Lin Hsiao-Wen, Jain Mohit Raja, Li Hong, Levison Steven W
Department of Neurology and Neurosciences, UMDNJ-New Jersey Medical School Cancer Center, Newark, NJ, USA.
J Neuroinflammation. 2009 Mar 6;6:7. doi: 10.1186/1742-2094-6-7.
Ciliary neurotrophic factor (CNTF) has been regarded as a potent trophic factor for motor neurons. However, recent studies have shown that CNTF exerts effects on glial cells as well as neurons. For instance, CNTF stimulates astrocytes to secrete FGF-2 and rat microglia to secrete glial cell line-derived neurotrophic factor (GDNF), which suggest that CNTF exerts effects on astrocytes and microglia to promote motor neuron survival indirectly. As CNTF is structurally related to IL-6, which can stimulate immune functions of microglia, we hypothesized that CNTF might exert similar effects.
We performed 2-D and 1-D proteomic experiments with western blotting and flow cytometry to examine effects of CNTF on primary microglia derived from neonatal mouse brains.
We show that murine microglia express CNTF receptor alpha (CNTFRalpha), which can be induced by interferon-gamma (IFNgamma). Whereas IL-6 activated STAT-3 and ERK phosphorylation, CNTF did not activate these pathways, nor did CNTF increase p38 MAP kinase phosphorylation. Using 2-D western blot analysis, we demonstrate that CNTF induced the dephosphorylation of a set of proteins and phosphorylation of a different set. Two proteins that were phosphorylated upon CNTF treatment were the LYN substrate-1 and beta-tubulin 5. CNTF weakly stimulated microglia, whereas a stronger response was obtained by adding exogenous soluble CNTFRalpha (sCNTFRalpha) as has been observed for IL-6. When used in combination, CNTF and sCNTFRalpha collaborated with IFNgamma to increase microglial surface expression of CD40 and this effect was quite pronounced when the microglia were differentiated towards dendritic-like cells. CNTF/sCNTFRalpha complex, however, failed to increase MHC class II expression beyond that induced by IFNgamma. The combination of CNTF and sCNTFRalpha, but not CNTF alone, enhanced microglial Cox-2 protein expression and PGE2 secretion (although CNTF was 30 times less potent than LPS). Surprisingly, Cox-2 production was enhanced 2-fold, rather than being inhibited, upon addition of a gp130 blocking antibody.
Our studies indicate that CNTF can activate microglia and dendritic-like microglia similar to IL-6; however, unlike IL-6, CNTF does not stimulate the expected signaling pathways in microglia, nor does it appear to require gp130.
睫状神经营养因子（CNTF）一直被视为运动神经元的一种强效营养因子。然而，最近的研究表明，CNTF对神经胶质细胞以及神经元均有作用。例如，CNTF刺激星形胶质细胞分泌成纤维细胞生长因子-2（FGF-2），刺激大鼠小胶质细胞分泌胶质细胞系源性神经营养因子（GDNF），这表明CNTF通过作用于星形胶质细胞和小胶质细胞间接促进运动神经元存活。由于CNTF在结构上与白细胞介素-6（IL-6）相关，而IL-6可刺激小胶质细胞的免疫功能，我们推测CNTF可能具有类似作用。
我们采用二维和一维蛋白质组学实验，并结合蛋白质印迹法和流式细胞术，来研究CNTF对新生小鼠脑源性原代小胶质细胞的作用。
我们发现鼠小胶质细胞表达CNTF受体α（CNTFRα），其可被γ干扰素（IFNγ）诱导。虽然IL-6可激活信号转导和转录激活因子3（STAT-3）以及细胞外信号调节激酶（ERK）的磷酸化，但CNTF并未激活这些信号通路，也未增加p38丝裂原活化蛋白激酶（MAP激酶）的磷酸化。通过二维蛋白质印迹分析，我们证明CNTF可诱导一组蛋白质的去磷酸化以及另一组蛋白质的磷酸化。经CNTF处理后发生磷酸化的两种蛋白质分别是LYN底物-1和β微管蛋白5。CNTF对小胶质细胞的刺激作用较弱，而添加外源性可溶性CNTFRα（sCNTFRα）可产生更强的反应，这与IL-6的情况类似。联合使用时，CNTF和sCNTFRα与IFNγ协同作用，增加小胶质细胞表面CD40的表达，当小胶质细胞分化为树突状细胞时，这种作用尤为明显。然而，CNTF/sCNTFRα复合物未能使主要组织相容性复合体II类分子（MHC II类分子）的表达增加超过IFNγ诱导的水平。CNTF和sCNTFRα联合使用可增强小胶质细胞环氧化酶-2（Cox-2）蛋白表达和前列腺素E2（PGE2）分泌（尽管CNTF的效力比脂多糖（LPS）低30倍）。令人惊讶的是，添加gp130阻断抗体后，Cox-2的产生增加了2倍，而非受到抑制。
我们的研究表明，CNTF可像IL-6一样激活小胶质细胞和树突状小胶质细胞；然而，与IL-6不同的是，CNTF不会刺激小胶质细胞中预期的信号通路，而且似乎也不需要gp130。
