Toll样受体信号对表皮黑素细胞天然免疫功能的影响和意义
摘要
白癜风是一种常见的原发性的、获得性的皮肤色素脱失性皮肤、粘膜疾病,以大小不等、数目不定的色素完全脱失斑为典型皮损。目前认为其发病是多基因遗传的、在多种内外因子的激发下表现为免疫功能紊乱,导致表皮黑素细胞破坏,终至色素脱失。彻底阐明白癜风的发病机制特别是免疫机制对于探索到治疗白癜风的新靶点有着非常重要的意义。
在白癜风中尽管存在自身免疫异常的证据,但是导致针对黑素细胞自身抗原的细胞和体液免疫是如何触发的问题仍有待解决。根据Matzinger的危险信号学说,体内的细胞损伤等危险信号可激活APC来活化适应性免疫反应,在白癜风自身免疫反应的触发期,往往存在皮肤黑素细胞生物学特性的异常,例如,外源性机械刺激、烧伤、过度紫外线照射以及化学物质的刺激会导致局部黑素细胞处于应激状态,而这种应激状态是否导致黑素细胞的免疫学特性发生改变,从而激发或者放大随之而来的针对黑素细胞抗原的适应性自身免疫反应,还需要的研究证实。但是毋庸置疑的是黑素细胞的天然免疫特性可能在联结黑素细胞应激反应与自身免疫免疫反应中起到了重要的桥梁作用。
近年来对黑素细胞的研究,特别是对其生物学特性及细胞生化方面的研究已经较为深入,许多证据都表明黑素细胞与皮肤免疫系统之间有密切联系。体外培养的黑素细胞表面可以表达MHC-Ⅰ/Ⅱ分子、粘附分子ICAM-1、VCAM-1及Fc受体等免疫分子,并且能够被IFN-γ和TNF-α上调表达；黑素细胞自身还能分泌细胞因子IL-1、IL-6、IL-8和TGF-β等细胞因子,认为黑素细胞是皮肤免疫系统的重要成员,并且可能以更加主动的方式参与并调节免疫反应。
TLRs被认为只表达在免疫细胞和多种炎症细胞表面,如树突状细胞(Dendritic cells, DC)、巨噬细胞和T、B淋巴细胞等,与其相应的配体结合后可以激活免疫细胞使其分泌大量的促炎性细胞因子和趋化因子,参与活化天然免疫应答和特异性免疫应答,构成机体免疫系统的第一道防线。近年来,有报道指出除免疫细胞外,多种组织细胞也表达TLRs,例如皮肤角质形成细胞表达不同的TLRs,与TLRs相应配体结合后被激活,分泌大量的细胞因子和趋化因子。
由上述关于白癜风发病机制和TLRs生物学功能的研究,我们设想：黑素细胞可以表达功能性TLRs, TLRs与其内外源性配体结合后引发黑素细胞生物学及免疫学表型和功能的变化,这种变化很可能与白癜风自身免疫反应的发生和发展有着密切的联系。尽管这种联系存在着诸多疑问。一旦这些疑问得到解决,我们将发现一个新颖的角度解释白癜风的发病机制,并有希望将该通路作为新的治疗靶点应用于白癜风的临床治疗。
原代培养细胞24h后开始贴壁,可见角质形成细胞间散在的多级树突状细胞,大部分细胞胞体圆而小,有两个对称树突,突起长短不等；少数细胞胞体大,呈多角形,有2个至数个树突。约14天后可传代,根据角质形成细胞和黑素细胞对胰酶的不同耐受性和成纤维细胞不能耐受G418的特性可分离出纯黑素细胞。显微镜下可见系胞质及树突呈棕褐色或黑色,多巴染色结果呈阳性。我们采用RT-PCR法对体外培养的第三代黑素细胞进行鉴定,结果显示,角质形成细胞特异性标记分子keratin 10和keratin 14,成纤维细胞的特异性标记分子ASO2均不能被扩增,证明用上述培养传代方法制备的第三代黑素细胞纯度较好,可用于进行下一步试验。
为了定性证明各TLRs成员在黑素细胞上的表达,我们以体外培养的第三代黑素细胞为对象,并且以正常人外周血PBMC作为阳性对照,采用RT-PCR法检测到mRNA水平表达的TLR1,2,3,4,6,7以及9,未检测到TLR5,8和10。我们采用间接免疫荧光标记和流式细胞术在蛋白水平证明黑素细胞上TLR2,4,7和9的表达。为了揭示黑素细胞上表达的TLR3的亚细胞定位,我们采用间接免疫荧光和流式细胞术对黑素细胞膜上和胞内可能存在的TLR3进行检测。结果证实：黑素细胞上表达的TLR3主要定位于细胞内。
为了进一步证实TLR3的表达,我们用Western blot方法对TLR3进行了免疫印迹检测。结果显示：PVDF膜在TLR3的位置上出现了特异性的染色条带,大小为100 kD左右,与文献报道的人TLR3分子量相符合。为排除黑素细胞培养条件对TLR3表达的影响,我们采用荧光免疫组织化学法检测了TLR3在皮肤组织中的原位表达。结果证实,NKI/beteb作为特异性标志显示黑素细胞位于表皮真皮交界处,并可以观察到黑素细胞上TLR3的表达。
上述结果显示,人表皮黑素细胞可以组成性表达TLR3,因此接下来讨论TLR3的表达是否可以被其特异性配体Poly(I:C)诱导。在不同浓度Poly(I:C)作用后6h,TLR3 mRNA开始上调表达,其上调倍数分别为：2.7(1μg/ml),4.2(10μg/ml)以及3.6 (100μg/ml)。在Poly I:C作用24小时后,TLR3上调表达倍数分别为3.8 (1μg/ml),13(10μg/ml)以及37(100μg/ml),并明显剂量依赖性。这些结果证明TLR3可以被其配体刺激上调表达,黑素细胞表达的TLR3具有一定生物学功能。
我们在前述工作中已证明黑素细胞表达TLR家族的众多成员,为了验证这些TLR是否也具有相应的功能,首先,我们检测了黑素细胞在TLR活化条件下促炎性细胞因子IL-8和IL-6的分泌情况。结果显示：针对TLR2,3,4,7和9的激活剂都可以诱导IL-8分泌量显著提高,其中以TLR3激活剂poly(I:C)最强烈。黑素细胞在用PGN,poly(I:C),LPS以及CpG 2006刺激时,IL-6分泌水平明显升高,但在用imiquimod作为刺激物刺激黑素细胞上表达的TLR7时,并没有发现IL-6分泌水平的变化。趋化因子在吸引免疫细胞浸润改变皮肤局部免疫格局中发挥重要作用。接下来我们采用real-time PCR法检测了TLR活化后趋化因子CCL2/MCP-1, CCL3/MIP-1和CCL5/RANTES。结果显示,在不同TLR激活剂刺激下,黑素细胞分泌各种趋化因子格局各有不同。所有的TLR配体都可以显著诱导CCL3 mRNA的表达,同时CCL2和CCL5也有不同程度的表达增高,但TLR7配体imiquimod无此效应。
在前面的结果中,我们证明了TLR活化可以诱导炎性细胞因子和趋化因子的释放,接下来我们将验证NF-κB信号通路的活化是否介导了TLR活化诱导黑素细胞炎性细胞因子和趋化因子的分泌。首先,我们用western blot检测磷酸化IκBα水平。结果证实：不同TLR配体处理黑素细胞后,磷酸化IκBα蛋白水平都有不同程度的升高。NF-κB活化后即发生核转位,由胞质转移至胞核。为了进一步证实NF-κB信号通路的激活,我们应用间接免疫荧光检测胞质及胞核内NF-κBp65水平。结果证实：未受刺激的黑素细胞,p65亚基几乎只分布在胞质内,TLR不同配体刺激后,黑素细胞胞质内p65蛋白明显下降,而胞核内p65蛋白则显著上调。
为观察TLR3配体dsRNA对黑素细胞的影响,我们利用phalloidin荧光染料对细胞骨架蛋白actin进行染色,结合相差显微镜观察黑素细胞形态变化。结果发现,用poly(I:C)作用于黑素细胞后,细胞形态由梭形或多角形变为扁平形,树突逐渐消失。台盼兰拒染实验显示,随着poly(I:C)剂量的增加,黑素细胞存活率逐渐降低,呈剂量依赖性。Hoechst33258细胞核染色结果显示,对照组细胞核荧光呈淡蓝色且着色较均一,而poly(I:C)作用后的黑素细胞核呈特征性亮蓝色荧光,并可观察到细胞核固缩及团块状的凋亡小体。收集poly(I:C)作用的黑素细胞,抽取细胞总DNA进行琼脂糖凝胶电泳,结果显示：与正常对照组细胞基因组DNA相比,poly(I:C)作用的黑素细胞DNA呈明显的ladder,提示有凋亡细胞的存在。进一步用Annexin-V/PI双染色结合流式细胞术检测黑素细胞凋亡情况,dsRNA作用后,显示早期凋亡信号(Annexin V+/PI-)的黑素细胞数量(22%)明显多于对照抗体组(2.5%),说明dsRNA在可以在体外诱导黑素细胞凋亡。
我们通过前期的结果证实了dsRNA对黑素细胞的促凋亡作用,为了进一步证实这种效应由TLR3介导,我们应用慢病毒载体稳定表达针对TLR3的shRNA,抑制原代培养的黑素细胞中TLR3的表达。采用western blot法检测TLR3蛋白水平的变化。结果显示：通过慢病毒载体稳定转染shTLR3的黑素细胞,其TLR3表达水平显著下降,而转染随机对照shRNA对TLR3表达水平并无影响。随后我们用流式细胞术检测下调TLR3表达的黑素细胞的凋亡情况。结果显示：稳定转染了shTLR3的黑素细胞受到poly(I:C)刺激后,其凋亡率明显降低,证明了TLR3介导了dsRNA对黑素细胞的促凋亡效应。
为验证NF-κB通路的活化是否与dsRNA诱导黑素细胞凋亡有关我们进而采用NF-κB特异性阻断剂Bay11-7082作用于TLR3激活下的黑素细胞,用westernblot检测磷酸化IκBα水平。结果显示：TLR3活化后上调的磷酸化IκBα水平,Bay11-7082有一定的抑制作用,进而检测dsRNA对黑素细胞的促凋亡效应。Bay11-7082虽然能够部分抑制NF-κB的活化,但对于dsRNA诱导的黑素细胞凋亡却没有抑制作用,同时我们用TLR4的配体LPS刺激黑素细胞,发现LPS不能明显抑制黑素细胞凋亡。因为LPS是已知的NF-κB强诱导剂,所以以上结果证实,NF-κB通路并没有参与TLR3活化诱导的黑素细胞凋亡。
TLR3活化诱导的干扰素释放对于机体抗病毒反应具有重要意义,同时有文献报道Ⅰ型干扰素,包括IFN-a和IFN-p,在某些细胞中可以诱导凋亡途径的激活,因此,在我们下一步研究中,我们分析了Ⅰ型干扰素在TLR3活化介导的黑素细胞凋亡中的可能作用。我们用TLR3配体dsRNA刺激黑素细胞后,取培养上清,用ELISA法分别检测Ⅰ型IFN即IFN-α和IFN-β的分泌。结果证实,受到dsRNA刺激进而TLR3活化之后,IFN-α和IFN-β的分泌水平显著提高。用western blot方法检测了TLR3活化后STAT1磷酸化的水平,结果显示：dsRNA处理黑素细胞2小时后,磷酸化STAT1水平显著升高。
为了进一步确证Ⅰ型IFN与黑素细胞凋亡的关系,我们使用特异性识别IFN-α和IFN-β的中和性抗体分别作用于黑素细胞,并用dsRNA刺激TLR3活化,结果发现：IFN-β中和性抗体能够强烈抑制poly(I:C)诱导下的黑素细胞凋亡,而IFN-α中和性抗体没有此效应。
为了进一步证明IFN-β分泌与黑素细胞凋亡之间的关系,我们运用不同浓度IFN-β中和性抗体作用于黑素细胞,结果发现,随着使用的抗体浓度增高,其抑制黑素细胞凋亡的效应也不断增强。
以上结果证明了黑素细胞自分泌的IFN-p在TLR3活化诱导的黑素细胞凋亡中的作用,进一步,我们分析了外源性加入的IFN-a或IFN-β是否能直接影响黑素细胞凋亡。结果显示：外源性加入的IFN-α、IFN-β没有显著影响黑素细胞的生存率。以上结果证明,IFN-β参与了TLR3活化诱导的黑素细胞凋亡,但IFN-β本身不能直接诱导黑素细胞凋亡。
细胞的凋亡在体内受到多条信号通路的影响,已报道在TLR活化中,MAPK途径发挥重要作用,所以在下一步的研究中我们将分析MAPK成员p38,ERK1／2和JNKl／2在dsRNA的促IFN-p分泌以及凋亡效应中的作用。我们用poly(I：C)与黑素细胞共孵育后,采用western blot检测p38,ERK1／2和JNKl／2的磷酸化情况。结果显示：poly(I:C)作用后,磷酸化p38、磷酸化ERK1／2和磷酸化JNK1／2水平都有不同程度上调。
为了证明活化的p38,ERK1／2和JNKl／2,是直接与dsRNA的促凋亡效应相关,还是仅为TLR3活化的伴随效应,我们采用特异性的阻断剂对p38, ERK1／2以及JNKl／2信号通路分别进行了阻断后,检测TLR3活化对黑素细胞凋亡的影响。结果显示：用p38特异性阻断剂SB203580与poly(I:C)同时作用后,黑素细胞凋亡率下降了73.6%,用ERK1／2特异性阻断剂U0126作用后,黑素细胞凋亡率下降了50%,而用JNK1／2阻断剂SP600125作用后,不影响黑素细胞的凋亡率,以上结果证明,在TLR3活化诱导的黑素细胞凋亡过程中,p38和ERK1／2通路可能发挥一定的作用。
为了进一步证实以上结果,我们将p38、ERK1／2以及JNKl／2通路阻断后,在形态学上观察TLR3活化对黑素细胞凋亡的影响,结果证实：对p38和ERK1／2的阻断可以部分逆转TLR3活化导致的黑素细胞凋亡,而阻断JNK1／2途径无此效应。阻断p38和ERK1／2都可以显著抑制poly(I:C)刺激下的IFN-β释放,阻断JNK1／2无明显作用。以上结果证实,p38和ERK1／2通路参与了TLR3活化介导的IFN-β分泌与黑素细胞凋亡。
综上所述,本课题证实人表皮黑素细胞在mRNA水平蛋白水平组成性表达TLR2,3,4,7和9的表达。这些TLR受其特异性配体刺激后,黑素细胞合成表达促炎性细胞因子和趋化因子的水平升高。我们发现黑素细胞表达的TLR3活化后可以诱导黑素细胞凋亡,这种凋亡效应依赖于IFN-β的自分泌,并受信号通路p38和ERK1／2的影响。基于以上结果,我们推测黑素细胞表达的TLR3很可能与白癜风自身免疫反应的发生和发展有着密切的联系。黑素细胞可以通过表达TLR3直接识别病毒代谢中产物dsRNA或坏死细胞释放的dsRNA,从而改变了黑素细胞的免疫学表性和生物学功能,激发或放大针对黑素细胞自身抗原的免疫反应。如果这一假设得到证实,将会帮助我们从一个新颖的角度解释白癜风的发病机制,并有希望从该通路入手寻找新的治疗靶点应用于白癜风的临床治疗。
