNA for the Th1 cytokines IFN-γ and TNF-α, the Th2 related IL-4 and IL-6, the cytolytic perforin and the immune response downregulating TGF-β was measured with in situ hybridization after culture of blood mononuclear cells (MNC) in the presence and absence of MBP. Numbers of cells expressing IFN-γ, TNF-α, perforin and IL-4 mRNA were significantly suppressed after culture with 10 U/ml IFN-β1b. No such effect was seen on MBP induced IL-6 or TGF-β mRNA expression. These observations suggest that one of the major effects of IFN-β1b is the induction of a shift in the cytokine mRNA profile towards a more immunosuppressive pattern. In parallel in vitro tests, the control substance dexametasone (40 μg/ml) reduced the numbers of cells expressing mRNA for all cytokines under study with the exception of TGF-β, to an extent equal to or even more pronounced than IFN-β1b.     

黄芩苷对海人酸致痫小鼠海马白细胞介素-1β及肿瘤坏死因子-α表达的影响

目的 探讨黄芩苷对海人酸诱导的小鼠癫痫持续状态后海马组织白细胞介素-1 β(IL-1β)、肿瘤坏死因子-α(TNF-α)表达的影响.方法 将54只ICR雄性小鼠随机分为对照组、癫痫持续状态(SE)组、黄芩苷治疗组,每组18只.采用侧脑室注入海人酸建立小鼠癫痫持续状态模型.HE染色观察黄芩苷对小鼠癫痫持续状态后海马神经细胞的形态学影响.通过RT-PCR和Western blot分别检测小鼠海马组织中IL-1β mRNA、TNF-α mRNA及IL-1β、TNF-α蛋白的表达量.结果 黄芩苷明显改善了SE后小鼠海马组织的病理形态学,并且可降低IL-1β、TNF-α的表达(P＜0.05).结论 黄芩苷可能通过降低癫痫鼠海马组织中IL-1 β、TNF-α的表达发挥抗炎作用,从而对脑组织进行保护.     

大鼠脑缺血再灌注后TNF—α，IL—β及细胞间粘附分子—1 mRNA的表达

目的：研究脑缺血再灌注后TNF－α,IL-β及ICAM－1在mRNA的表达变化,以探讨它们在脑再灌注损伤中的作用。／方法：提取局灶性脑缺血再灌注大鼠的RNA,用半定量逆转录－多聚酶链反应（RTPCR）测定再灌注不同时间点TNF－α,IL-β及ICAM－1在mRNA的表达水平,结果：在缺血侧,TNF－α,IL-β mRNA的表达变化相似,缺血及再灌注后表达增加,并在再灌注4h后达到顶峰,而CAM－1 mRNA在再灌注10h后达到顶峰,三类mRNA水平的升高可以保持到再灌注后3d,在缺血对侧,与假手术组比较,这三类mRNA水平有所增加,但是远低于缺血侧的mRNA水平。结论：TNF－α,IL-β及ICAM－1 mRNA在再灌注后不同时间的表达变化表明它们在缺血再灌注损伤中起重要作用。     

凝血酶诱发的脑积水中TNF-α与IL-1β对水孔蛋白-1与水孔蛋白-4表达水平的影响

目的探讨蛛网膜下腔出血(Subarachnoid hemorrhage, SAH)中凝血酶(Thrombin, TH)诱导脑积水形成时肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-1β(Interleukin-1β,IL-1β)在脑积水病理发展过程中对水孔蛋白1(Aquaporin 1,AQP1)和水孔蛋白4(Aquaporin 4,AQP4)表达水平的调控作用。方法选用健康雄性远交群(Sprague Dawley, SD)大鼠40只,随机分成4组(n=10):假手术组、凝血酶(Thrombin, TH)组、TH/TNF-α抑制剂组、TH/IL-1β抑制剂组;假手术组大鼠枕大池注入0.3 mL生理盐水;TH组大鼠枕大池注入0.3 mL(10 U/mL)凝血酶;TH/TNF-α抑制剂组大鼠枕大池注入0.3 mL(10 U/mL)凝血酶+(0.25μM)TNF-α抑制剂Simponi, TH/IL-1β抑制剂组大鼠枕大池注入0.3 mL(10 U/mL)凝血酶+(0.25μM)TH/IL-1β抑制剂GIBH-130;每组大鼠至少5只以上,按照缺...     

几种激素和IL-1β对人脑星形胶质细胞IL-6 、TNF-α分泌的影响

目的 观察T3等因素对体外培养人胎大脑星形胶质细胞分泌IL-6、TNF-α的调节作用。方法 纯化培养人胎大脑星形胶质细胞,应用酶联免疫分析(ELISA)方法检测培养上清液中IL-6、TNF-α的水平。结果 (1)星形胶质细胞(AC)在体外培养条件下可自发分泌IL-6,而TNF-α则几乎检测不到。(2)LPS(0.1μg/mL)即可诱导AC产生IL-6和TNF-α。(3)IL-1β是IL-6分泌的主要诱导剂,但不诱导TNF-α分泌。(4)氢化可的松可明显抑制AC分泌IL-6、TNF-α。(5)T3在72h可刺激IL-6的分泌。(6)胰岛素对IL-6的分泌没有明显的调节作用。结论 AC可通过分泌细胞因子参与炎症反应等病理过程并维持中枢神经系统的正常发育、内环境的稳定,且受多种因素的调节。在中枢神经系统中T3、胰岛素主要参与调节发育和代谢,可能不直接参与炎症和免疫机制调节。     