The human epidermis provides a first defense barrier to a potentially hostile environment. It has been shown that within the epidermis, not only professional immune cells (e.g., Langerhans cells) but also non-immune cells (e.g., keratinocytes and melanocytes) are involved in the immune protection of the host. Although originally identified as a professional producer of melanin, recent studies have revealed that melanocytes exhibit a variety of functions. For example, human melanocytes have the capacity to express HLA-DR, CD40 and adhesion molecules such as ICAM-1 and VCAM-1. In addition, they can produce various soluble mediators of inflammation such as IL-1, IL-6 and IL-8. These studies suggest that melanocytes are not simply pigment-producing cells but also immunocompetent cells. However, it is unclear whether melanocytes can recognize pathogens and play an active role in the skin's local immune defense system.
Recognition of pathogens by innate immune cells is mediated by pattern-recognition receptors that recognize conserved pathogen-associated molecular patterns (PAMPs). One major group of pattern-recognition receptors is the Toll-like receptors (TLRs), which transduce signals leading to the activation of NF-κB, which subsequently drive the induction of several pro-inflammatory cytokines and chemokines. Apart from sensing exogenous ligands from microbial components, which is critical for pathogen elimination, TLRs are able to recognize endogenous ligands, such as heat shock proteins (HSPs) and extracellular matrix components, which may be called danger-associated molecular patterns (DAMPs), in analogy to PAMPs.
Thus far, more than 10 different TLRs with distinct ligand specificity have been identified. In human skin, the distribution of TLRs is incompletely defined. Several studies demonstrated that keratinocytes display TLRs and respond to corresponding PAMPs by producing pro-inflammatory cytokines. In contrast, the TLR expression pattern of primary human melanocytes has never been systematically examined.
Vitiligo is characterized by a loss of melanin-producing melanocytes from the epidermis of patients, which results in skin depigmentation. Both genetic and environmental factors have been implicated to explain the loss of epidermal melanocytes in this disorder. Genetic risk factors are important, but are not sufficient for the disease to occur, and environmental triggers may also contribute to the initiation and/or progression of vitiligo. Human skin is known to be constantly exposed to various pathogens of prokaryotic, eukaryotic, and viral origin. Indeed, a variety of viruses including cytomegalovirus, hepatitis virus, and AIDS virus, have been implicated in the pathogenesis of the vitiligo. In addition, a strong causative link between herpes virus and vitiligo has been noted in the Smyth chicken model. Finally, viral infections have been long associated with a variety or autoimmune disorders. Interestingly, some of them including thyroid disease and typeⅠdiabetes mellitus, occur with increased frequency in patients with vitiligo. These observations suggest that virus might be an important environmental factor that participates in the initiation or acceleration of vitiligo in genetically predisposed individuals.
Toll-like receptors (TLRs) play a crucial role in pathogen detection and in the mounting of antimicrobial immune responses. Although initial studies have focused on the TLR expression and function in the immune system, more recent studies have shown that TLR expression is shared by many different cell types including human epidermal melanocytes. We have previously shown that TLRs expressed in melanocytes are functional and respond to their respective ligands by activating the central transcription factor NF-κB and by up-regulating pro-inflammatory mediators. In respect of antiviral responses, TLR3 was shown to be a receptor for double-stranded RNA (dsRNA), which represents either genomic or life cycle intermediate material of many viruses. In the process of viral infection, dsRNA stimulates cellular antiviral activities, and occasionally, cell death, which is another way of protecting the host against virus spreading. In our preliminary study, we found in vitro treatment with poly(I:C), a synthetic analogue dsRNA, resulted in massive death and detachment of cultured human melanocytes. Could human melanocytes utilize TLR3 as an initiator of signaling pathway leading to destruction of themselves in response to viral infections? This hypothesis, as well as its implications in the pathogenesis of vitiligo, needs more evidential supports from both clinical observations and experimental investigations.
In the present study, we provided laboratory evidences that melanocytes expressed TLR3. Poly(I:C) treatment induced apoptosis in human melanocytes in a TLR3-dependent manner. This phenomenon required the involvement of IFN-βautocrine signaling but not NF-κB activation. Moreover, dsRNA stimulation led to the activation of downstream signaling pathways, including p38, ERK1/2 and JNK1/2, among which p38 and ERK1/2 controlled both IFN-βsecretion and IFN-βmediated cell death. The fact that human melanocytes dramatically respond to TLR3 ligand indicates that viral infection may play a part in the initiation/amplification of vitiligo by directly triggering melanocyte apoptosis.
Primary human melanocyte cultures were obtained from neonatal foreskin. All samples obtained were from surgical procedures with the patients'informed consent following a protocol approved by the Huashan Hospital Fudan University Institutional Review Board. The epidermis was separated from the dermis after an overnight incubation of skin samples in a 0.25% Dispase solution in PBS at 4℃. In order to separate cellular elements, epidermal sheets were incubated at 37℃in a solution of 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in PBS, for 10 minutes. Cellular suspension was then filtered through 70μm cell strainer and then centrifuged at 1500 rpm for 7 minutes to harvest cells. Then, melanocytes were selectively grown in a defined medium M254 with PMA-free human melanocyte growth supplements. The cells were maintained in a humidified incubator with 5% CO2 at 37℃and were fed every 3-4 d, and further passaged at 1:2 when they became 80% confluent, with experiments being carried out from 3 to 4 passages.
To investigate the expression profile of TLRs in human melanocytes, in vitro-expanded melanocytes obtained from healthy subjects were assessed by reverse transcription (RT)-PCR for TLR mRNA using a panel of specific primers for TLR1-TLR10. We found the constitutive mRNA expression of TLRs 1,2,3,4,6,7 and 9 in human melanocytes but not TLRs 5,8, and 10. As a positive control, human PBMC were shown to express all mRNA of TLRs 1-10. The purity of the cultured human melanocytes was verified by RT-PCR analysis for the keratinocyte markers, such as keratin 10 and keratin 14, and the fibroblast marker ASO2, showing that contamination from keratinocytes or fibroblasts was negligible. To measure the TLR protein expression in human melanocytes, flow cytometry and immunofluorescence assays were performed with specific antibodies to human TLR proteins. Ample amounts of TLRs 2,4,7 and 9 were detected in human melanocytes indicated by flow cytometry analysis. TLR3 is generally recognized as an intracellular receptor, but a recent study showed a surface TLR3 on human skin and lung fibroblasts. Thus, we analyzed the specific location of TLR3 expression in human melanocytes. Using the same mAb specific to TLR3, we demonstrated the detection of intracellular TLR3 in human melanocytes but not on the cell surface. The protein expression of TLRs on in vitro-cultured human melanocytes was also demonstrated by immunofluorescence assays, and consistent findings were noted. Compared to the isotype control, a clear positive staining for TLRs 2,3,4,7 and 9 was obtained. These results paralleled our RT-PCR endings. Thus human melanocytes not only expressed TLRs mRNA but also expressed TLRs protein. 3. Regulation of TLR3 in human melanocytes
To determine whether the TLR3 expression was inducible by its activation with poly(I:C), a synthetic surrogate of viral dsRNA, the kinetic of TLR3 mRNA was analyzed by real-time RT-PCR. In a dose-dependent fashion, TLR3 mRNA increased at 6 hours after treatment with poly(I:C) (2.7-fold at 1μg/ml,4.2-fold at 10μg/ml, and 3.6-fold at 100μg/ml) and at 24 hours after stimulation (3.8-fold at 1μg/ml, 13-fold at 10μg/ml and 37-fold at 100μg/ml). These data show that human melanocytes constitutively and inducibly express TLR3, suggesting that melanocytes have the potential to recognize virus-derived pathogenic motifs via TLR3.
To characterize the functional relevance of TLRs in human melanocytes, the production of inflammatory cytokines by melanocytes in response to TLR ligands were determined. Human melanocytes were stimulated with various TLR ligands, for example, The TLR2 ligand, peptidoglycan (PGN), the TLR3 ligand, poly (I:C), the TLR4 ligand, LPS, the TLR7 ligand, imiquimod, and CpG 2006, which ligates TLR9. Subsequently, the secretion level of IL-8 and IL-6 were analyzed by ELISA. The maximal concentration of poly (I:C) used in our analysis was restricted to 10μg/ml, as larger dosage led to massive death and detachment of melanocytes. All of the TLR ligands examined potently induced the production of IL-8 in a dose-dependent manner, with maximal secretion being observed at 1μg/ml poly (I:C). Melanocytes also released substantial amounts of IL-6 when stimulated with PGN, poly (I:C), LPS and CpG 2006. On the contrary, no IL-6 production was observed in melanocytes treated with imiquimod, even though the cells expressed TLR7 mRNA and protein.
Activated epidermal melanocytes may modulate the recruitment and activation of different immune cells at least in part through the expression of the chemokines. We next analyzed accumulation of mRNAs of inflammatory chemokines such as CCL2/MCP-1, CCL3/MIP-1 and CCL5/RANTES upon different TLR activation using real-time RT-PCR. Here we showed that each TLR ligand induced the release of CCL2, CCL3 or CCL5 with a different kinetics. All the TLR ligands induced significant amounts of CCL3 mRNA in 6-hour stimulated melanocytes, whereas mRNA for CCL2 and CCL5 were significantly induced by all the TLR ligands except imiquimod, the TLR7 ligand. Although some basal expression of CCL2, CCL3 and CCL5 could be observed in nonstimulated cells, there was at least 1-fold increase in their mRNA levels after treatment with TLR ligands.
Collectively, these data demonstrated that TLR ligand pretreatment of epidermal melanocytes created a pro-inflammatory milieu by showing elevated cytokines and chemokines.
Activation of NF-κB plays a central role in TLR-mediated cellular activation and gene expression in a variety of cell types. Therefore, we tested whether triggering TLRs in human melanocytes can give rise to the activation of NF-κB signaling pathway. TLRs within human melanocytes were stimulated for 6 hour by various ligands and assessed by Western blot analysis to examine phosphorylation of IκBα, which is one of the markers of NF-κB activity. No phosphorylated-IκBαwas observed in unstimulated melanocytes. On the contrary, the clear bands of phosphorylated-IκBαcould be induced by PGN (100μg/ml), poly (I:C) (10μg/ml), LPS (100μg/ml), imiquimod (100μg/ml) or CpG 2006 (5μM) to different degrees, indicating that NF-κB pathway participated in TLR2,3,4,7 and 9 signaling in response to the respective PAMPs.
To further substantiate the activation of NF-κB pathway in TLR ligand-treated melanocytes, translocation to the nucleus of the NF-KBp65 was analyzed. Human melanocytes were cultured in slide chambers in the presence of TLR ligands and stained for the NF-KBp65 as described in Materials and methods. The slides were then analyzed for nuclear localization by fluorescence microscopy. In unstimulated melanocytes, the stainings for the NF-κBp65 were mainly visible in the cytoplasm. In contrast, when melanocytes were stimulated with PGN (100μg/ml), poly (I:C) (10μg/ml), LPS (100μg/ml), imiquimod (100μg/ml) or CpG 2006 (5μM), a clear nuclear translocation of the p65 subunit was observed in all the stimulated groups. These data indicated that TLR 2,3,4,7 or 9 ligand engagement led to the activation of NF-κB, which explained the numerous pro-inflammatory genes being activated.
We further investigated the responsiveness of melanocytes to dsRNA. Melanocytes exposed to 100 mg/ml poly(I:C) underwent striking changes in cell appearance:cytoplasmic spreading and flattening of the cells and their nuclei were illustrated by phase contrast and phalloidin staining at 8 hours and 24 hours. A more prolonged exposure to poly(I:C) for 48 hours led to a decrease in the melanocyte viability in a dose-dependent way, measured by trypan blue exclusion analysis. The detrimental effect of poly(I:C) on the viability of melanocytes was further quantified by Annexin V/PI staining.22%,47% apoptosis (Annexin V+) were achieved following the treatment with 50,100μg/ml poly(I:C) compared with the 2.5% apoptosis for the control group, indicating that cytotoxicity of poly(I:C) to the melanocytes is largely due to the induction of apoptosis.
Several intracellular receptors have been described to bind to dsRNA, such as TLR3, PKR, and MDA-5. To determine whether the apoptosis-inducing effect of poly(I:C) was mediated by TLR3, the receptor expression was efficiently suppressed through infection of a lentivirus designed to stably express a TLR3-specific shRNA. The effect of poly(I:C) on melanocyte apoptosis was then analyzed in control and TLR3 knockdown cells by Annexin V staining. Similar to nontransduced cells, the apoptosis-inducing effect of poly(I:C) was apparent in non-sense shRNA cells, used as control. In contrast, shTLR3 melanocytes were greatly resistant to poly(I:C) toxicity, clearly indicating that the apoptosis-inducing effect of dsRNA depends upon TLR3.
Previously, we have demonstrated the NF-κB activation status through IκBα□phosphorylation in melanocytes upon poly(I:C) stimulation. To determine whether the activation of NF-κB pathway was responsible for melanocyte apoptosis induced by poly(I:C), Bay11-7082, a specific pharmacological inhibitor of IκBαphosphorylation was utilized. The poly(I:C)-induced phosphorylation of IκBαwas significantly inhibited by Bay11-7082. However, culture of melanocytes in the presence of Bay11-7082 failed to prevent poly(I:C)-induced apoptosis, even when dose as high as 0.5μM was used. Of note, we found that stimulation of melanocytes with LPS, a strong NF-κB activator, did not alter their apoptosis levels significantly, suggesting that NF-κB activation is not a key mediator of dsRNA-triggered melanocyte death.
Because the production of type I IFNs (IFN-a and IFN-(3) induced by TLR3 activation has been reported to trigger apoptosis in several cell types, the role of typeⅠIFNs in dsRNA-induced melanocyte apoptosis was evaluated. Both IFN-αand IFN-Pproduction were strongly induced in melanocytes upon poly(I:C) treatment, and STAT1 phosphorylation was observed, indicative of typeⅠIFN signaling. Neutralization of IFN-βwith specific antibodies strongly inhibited the poly(I:C)-induced apoptosis of melanocytes, while neutralization of IFN-βdid not modify the effect. Phase-contrast observation also confirmed that the rescue of melanocytes from apoptosis was dependent upon the dose of antibodies adopted in the neutralization experiments, demonstrating that IFN-βwas necessary for TLR3-mediated cell death. However, treatment of melanocytes with IFN-αor IFN-β, alone or in a mixture of both for less than 72 hours did not significantly affect melanocyte viability. These results establish that IFN-βsignaling is required for TLR3-triggered cytotoxicity, although it is insufficient to induce cell death by itself.