Cytokine modulation of murine microglial cell superoxide production

Activated microglia may contribute to two opposite effects during inflammation within the central nervous system: host defense against microorganisms and neuronal injury. Each of these processes may be mediated by the generation of reactive oxygen intermediates by activated microglia. We investigated the effects of two proinflammatory cytokines, interferon (IFN)-β and tumor necrosis factor (TNF)-α, and of the anti-inflammatory cytokine, transforming growth factor (TGF)-β, on murine microglial cell superoxide (O₂) production upon stimulation with phorbol myristate acetate (PMA). Priming of microglia with IFN-β or TNF-α resulted in a dose-dependent enhancement of O₂ release in response to PMA. The priming effects of these two cytokines were additive, suggesting that they acted by independent mechanisms. We also found that IFN-β and TNF-β stimulated the release of bioactive TGF-β and that treatment of microglial cell cultures with TGF-β antagonized the priming effects of IFN-β and TNF-α on O₂ production. The results of this study have implications for understanding the mechanisms by which cytokines and microglia may contribute to host defense as well as to injury of the brain. © 1995 Wiley-Liss, Inc.     

IL‐1β enhances the antibacterial activity of astrocytes by activation of NF‐κB

Astrocytes have important immune functions in CNS, and astrocytes stimulated by interferon‐γ were showed to have direct antimicrobial function. However whether astrocytes without the stimulation of cytokines have antibacterial function, and how this function is regulated are still largely unknown. In this study, we found that primary cultured astrocytes inhibited the growth of both gram‐negative and gram‐positive bacteria. Further more, we showed that interleukin‐1β (IL‐1β) enhanced the antibacterial effect in a dose‐dependent manner, and the antibacterial effect of astrocytes from IL‐1β receptor‐deficient mice failed to be enhanced by IL‐1β. IL‐1β stimulated IκBα degradation, NF‐κB nuclear translocation, and transactivation in astrocytes. NF‐κB inhibitors blocked NF‐κB activation and the enhanced antibacterial effect induced by IL‐1β. In addition, overexpression of dominant negative IκBα in astrocytes inhibited IκBα degradation and NF‐κB transactivation, and also inhibited the enhanced antibacterial effect induced by IL‐1β. All these data demonstrated that IL‐1β enhanced the antibacterial activity of astrocytes by activation of NF‐κB. © 2009 Wiley‐Liss, Inc.     

Restricted Infection and Cytokine Expression in Primary Murine Astrocytes Induced by the H5N1 Influenza Virus

Central nervous system (CNS) dysfunction caused by infection with neurovirulent viruses is of interest, and may play an important role in many neurodegenerative diseases. Since neuronal functions are believed to be partly regulated by cytokines produced by astrocytes, neuroinflammation and neurodegeneration caused by H5N1 influenza virus infection could be due to abnormal cytokine production of those infected astrocytes. In the present study, cytokine responses of murine astrocytes following H5N1 virus infection were investigated. Primary astrocytes from neonatal outbred ICR mice were isolated and used to investigate cytopathology upon infection with the H5N1 virus (A/Chicken/Thailand/CUK2/04). Thereafter, cell lysates at 6, 24, 48, and 72 hours-post infection (hpi) were collected and subjected to quantification of cytokine gene expression, including TNF-α, IL-1β, IL-2, IL-6 and IL-12β, by quantitative RT-PCR. The results revealed that infection with the H5N1 virus in primary murine astrocytes was restricted, and resulted in abortive virus infection. However, this abortive infection in the astrocytes was found to result in significant upregulation of IL-1β mRNA expression at 72 hpi compared with the mock-infected group, while the mRNA expression of IL-2 and IL-12β was observed to have undergone significant down-regulation at 6-72 hpi and 24-48 hpi, respectively. The results of the present study could support the role of astrocytes in neuroinflammation and neurodegeneration.     

Effects of interleukin-1β and tumor necrosis factor-α on the expression of glial fibrillary acidic protein and transferrin in cultured astrocytes

Recent evidence suggests that interleukin (IL)-1 and tumor necrosis factor (TNF) may play a role in astrogliosis following injury to the CNS. The short-term biochemical effects of these immune-related cytokines were determined on cultured rat polygonal and process-bearing astrocytes. Both IL-1 and TNF stimulated the rate of thymidine incorporation in polygonal astrocytes up to 137% and 215%, respectively, over the level observed in untreated controls. By contrast, thymidine incorporation was relatively unaffected by these cytokines in process-bearing astrocytes. The cytokines did not significantly affect the level of glial fibrillary acidic protein (GFAP) within polygonal astrocytes, even though they appeared to downregulate the expression of GFAP mRNA by as much as 62%. Both cytokines increased the intracellular expression of transferrin (Tf) within some polygonal astrocytes. In untreated control cultures, fewer than 2% of polygonal astrocytes were immunoreactive for Tf. By contrast, approximately 30% of polygonal astrocytes treated with IL-1 or TNF-α became strongly immunoreactive for Tf. Neither IL-2 nor a number of other known growth factors appeared to alter the level of immunoreactive Tf in these cells. Process-bearing astrocytes were negative for Tf, regardless of the treatment used. Northern blot analysis demonstrated that the level of Tf mRNA in cultures of polygonal astrocytes increased 148% above the level observed in untreated controls following treatment with either IL-1 or TNF, whereas no change was observed following treatment with IL-2. These results suggest that increased levels of particular cytokines known to be present in injured CNS can produce pronounced biochemical alterations within a subtype of cultured astrocytes. © 1993 Wiley-Liss, Inc.     

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.