To identify the mechanism of IFN-(3 production upon poly(I:C) stimulation, we first determined the signaling pathways activated by poly(I:C) in melanocytes. Significant phosphorylation of p38 MAPK was detected at 30 minutes after poly(I:C) stimulation, whereas increase of ERK1/2 phosphorylation was observed at 2 hours in melanocytes. Finally, a significant increase of phosphorylation of JNK1/2 was detected at 1 hour after stimulation. To further address the role of activation of these signaling molecules in dsRNA-induced IFN-βproduction and melanocyte apoptosis, we utilized pharmacological inhibitors to p38 MAPK, ERK1/2 and JNK1/2. As shown by Annexin V staining, apoptotic cells in the presence of poly(I:C) strongly decreased when the p38 MAPK or ERK1/2 signaling was blocked (mean of death reduction:73.6% and 50% for p38 inhibition and ERK1/2 inhibition). Accordingly, morphological analysis suggested that the blockade of p38 MAPK or ERK1/2, but not JNK1/2, partially rescued the melanocytes from poly(I:C)-induced death. Furthermore, blockade of p38 MAPK and ERK1/2, inhibited poly(I:C)-stimulated IFN-βsecretion significantly, whereas JNK1/2 blockade had limited effects. Taken together, these results suggest that p38 MAPK and ERK1/2 are involved in both the autocrine secretion of IFN-βand the induction of apoptosis.
In summary, this study provides evidence for a TLR expression and response profile of normal human melanocytes, which stress the importance of the melanocytes not only as pigment cells but also as sentinels of skin homeostasis. The present work represents the first detailed mechanistic study on innate immunity signaling pathways activated by dsRNA in melanocytes, and clarifies part of complex molecular responses of melanocyte faced with viral components. On the basis of our results, a possible pathogenetic hypothesis could be that viral dsRNA stimulates TLR3 in human melanocytes and triggers the cellular apoptosis through IFN-β. These findings may add new insight into the possible etiopathogenetic mechanisms leading to melanocyte loss in vitiligo and propose IFN-βas a potential therapeutic target.
在白癜风中尽管存在自身免疫异常的证据,但是导致针对黑素细胞自身抗原的细胞和体液免疫是如何触发的问题仍有待解决。根据Matzinger的危险信号学说,体内的细胞损伤等危险信号可激活APC来活化适应性免疫反应,在白癜风自身免疫反应的触发期,往往存在皮肤黑素细胞生物学特性的异常,例如,外源性机械刺激、烧伤、过度紫外线照射以及化学物质的刺激会导致局部黑素细胞处于应激状态,而这种应激状态是否导致黑素细胞的免疫学特性发生改变,从而激发或者放大随之而来的针对黑素细胞抗原的适应性自身免疫反应,还需要的研究证实。但是毋庸置疑的是黑素细胞的天然免疫特性可能在联结黑素细胞应激反应与自身免疫免疫反应中起到了重要的桥梁作用。
近年来对黑素细胞的研究,特别是对其生物学特性及细胞生化方面的研究已经较为深入,许多证据都表明黑素细胞与皮肤免疫系统之间有密切联系。体外培养的黑素细胞表面可以表达MHC-Ⅰ/Ⅱ分子、粘附分子ICAM-1、VCAM-1及Fc受体等免疫分子,并且能够被IFN-γ和TNF-α上调表达；黑素细胞自身还能分泌细胞因子IL-1、IL-6、IL-8和TGF-β等细胞因子,认为黑素细胞是皮肤免疫系统的重要成员,并且可能以更加主动的方式参与并调节免疫反应。
TLRs被认为只表达在免疫细胞和多种炎症细胞表面,如树突状细胞(Dendritic cells, DC)、巨噬细胞和T、B淋巴细胞等,与其相应的配体结合后可以激活免疫细胞使其分泌大量的促炎性细胞因子和趋化因子,参与活化天然免疫应答和特异性免疫应答,构成机体免疫系统的第一道防线。近年来,有报道指出除免疫细胞外,多种组织细胞也表达TLRs,例如皮肤角质形成细胞表达不同的TLRs,与TLRs相应配体结合后被激活,分泌大量的细胞因子和趋化因子。
由上述关于白癜风发病机制和TLRs生物学功能的研究,我们设想：黑素细胞可以表达功能性TLRs, TLRs与其内外源性配体结合后引发黑素细胞生物学及免疫学表型和功能的变化,这种变化很可能与白癜风自身免疫反应的发生和发展有着密切的联系。尽管这种联系存在着诸多疑问。一旦这些疑问得到解决,我们将发现一个新颖的角度解释白癜风的发病机制,并有希望将该通路作为新的治疗靶点应用于白癜风的临床治疗。
原代培养细胞24h后开始贴壁,可见角质形成细胞间散在的多级树突状细胞,大部分细胞胞体圆而小,有两个对称树突,突起长短不等；少数细胞胞体大,呈多角形,有2个至数个树突。约14天后可传代,根据角质形成细胞和黑素细胞对胰酶的不同耐受性和成纤维细胞不能耐受G418的特性可分离出纯黑素细胞。显微镜下可见系胞质及树突呈棕褐色或黑色,多巴染色结果呈阳性。我们采用RT-PCR法对体外培养的第三代黑素细胞进行鉴定,结果显示,角质形成细胞特异性标记分子keratin 10和keratin 14,成纤维细胞的特异性标记分子ASO2均不能被扩增,证明用上述培养传代方法制备的第三代黑素细胞纯度较好,可用于进行下一步试验。
为了定性证明各TLRs成员在黑素细胞上的表达,我们以体外培养的第三代黑素细胞为对象,并且以正常人外周血PBMC作为阳性对照,采用RT-PCR法检测到mRNA水平表达的TLR1,2,3,4,6,7以及9,未检测到TLR5,8和10。我们采用间接免疫荧光标记和流式细胞术在蛋白水平证明黑素细胞上TLR2,4,7和9的表达。为了揭示黑素细胞上表达的TLR3的亚细胞定位,我们采用间接免疫荧光和流式细胞术对黑素细胞膜上和胞内可能存在的TLR3进行检测。结果证实：黑素细胞上表达的TLR3主要定位于细胞内。
为了进一步证实TLR3的表达,我们用Western blot方法对TLR3进行了免疫印迹检测。结果显示：PVDF膜在TLR3的位置上出现了特异性的染色条带,大小为100 kD左右,与文献报道的人TLR3分子量相符合。为排除黑素细胞培养条件对TLR3表达的影响,我们采用荧光免疫组织化学法检测了TLR3在皮肤组织中的原位表达。结果证实,NKI/beteb作为特异性标志显示黑素细胞位于表皮真皮交界处,并可以观察到黑素细胞上TLR3的表达。
上述结果显示,人表皮黑素细胞可以组成性表达TLR3,因此接下来讨论TLR3的表达是否可以被其特异性配体Poly(I:C)诱导。在不同浓度Poly(I:C)作用后6h,TLR3 mRNA开始上调表达,其上调倍数分别为：2.7(1μg/ml),4.2(10μg/ml)以及3.6 (100μg/ml)。在Poly I:C作用24小时后,TLR3上调表达倍数分别为3.8 (1μg/ml),13(10μg/ml)以及37(100μg/ml),并明显剂量依赖性。这些结果证明TLR3可以被其配体刺激上调表达,黑素细胞表达的TLR3具有一定生物学功能。
我们在前述工作中已证明黑素细胞表达TLR家族的众多成员,为了验证这些TLR是否也具有相应的功能,首先,我们检测了黑素细胞在TLR活化条件下促炎性细胞因子IL-8和IL-6的分泌情况。结果显示：针对TLR2,3,4,7和9的激活剂都可以诱导IL-8分泌量显著提高,其中以TLR3激活剂poly(I:C)最强烈。黑素细胞在用PGN,poly(I:C),LPS以及CpG 2006刺激时,IL-6分泌水平明显升高,但在用imiquimod作为刺激物刺激黑素细胞上表达的TLR7时,并没有发现IL-6分泌水平的变化。趋化因子在吸引免疫细胞浸润改变皮肤局部免疫格局中发挥重要作用。接下来我们采用real-time PCR法检测了TLR活化后趋化因子CCL2/MCP-1, CCL3/MIP-1和CCL5/RANTES。结果显示,在不同TLR激活剂刺激下,黑素细胞分泌各种趋化因子格局各有不同。所有的TLR配体都可以显著诱导CCL3 mRNA的表达,同时CCL2和CCL5也有不同程度的表达增高,但TLR7配体imiquimod无此效应。
在前面的结果中,我们证明了TLR活化可以诱导炎性细胞因子和趋化因子的释放,接下来我们将验证NF-κB信号通路的活化是否介导了TLR活化诱导黑素细胞炎性细胞因子和趋化因子的分泌。首先,我们用western blot检测磷酸化IκBα水平。结果证实：不同TLR配体处理黑素细胞后,磷酸化IκBα蛋白水平都有不同程度的升高。NF-κB活化后即发生核转位,由胞质转移至胞核。为了进一步证实NF-κB信号通路的激活,我们应用间接免疫荧光检测胞质及胞核内NF-κBp65水平。结果证实：未受刺激的黑素细胞,p65亚基几乎只分布在胞质内,TLR不同配体刺激后,黑素细胞胞质内p65蛋白明显下降,而胞核内p65蛋白则显著上调。
为观察TLR3配体dsRNA对黑素细胞的影响,我们利用phalloidin荧光染料对细胞骨架蛋白actin进行染色,结合相差显微镜观察黑素细胞形态变化。结果发现,用poly(I:C)作用于黑素细胞后,细胞形态由梭形或多角形变为扁平形,树突逐渐消失。台盼兰拒染实验显示,随着poly(I:C)剂量的增加,黑素细胞存活率逐渐降低,呈剂量依赖性。Hoechst33258细胞核染色结果显示,对照组细胞核荧光呈淡蓝色且着色较均一,而poly(I:C)作用后的黑素细胞核呈特征性亮蓝色荧光,并可观察到细胞核固缩及团块状的凋亡小体。收集poly(I:C)作用的黑素细胞,抽取细胞总DNA进行琼脂糖凝胶电泳,结果显示：与正常对照组细胞基因组DNA相比,poly(I:C)作用的黑素细胞DNA呈明显的ladder,提示有凋亡细胞的存在。进一步用Annexin-V/PI双染色结合流式细胞术检测黑素细胞凋亡情况,dsRNA作用后,显示早期凋亡信号(Annexin V+/PI-)的黑素细胞数量(22%)明显多于对照抗体组(2.5%),说明dsRNA在可以在体外诱导黑素细胞凋亡。
我们通过前期的结果证实了dsRNA对黑素细胞的促凋亡作用,为了进一步证实这种效应由TLR3介导,我们应用慢病毒载体稳定表达针对TLR3的shRNA,抑制原代培养的黑素细胞中TLR3的表达。采用western blot法检测TLR3蛋白水平的变化。结果显示：通过慢病毒载体稳定转染shTLR3的黑素细胞,其TLR3表达水平显著下降,而转染随机对照shRNA对TLR3表达水平并无影响。随后我们用流式细胞术检测下调TLR3表达的黑素细胞的凋亡情况。结果显示：稳定转染了shTLR3的黑素细胞受到poly(I:C)刺激后,其凋亡率明显降低,证明了TLR3介导了dsRNA对黑素细胞的促凋亡效应。
为验证NF-κB通路的活化是否与dsRNA诱导黑素细胞凋亡有关我们进而采用NF-κB特异性阻断剂Bay11-7082作用于TLR3激活下的黑素细胞,用westernblot检测磷酸化IκBα水平。结果显示：TLR3活化后上调的磷酸化IκBα水平,Bay11-7082有一定的抑制作用,进而检测dsRNA对黑素细胞的促凋亡效应。Bay11-7082虽然能够部分抑制NF-κB的活化,但对于dsRNA诱导的黑素细胞凋亡却没有抑制作用,同时我们用TLR4的配体LPS刺激黑素细胞,发现LPS不能明显抑制黑素细胞凋亡。因为LPS是已知的NF-κB强诱导剂,所以以上结果证实,NF-κB通路并没有参与TLR3活化诱导的黑素细胞凋亡。
TLR3活化诱导的干扰素释放对于机体抗病毒反应具有重要意义,同时有文献报道Ⅰ型干扰素,包括IFN-a和IFN-p,在某些细胞中可以诱导凋亡途径的激活,因此,在我们下一步研究中,我们分析了Ⅰ型干扰素在TLR3活化介导的黑素细胞凋亡中的可能作用。我们用TLR3配体dsRNA刺激黑素细胞后,取培养上清,用ELISA法分别检测Ⅰ型IFN即IFN-α和IFN-β的分泌。结果证实,受到dsRNA刺激进而TLR3活化之后,IFN-α和IFN-β的分泌水平显著提高。用western blot方法检测了TLR3活化后STAT1磷酸化的水平,结果显示：dsRNA处理黑素细胞2小时后,磷酸化STAT1水平显著升高。
为了进一步确证Ⅰ型IFN与黑素细胞凋亡的关系,我们使用特异性识别IFN-α和IFN-β的中和性抗体分别作用于黑素细胞,并用dsRNA刺激TLR3活化,结果发现：IFN-β中和性抗体能够强烈抑制poly(I:C)诱导下的黑素细胞凋亡,而IFN-α中和性抗体没有此效应。
为了进一步证明IFN-β分泌与黑素细胞凋亡之间的关系,我们运用不同浓度IFN-β中和性抗体作用于黑素细胞,结果发现,随着使用的抗体浓度增高,其抑制黑素细胞凋亡的效应也不断增强。
以上结果证明了黑素细胞自分泌的IFN-p在TLR3活化诱导的黑素细胞凋亡中的作用,进一步,我们分析了外源性加入的IFN-a或IFN-β是否能直接影响黑素细胞凋亡。结果显示：外源性加入的IFN-α、IFN-β没有显著影响黑素细胞的生存率。以上结果证明,IFN-β参与了TLR3活化诱导的黑素细胞凋亡,但IFN-β本身不能直接诱导黑素细胞凋亡。
细胞的凋亡在体内受到多条信号通路的影响,已报道在TLR活化中,MAPK途径发挥重要作用,所以在下一步的研究中我们将分析MAPK成员p38,ERK1／2和JNKl／2在dsRNA的促IFN-p分泌以及凋亡效应中的作用。我们用poly(I：C)与黑素细胞共孵育后,采用western blot检测p38,ERK1／2和JNKl／2的磷酸化情况。结果显示：poly(I:C)作用后,磷酸化p38、磷酸化ERK1／2和磷酸化JNK1／2水平都有不同程度上调。
为了证明活化的p38,ERK1／2和JNKl／2,是直接与dsRNA的促凋亡效应相关,还是仅为TLR3活化的伴随效应,我们采用特异性的阻断剂对p38, ERK1／2以及JNKl／2信号通路分别进行了阻断后,检测TLR3活化对黑素细胞凋亡的影响。结果显示：用p38特异性阻断剂SB203580与poly(I:C)同时作用后,黑素细胞凋亡率下降了73.6%,用ERK1／2特异性阻断剂U0126作用后,黑素细胞凋亡率下降了50%,而用JNK1／2阻断剂SP600125作用后,不影响黑素细胞的凋亡率,以上结果证明,在TLR3活化诱导的黑素细胞凋亡过程中,p38和ERK1／2通路可能发挥一定的作用。
为了进一步证实以上结果,我们将p38、ERK1／2以及JNKl／2通路阻断后,在形态学上观察TLR3活化对黑素细胞凋亡的影响,结果证实：对p38和ERK1／2的阻断可以部分逆转TLR3活化导致的黑素细胞凋亡,而阻断JNK1／2途径无此效应。阻断p38和ERK1／2都可以显著抑制poly(I:C)刺激下的IFN-β释放,阻断JNK1／2无明显作用。以上结果证实,p38和ERK1／2通路参与了TLR3活化介导的IFN-β分泌与黑素细胞凋亡。
综上所述,本课题证实人表皮黑素细胞在mRNA水平蛋白水平组成性表达TLR2,3,4,7和9的表达。这些TLR受其特异性配体刺激后,黑素细胞合成表达促炎性细胞因子和趋化因子的水平升高。我们发现黑素细胞表达的TLR3活化后可以诱导黑素细胞凋亡,这种凋亡效应依赖于IFN-β的自分泌,并受信号通路p38和ERK1／2的影响。基于以上结果,我们推测黑素细胞表达的TLR3很可能与白癜风自身免疫反应的发生和发展有着密切的联系。黑素细胞可以通过表达TLR3直接识别病毒代谢中产物dsRNA或坏死细胞释放的dsRNA,从而改变了黑素细胞的免疫学表性和生物学功能,激发或放大针对黑素细胞自身抗原的免疫反应。如果这一假设得到证实,将会帮助我们从一个新颖的角度解释白癜风的发病机制,并有希望从该通路入手寻找新的治疗靶点应用于白癜风的临床治疗。
The human epidermis provides a first defense barrier to a potentially hostile environment. It has been shown that within the epidermis, not only professional immune cells (e.g., Langerhans cells) but also non-immune cells (e.g., keratinocytes and melanocytes) are involved in the immune protection of the host. Although originally identified as a professional producer of melanin, recent studies have revealed that melanocytes exhibit a variety of functions. For example, human melanocytes have the capacity to express HLA-DR, CD40 and adhesion molecules such as ICAM-1 and VCAM-1. In addition, they can produce various soluble mediators of inflammation such as IL-1, IL-6 and IL-8. These studies suggest that melanocytes are not simply pigment-producing cells but also immunocompetent cells. However, it is unclear whether melanocytes can recognize pathogens and play an active role in the skin's local immune defense system.
Recognition of pathogens by innate immune cells is mediated by pattern-recognition receptors that recognize conserved pathogen-associated molecular patterns (PAMPs). One major group of pattern-recognition receptors is the Toll-like receptors (TLRs), which transduce signals leading to the activation of NF-κB, which subsequently drive the induction of several pro-inflammatory cytokines and chemokines. Apart from sensing exogenous ligands from microbial components, which is critical for pathogen elimination, TLRs are able to recognize endogenous ligands, such as heat shock proteins (HSPs) and extracellular matrix components, which may be called danger-associated molecular patterns (DAMPs), in analogy to PAMPs.
Thus far, more than 10 different TLRs with distinct ligand specificity have been identified. In human skin, the distribution of TLRs is incompletely defined. Several studies demonstrated that keratinocytes display TLRs and respond to corresponding PAMPs by producing pro-inflammatory cytokines. In contrast, the TLR expression pattern of primary human melanocytes has never been systematically examined.
Vitiligo is characterized by a loss of melanin-producing melanocytes from the epidermis of patients, which results in skin depigmentation. Both genetic and environmental factors have been implicated to explain the loss of epidermal melanocytes in this disorder. Genetic risk factors are important, but are not sufficient for the disease to occur, and environmental triggers may also contribute to the initiation and/or progression of vitiligo. Human skin is known to be constantly exposed to various pathogens of prokaryotic, eukaryotic, and viral origin. Indeed, a variety of viruses including cytomegalovirus, hepatitis virus, and AIDS virus, have been implicated in the pathogenesis of the vitiligo. In addition, a strong causative link between herpes virus and vitiligo has been noted in the Smyth chicken model. Finally, viral infections have been long associated with a variety or autoimmune disorders. Interestingly, some of them including thyroid disease and typeⅠdiabetes mellitus, occur with increased frequency in patients with vitiligo. These observations suggest that virus might be an important environmental factor that participates in the initiation or acceleration of vitiligo in genetically predisposed individuals.
Toll-like receptors (TLRs) play a crucial role in pathogen detection and in the mounting of antimicrobial immune responses. Although initial studies have focused on the TLR expression and function in the immune system, more recent studies have shown that TLR expression is shared by many different cell types including human epidermal melanocytes. We have previously shown that TLRs expressed in melanocytes are functional and respond to their respective ligands by activating the central transcription factor NF-κB and by up-regulating pro-inflammatory mediators. In respect of antiviral responses, TLR3 was shown to be a receptor for double-stranded RNA (dsRNA), which represents either genomic or life cycle intermediate material of many viruses. In the process of viral infection, dsRNA stimulates cellular antiviral activities, and occasionally, cell death, which is another way of protecting the host against virus spreading. In our preliminary study, we found in vitro treatment with poly(I:C), a synthetic analogue dsRNA, resulted in massive death and detachment of cultured human melanocytes. Could human melanocytes utilize TLR3 as an initiator of signaling pathway leading to destruction of themselves in response to viral infections? This hypothesis, as well as its implications in the pathogenesis of vitiligo, needs more evidential supports from both clinical observations and experimental investigations.
In the present study, we provided laboratory evidences that melanocytes expressed TLR3. Poly(I:C) treatment induced apoptosis in human melanocytes in a TLR3-dependent manner. This phenomenon required the involvement of IFN-βautocrine signaling but not NF-κB activation. Moreover, dsRNA stimulation led to the activation of downstream signaling pathways, including p38, ERK1/2 and JNK1/2, among which p38 and ERK1/2 controlled both IFN-βsecretion and IFN-βmediated cell death. The fact that human melanocytes dramatically respond to TLR3 ligand indicates that viral infection may play a part in the initiation/amplification of vitiligo by directly triggering melanocyte apoptosis.
Primary human melanocyte cultures were obtained from neonatal foreskin. All samples obtained were from surgical procedures with the patients'informed consent following a protocol approved by the Huashan Hospital Fudan University Institutional Review Board. The epidermis was separated from the dermis after an overnight incubation of skin samples in a 0.25% Dispase solution in PBS at 4℃. In order to separate cellular elements, epidermal sheets were incubated at 37℃in a solution of 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in PBS, for 10 minutes. Cellular suspension was then filtered through 70μm cell strainer and then centrifuged at 1500 rpm for 7 minutes to harvest cells. Then, melanocytes were selectively grown in a defined medium M254 with PMA-free human melanocyte growth supplements. The cells were maintained in a humidified incubator with 5% CO2 at 37℃and were fed every 3-4 d, and further passaged at 1:2 when they became 80% confluent, with experiments being carried out from 3 to 4 passages.
To investigate the expression profile of TLRs in human melanocytes, in vitro-expanded melanocytes obtained from healthy subjects were assessed by reverse transcription (RT)-PCR for TLR mRNA using a panel of specific primers for TLR1-TLR10. We found the constitutive mRNA expression of TLRs 1,2,3,4,6,7 and 9 in human melanocytes but not TLRs 5,8, and 10. As a positive control, human PBMC were shown to express all mRNA of TLRs 1-10. The purity of the cultured human melanocytes was verified by RT-PCR analysis for the keratinocyte markers, such as keratin 10 and keratin 14, and the fibroblast marker ASO2, showing that contamination from keratinocytes or fibroblasts was negligible. To measure the TLR protein expression in human melanocytes, flow cytometry and immunofluorescence assays were performed with specific antibodies to human TLR proteins. Ample amounts of TLRs 2,4,7 and 9 were detected in human melanocytes indicated by flow cytometry analysis. TLR3 is generally recognized as an intracellular receptor, but a recent study showed a surface TLR3 on human skin and lung fibroblasts. Thus, we analyzed the specific location of TLR3 expression in human melanocytes. Using the same mAb specific to TLR3, we demonstrated the detection of intracellular TLR3 in human melanocytes but not on the cell surface. The protein expression of TLRs on in vitro-cultured human melanocytes was also demonstrated by immunofluorescence assays, and consistent findings were noted. Compared to the isotype control, a clear positive staining for TLRs 2,3,4,7 and 9 was obtained. These results paralleled our RT-PCR endings. Thus human melanocytes not only expressed TLRs mRNA but also expressed TLRs protein. 3. Regulation of TLR3 in human melanocytes
To determine whether the TLR3 expression was inducible by its activation with poly(I:C), a synthetic surrogate of viral dsRNA, the kinetic of TLR3 mRNA was analyzed by real-time RT-PCR. In a dose-dependent fashion, TLR3 mRNA increased at 6 hours after treatment with poly(I:C) (2.7-fold at 1μg/ml,4.2-fold at 10μg/ml, and 3.6-fold at 100μg/ml) and at 24 hours after stimulation (3.8-fold at 1μg/ml, 13-fold at 10μg/ml and 37-fold at 100μg/ml). These data show that human melanocytes constitutively and inducibly express TLR3, suggesting that melanocytes have the potential to recognize virus-derived pathogenic motifs via TLR3.
To characterize the functional relevance of TLRs in human melanocytes, the production of inflammatory cytokines by melanocytes in response to TLR ligands were determined. Human melanocytes were stimulated with various TLR ligands, for example, The TLR2 ligand, peptidoglycan (PGN), the TLR3 ligand, poly (I:C), the TLR4 ligand, LPS, the TLR7 ligand, imiquimod, and CpG 2006, which ligates TLR9. Subsequently, the secretion level of IL-8 and IL-6 were analyzed by ELISA. The maximal concentration of poly (I:C) used in our analysis was restricted to 10μg/ml, as larger dosage led to massive death and detachment of melanocytes. All of the TLR ligands examined potently induced the production of IL-8 in a dose-dependent manner, with maximal secretion being observed at 1μg/ml poly (I:C). Melanocytes also released substantial amounts of IL-6 when stimulated with PGN, poly (I:C), LPS and CpG 2006. On the contrary, no IL-6 production was observed in melanocytes treated with imiquimod, even though the cells expressed TLR7 mRNA and protein.
Activated epidermal melanocytes may modulate the recruitment and activation of different immune cells at least in part through the expression of the chemokines. We next analyzed accumulation of mRNAs of inflammatory chemokines such as CCL2/MCP-1, CCL3/MIP-1 and CCL5/RANTES upon different TLR activation using real-time RT-PCR. Here we showed that each TLR ligand induced the release of CCL2, CCL3 or CCL5 with a different kinetics. All the TLR ligands induced significant amounts of CCL3 mRNA in 6-hour stimulated melanocytes, whereas mRNA for CCL2 and CCL5 were significantly induced by all the TLR ligands except imiquimod, the TLR7 ligand. Although some basal expression of CCL2, CCL3 and CCL5 could be observed in nonstimulated cells, there was at least 1-fold increase in their mRNA levels after treatment with TLR ligands.
Collectively, these data demonstrated that TLR ligand pretreatment of epidermal melanocytes created a pro-inflammatory milieu by showing elevated cytokines and chemokines.
Activation of NF-κB plays a central role in TLR-mediated cellular activation and gene expression in a variety of cell types. Therefore, we tested whether triggering TLRs in human melanocytes can give rise to the activation of NF-κB signaling pathway. TLRs within human melanocytes were stimulated for 6 hour by various ligands and assessed by Western blot analysis to examine phosphorylation of IκBα, which is one of the markers of NF-κB activity. No phosphorylated-IκBαwas observed in unstimulated melanocytes. On the contrary, the clear bands of phosphorylated-IκBαcould be induced by PGN (100μg/ml), poly (I:C) (10μg/ml), LPS (100μg/ml), imiquimod (100μg/ml) or CpG 2006 (5μM) to different degrees, indicating that NF-κB pathway participated in TLR2,3,4,7 and 9 signaling in response to the respective PAMPs.
To further substantiate the activation of NF-κB pathway in TLR ligand-treated melanocytes, translocation to the nucleus of the NF-KBp65 was analyzed. Human melanocytes were cultured in slide chambers in the presence of TLR ligands and stained for the NF-KBp65 as described in Materials and methods. The slides were then analyzed for nuclear localization by fluorescence microscopy. In unstimulated melanocytes, the stainings for the NF-κBp65 were mainly visible in the cytoplasm. In contrast, when melanocytes were stimulated with PGN (100μg/ml), poly (I:C) (10μg/ml), LPS (100μg/ml), imiquimod (100μg/ml) or CpG 2006 (5μM), a clear nuclear translocation of the p65 subunit was observed in all the stimulated groups. These data indicated that TLR 2,3,4,7 or 9 ligand engagement led to the activation of NF-κB, which explained the numerous pro-inflammatory genes being activated.
We further investigated the responsiveness of melanocytes to dsRNA. Melanocytes exposed to 100 mg/ml poly(I:C) underwent striking changes in cell appearance:cytoplasmic spreading and flattening of the cells and their nuclei were illustrated by phase contrast and phalloidin staining at 8 hours and 24 hours. A more prolonged exposure to poly(I:C) for 48 hours led to a decrease in the melanocyte viability in a dose-dependent way, measured by trypan blue exclusion analysis. The detrimental effect of poly(I:C) on the viability of melanocytes was further quantified by Annexin V/PI staining.22%,47% apoptosis (Annexin V+) were achieved following the treatment with 50,100μg/ml poly(I:C) compared with the 2.5% apoptosis for the control group, indicating that cytotoxicity of poly(I:C) to the melanocytes is largely due to the induction of apoptosis.
Several intracellular receptors have been described to bind to dsRNA, such as TLR3, PKR, and MDA-5. To determine whether the apoptosis-inducing effect of poly(I:C) was mediated by TLR3, the receptor expression was efficiently suppressed through infection of a lentivirus designed to stably express a TLR3-specific shRNA. The effect of poly(I:C) on melanocyte apoptosis was then analyzed in control and TLR3 knockdown cells by Annexin V staining. Similar to nontransduced cells, the apoptosis-inducing effect of poly(I:C) was apparent in non-sense shRNA cells, used as control. In contrast, shTLR3 melanocytes were greatly resistant to poly(I:C) toxicity, clearly indicating that the apoptosis-inducing effect of dsRNA depends upon TLR3.
Previously, we have demonstrated the NF-κB activation status through IκBα□phosphorylation in melanocytes upon poly(I:C) stimulation. To determine whether the activation of NF-κB pathway was responsible for melanocyte apoptosis induced by poly(I:C), Bay11-7082, a specific pharmacological inhibitor of IκBαphosphorylation was utilized. The poly(I:C)-induced phosphorylation of IκBαwas significantly inhibited by Bay11-7082. However, culture of melanocytes in the presence of Bay11-7082 failed to prevent poly(I:C)-induced apoptosis, even when dose as high as 0.5μM was used. Of note, we found that stimulation of melanocytes with LPS, a strong NF-κB activator, did not alter their apoptosis levels significantly, suggesting that NF-κB activation is not a key mediator of dsRNA-triggered melanocyte death.
Because the production of type I IFNs (IFN-a and IFN-(3) induced by TLR3 activation has been reported to trigger apoptosis in several cell types, the role of typeⅠIFNs in dsRNA-induced melanocyte apoptosis was evaluated. Both IFN-αand IFN-Pproduction were strongly induced in melanocytes upon poly(I:C) treatment, and STAT1 phosphorylation was observed, indicative of typeⅠIFN signaling. Neutralization of IFN-βwith specific antibodies strongly inhibited the poly(I:C)-induced apoptosis of melanocytes, while neutralization of IFN-βdid not modify the effect. Phase-contrast observation also confirmed that the rescue of melanocytes from apoptosis was dependent upon the dose of antibodies adopted in the neutralization experiments, demonstrating that IFN-βwas necessary for TLR3-mediated cell death. However, treatment of melanocytes with IFN-αor IFN-β, alone or in a mixture of both for less than 72 hours did not significantly affect melanocyte viability. These results establish that IFN-βsignaling is required for TLR3-triggered cytotoxicity, although it is insufficient to induce cell death by itself.
To identify the mechanism of IFN-(3 production upon poly(I:C) stimulation, we first determined the signaling pathways activated by poly(I:C) in melanocytes. Significant phosphorylation of p38 MAPK was detected at 30 minutes after poly(I:C) stimulation, whereas increase of ERK1/2 phosphorylation was observed at 2 hours in melanocytes. Finally, a significant increase of phosphorylation of JNK1/2 was detected at 1 hour after stimulation. To further address the role of activation of these signaling molecules in dsRNA-induced IFN-βproduction and melanocyte apoptosis, we utilized pharmacological inhibitors to p38 MAPK, ERK1/2 and JNK1/2. As shown by Annexin V staining, apoptotic cells in the presence of poly(I:C) strongly decreased when the p38 MAPK or ERK1/2 signaling was blocked (mean of death reduction:73.6% and 50% for p38 inhibition and ERK1/2 inhibition). Accordingly, morphological analysis suggested that the blockade of p38 MAPK or ERK1/2, but not JNK1/2, partially rescued the melanocytes from poly(I:C)-induced death. Furthermore, blockade of p38 MAPK and ERK1/2, inhibited poly(I:C)-stimulated IFN-βsecretion significantly, whereas JNK1/2 blockade had limited effects. Taken together, these results suggest that p38 MAPK and ERK1/2 are involved in both the autocrine secretion of IFN-βand the induction of apoptosis.
In summary, this study provides evidence for a TLR expression and response profile of normal human melanocytes, which stress the importance of the melanocytes not only as pigment cells but also as sentinels of skin homeostasis. The present work represents the first detailed mechanistic study on innate immunity signaling pathways activated by dsRNA in melanocytes, and clarifies part of complex molecular responses of melanocyte faced with viral components. On the basis of our results, a possible pathogenetic hypothesis could be that viral dsRNA stimulates TLR3 in human melanocytes and triggers the cellular apoptosis through IFN-β. These findings may add new insight into the possible etiopathogenetic mechanisms leading to melanocyte loss in vitiligo and propose IFN-βas a potential therapeutic target.
引文
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[30]Sumikawa Y, Asada H, Hoshino K, et al. Induction of beta-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2. Microbes Infect,2006,8(6):1513-1521.
[31]Lebre MC, van der Aar AM, van Baarsen L, et al. Human keratinocytes express functional Toll-like receptor 3,4,5, and 9. J Invest Dermatol,2007,127(2): 331-341.
[32]Miller LS, Modlin RL. Toll-like receptors in the skin. Semin Immunopathol,2007, 29(1):15-26.
[33]Klein Klouwenberg P, Tan L, Werkman W, et al. The role of Toll-like receptors in regulating the immune response against respiratory syncytial virus. Crit Rev Immunol,2009,29(6):531-550.
[34]Yoneyama M, Fujita T. Recognition of viral nucleic acids in innate immunity. Rev Med Virol,2010,20(1):4-22.
[35]Kupper TS, Fuhlbrigge RC. Immune surveillance in the skin:mechanisms and clinical consequences. Nat Rev Immunol,2004,4:211-222.
[36]Miller LS, Modlin RL. Human keratinocyte Toll-like receptors promote distinct immune responses. J Invest Dermatol,2007,127:262-263.
[37]Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol,2001,2:675-680.
[38]Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol,2001,1: 135-145.
[39]Barton GM, Medzhitov R. Toll-like receptor signaling pathways. Science,2003, 300:1524-1525.
[40]Vabulas RM, Ahmad-Nejad P, Ghose S, et al. HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J Biol Chem,2002,277: 15107-15112.
[41]Ohashi K, Burkart V, Flohe S, et al. Cutting edge:heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol,2000, 164:558-561.
[42]Johnson GB, Brunn GJ, Kodaira Y, et al. Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. J Immunol,2002,168:5233-5239.
[43]Okamura Y, Watari M, Jerud ES, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem,2001,276:12-14.
[44]Kawai K, Shimura H, Minagawa M, et al. Expression of functional Toll-like receptor 2 on human epidermal keratinocytes. J Dermatol Sci,2002,30:185-194.
[45]Song PI, Park YM, Abraham T, et al. Human keratinocytes express functional CD14 and toll-like receptor 4. J Invest Dermatol,2002,119:424-432.
[46]Mempel M, Voelcker V, Kollisch G, et al. Toll-like receptor expression in human keratinocytes:nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent. J Invest Dermatol,2003,121:1389-1396.
[47]Lebre MC, van der Aar AM, van Baarsen L, et al. Human keratinocytes express functional Toll-like receptor 3,4,5, and 9. J Invest Dermatol,2007,127: 331-341.
[48]Monie TP, Bryant CE, Gay NJ. Activating immunity:lessons from the TLRs and NLRs. Trends Biochem Sci,2009,34(11):553-561.
[49]Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol,2009,21(4):317-337.
[50]Verstrepen L, Bekaert T, Chau TL, et al. TLR-4, IL-1R and TNF-R signaling to NF-kappaB:variations on a common theme. Cell Mol Life Sci,2008,65(19): 2964-2978.
[51]Kawai T, Akira S. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med,2007,13(11):460-469.
[52]Esen N, Tanga FY, DeLeo JA, et al. Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J Neurochem,2004,88:746-758.
[53]Hajjar AM, O'Mahony DS, Ozinsky A, et al. Cutting edge:functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J Immunol,2001,166:15-19.
[54]Satta N, Kruithof EK, Reber G, et al. Induction of TLR2 expression by inflammatory stimuli is required for endothelial cell responses to lipopeptides. Mol Immunol,2008,46:145-157.
[55]Quesniaux VJ, Nicolle DM, Torres D, et al. Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans. J Immunol,2004,172: 4425-4434.
[56]Gray JS, Bae HK, Li JC, et al. Double-stranded RNA-activated protein kinase mediates induction of interleukin-8 expression by deoxynivalenol, Shiga toxin 1, and ricin in monocytes. Toxicol Sci,2008,105:322-330.
[57]Kuyama M, Nakanishi G, Arata J, et al. Expression of double-stranded RNA-activated protein kinase in keratinocytes and keratinocytic neoplasia. J Dermatol,2003,30:579-589.
[58]Ahn JH, Park TJ, Jin SH, Kang HY. Human melanocytes express functional Toll-like receptor 4. Exp Dermatol,2008,17:412-417.
[59]Ahn JH, Jin SH, Kang HY. LPS induces melanogenesis through p38 MAPK activation in human melanocytes. Arch Dermatol Res,2008,300:325-329.
[60]Kroll TM, Bommiasamy H, Boissy RE, et al.4-Tertiary butyl phenol exposure sensitizes human melanocytes to dendritic cell-mediated killing:relevance to vitiligo. J Invest Dermatol,2005,124:798-806.
[61]Kang HY, Park TJ, Jin SH. Imiquimod, a Toll-like receptor 7 agonist, inhibits melanogenesis and proliferation of human melanocytes. J Invest Dermatol,2009, 129:243-246.
[62]Dorn A, Ludwig RJ, Bock A, et al. Oligonucleotides suppress IL-8 in skin keratinocytes in vitro and offer anti-inflammatory properties in vivo. J Invest Dermatol,2007,127:846-854.
[63]Kim HS, Lee MS. Role of innate immunity in triggering and tuning of autoimmune diabetes. Curr Mol Med,2009,9(1):30-44.
[64]Li M, Zheng DJ, Field LL, et al. Murine pancreatic beta TC3 cells show greater 2', 5'-oligoadenylate synthetase (2'5'AS) antiviral enzyme activity and apoptosis following IFN-alpha or poly(I:C) treatment than pancreatic alpha TC3 cells. Exp Diabetes Res,2009,2009:631026.
[65]Garcia M, Dogusan Z, Moore F, et al. Regulation and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells. Biochim Biophys Acta.2009, 1793(11):1768-1775.
[66]Wong FS, Wen L. Toll-like receptors and diabetes. Ann N Y Acad Sci,2008,1150: 123-132.
[1]Berardesca E, Maibach H. Racial differences in skin pathophysiology. J Am Acad Dermatol. 1996; 34(4):667-72.
[2]Wesley NO, Maibach HI.Racial (ethnic) differences in skin properties:the objective data. Am J Clin Dermatol.2003;4(12):843-60.
[3]Schallreuter, K.U., Kothari, et al. Regulation of melanogenesis-controversies and new concepts. Exp. Dermatol.2008; 17,395-404.
[4]Gunathilake, R., Schurer, N.Y., Shoo, B.A., et al. H-regulated mechanisms account for pigmenttype differences in epidermal barrier function. J. Invest. Dermatol.2009; 129, 1719-1729.
[5]Hachem, J.P., Man, et. Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity. J. Invest. Dermatol.2005; 125,510-520.
[6]SCHRAERMEYER, U., PETERS, S., THUMANN, G., KOCIOK, N.& HEIMANN, K. Melanin granules of retinalpigment epithelium are connected with the lysosomaldegradation pathway. Exp. Eye Res.1999; 68,237-245.
[7]DELL'ANGELICA, E. C., MULLINS, et al. Lysosome-related organelles. FASEB J.2000; 14,1265-1278.
[8]LE POOLE, I. C., MUTIS, T., et al. A novel antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders. J. Immunol.1993; 151,7284-7292.
[9]Swope VB, Sauder DN, McKenzie RC, et al. Synthesis of interleukin-1 alpha and beta by normal human melanocytes. J Invest Dermatol.1994; 102:749-53.
[10]Kruger-Krasagakes S, Krasagakis K, Garbe C, et al. Production of cytokines by human melanoma cells and melanocytes. Recent Results Cancer Res.1995; 139:155-68.
[11]Lu Y, Zhu WY, Tan C,et al. Melanocytes are potential immunocompetent cells:evidence from recognition of immunological characteristics of cultured human melanocytes. Pigment Cell Res.2002; 15:454-60.
[12]Ahn JH, Park TJ, Jin SH, et al. Human melanocytes express functional Toll-like receptor 4. Exp Dermatol.2008; 17(5):412-7.
[13]Ahn JH, Jin SH, Kang HY. LPS induces melanogenesis through p38 MAPK activation in human melanocytes. Arch Dermatol Res.2008; 300(6):325-9.
[14]Kang HY, Park TJ, Jin SH. Imiquimod, a Toll-like receptor 7 agonist, inhibits melanogenesis and proliferation of human melanocytes. J Invest Dermatol.2009; 129(1):243-6.
[15]Denman CJ, McCracken J, Hariharan V, et al. HSP70i Accelerates Depigmentation in a Mouse Model of Autoimmune Vitiligo. J Invest Dermatol.2008; 13.
[16]TSATMALI, M., GRAHAM, A., SZATKOWSKI, D., et al. Alpha-melanocyte-stimulating hormone modulates nitric oxide production in melanocytes. J. Invest. Dermatol.2000; 114, 520-526.
[17]SASAKI, M., HORIKOSHI, T., UCHIWA, H.& MIYACHI, Y. Up-regulation of tyrosinase gene by nitric oxide in human melanocytes. Pigment Cell Res.2000; 13,248-252.
[18]LUGER, T. A., SCHOLZEN, T.& GRABBE, S. The role of a-melanocyte stimulating hormone in cutaneous biology. J. Invest. Dermatol. Symp. Proc.1997; 2,87-93.
[19]YOSHIDA, M., TAKAHASHI, Y.& SHINTARO, I. Histamine induces melanogenesis and morphologic changes by Protein Kinase A activation via H2 receptors in human normal melanocytes. J. Invest. Dermatol.2000; 114,334-342.
[2]Kakourou T. Vitiligo in children. World J Pediatry,2009,5(4):265-268
[3]Gawkrodger DJ. Vitiligo:what general physicians need to know. Clin Med,2009, 9(5):408-409.
[4]Khaled A, Zeglaoui F, Ezzine N, et al. What is new in the treatment of vitiligo. Tunis Med,2008,86(4):307-311.
[5]Fenton JS, Bergstrom KG. Vitiligo:nonsurgical treatment options and the evidence behind their use. J Drugs Dermatol,2008,7(7):705-711.
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[7]Sehgal VN, Srivastava G. Vitiligo treatment options:an evolving scenario. J Dermatolog Treat,2006,17(5):262-275.
[8]Le Poole IC, Luiten RM. Autoimmune etiology of generalized vitiligo. Curr Dir Autoimmun,2008,10:2207-243.
[9]Westerhof W, d'Ischia M. Vitiligo puzzle:the pieces fall in place. Pigment Cell Res,2007,20(5):345-359.
[10]Rezaei N, Gavalas NG, Weetman AP, et al. Autoimmunity as an aetiological factor in vitiligo. J Eur Acad Dermatol Venereol,2007,21(7):865-876.
[11]Dell' anna ML, Picardo M. A review and a new hypothesis for non-immunological pathogenetic mechanisms in vitiligo. Pigment Cell Res,2006, 19(5):406-411.
[12]Passeron T, Ortonne JP. Physiopathology and genetics of vitiligo. J Autoimmun, 2005,25 Suppl:63-68.
[13]Marks MS, Theos AC, Raposo G. Melanosomes and MHC class Ⅱ antigen-processing compartments:a tinted view of intracellular trafficking and immunity. Immunol Res,2003,27(2-3):409-426.
[14]Li YL, Yu CL, Yu HS. IgG anti-melanocyte antibodies purified from patients with active vitiligo induce HLA-DR and intercellular adhesion molecule-1 expression and an increase in interleukin-8 release by melanocytes. J Invest Dermatol,2000, 115(6):969-973.
[15]Dawn G, MadKie RM. Expression of endoglin in human melanocytic lesions. Clin Exp Dermatol,2002,27(2):153-156.
[16]Trcka J, Moroi Y, Clynes RA Redundant and alternative roles for activating Fc receptors and complement in an antibody-dependent model of autoimmune vitiligo. Immunity,2002,16(6):861-868.
[17]Hirobe T, Ootaka H. Interleukin-1 alpha stimulates the differentiation of melanocytes but inhibits the proliferation of melanoblasts from neonatal mouse epidermis. Zoolog Sci,2007,24(10):959-970.
[18]Moretti S, Fabbri P, Baroni G, et al. Keratinocyte dysfunction in vitiligo epidermis:cytokine microenvironment and correlation to keratinocyte apoptosis. Histol Histopathol,2009,24(7):849-857.
[19]Mangahas CR, dela Cruz GV, Friedman-Jimenez G, et al. Endothelin-1 induces CXCL1 and CXCL8 secretion in human melanoma cells. J Invest Dermatol,2005, 125(2):307-311.
[20]Le Poole IC, Mutis T, van den Wijngaard RM, et al. A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders. J Immunol,1993,151(12):7284-7292.
[21]Iftikhar N, Rahman A, Janjua SA. Vitiligo appearing in striae distensae as a Koebner phenomenon. J Coll Physicians Surg Pak,2009,19(12):796-797.
[22]Mulekar SV, Asaad M, Ghwish B, et al. Koebner phenomenon in vitiligo:not always an indication of surgical failure. Arch Dermatol.2007,143(6):801-802.
[23]Polat M, Yal(?)in B, Alli N. Vitiligo at the site of radiotherapy for nasopharyngeal carcinoma. Am J Clin Dermatol,2007,8(4):247-249.
[24]Fischer M, Ehlers M. Toll-like receptors in autoimmunity. Ann N Y Acad Sci, 2008,1143:21-34.
[25]Bianchi ME. DAMPs, PAMPs and alarmins:all we need to know about danger. J Leukoc Biol,2007,81(1):1-5.
[26]Lai Y, Di Nardo A, Nakatsuji T, et al. Commensal bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury. Nat Med,2009,15(12): 1377-1382.
[27]Nozaki N, Takeishi Y, Kubota I. Toll-like receptor (TLR). Nippon Rinsho,2007, 28,65 Suppl 4:229-232.
[28]Sabroe I, Whyte MK. Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation in respiratory disease. Biochem Soc Trans.2007,35(Pt 6):1492-1495.
[29]Wagner H. Endogenous TLR ligands and autoimmunity. Adv Immunol.2006,91: 159-173.
[30]Sumikawa Y, Asada H, Hoshino K, et al. Induction of beta-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2. Microbes Infect,2006,8(6):1513-1521.
[31]Lebre MC, van der Aar AM, van Baarsen L, et al. Human keratinocytes express functional Toll-like receptor 3,4,5, and 9. J Invest Dermatol,2007,127(2): 331-341.
[32]Miller LS, Modlin RL. Toll-like receptors in the skin. Semin Immunopathol,2007, 29(1):15-26.
[33]Klein Klouwenberg P, Tan L, Werkman W, et al. The role of Toll-like receptors in regulating the immune response against respiratory syncytial virus. Crit Rev Immunol,2009,29(6):531-550.
[34]Yoneyama M, Fujita T. Recognition of viral nucleic acids in innate immunity. Rev Med Virol,2010,20(1):4-22.
[35]Kupper TS, Fuhlbrigge RC. Immune surveillance in the skin:mechanisms and clinical consequences. Nat Rev Immunol,2004,4:211-222.
[36]Miller LS, Modlin RL. Human keratinocyte Toll-like receptors promote distinct immune responses. J Invest Dermatol,2007,127:262-263.
[37]Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol,2001,2:675-680.
[38]Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol,2001,1: 135-145.
[39]Barton GM, Medzhitov R. Toll-like receptor signaling pathways. Science,2003, 300:1524-1525.
[40]Vabulas RM, Ahmad-Nejad P, Ghose S, et al. HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J Biol Chem,2002,277: 15107-15112.
[41]Ohashi K, Burkart V, Flohe S, et al. Cutting edge:heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol,2000, 164:558-561.
[42]Johnson GB, Brunn GJ, Kodaira Y, et al. Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. J Immunol,2002,168:5233-5239.
[43]Okamura Y, Watari M, Jerud ES, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem,2001,276:12-14.
[44]Kawai K, Shimura H, Minagawa M, et al. Expression of functional Toll-like receptor 2 on human epidermal keratinocytes. J Dermatol Sci,2002,30:185-194.
[45]Song PI, Park YM, Abraham T, et al. Human keratinocytes express functional CD14 and toll-like receptor 4. J Invest Dermatol,2002,119:424-432.
[46]Mempel M, Voelcker V, Kollisch G, et al. Toll-like receptor expression in human keratinocytes:nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent. J Invest Dermatol,2003,121:1389-1396.
[47]Lebre MC, van der Aar AM, van Baarsen L, et al. Human keratinocytes express functional Toll-like receptor 3,4,5, and 9. J Invest Dermatol,2007,127: 331-341.
[48]Monie TP, Bryant CE, Gay NJ. Activating immunity:lessons from the TLRs and NLRs. Trends Biochem Sci,2009,34(11):553-561.
[49]Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol,2009,21(4):317-337.
[50]Verstrepen L, Bekaert T, Chau TL, et al. TLR-4, IL-1R and TNF-R signaling to NF-kappaB:variations on a common theme. Cell Mol Life Sci,2008,65(19): 2964-2978.
[51]Kawai T, Akira S. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med,2007,13(11):460-469.
[52]Esen N, Tanga FY, DeLeo JA, et al. Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J Neurochem,2004,88:746-758.
[53]Hajjar AM, O'Mahony DS, Ozinsky A, et al. Cutting edge:functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J Immunol,2001,166:15-19.
[54]Satta N, Kruithof EK, Reber G, et al. Induction of TLR2 expression by inflammatory stimuli is required for endothelial cell responses to lipopeptides. Mol Immunol,2008,46:145-157.
[55]Quesniaux VJ, Nicolle DM, Torres D, et al. Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans. J Immunol,2004,172: 4425-4434.
[56]Gray JS, Bae HK, Li JC, et al. Double-stranded RNA-activated protein kinase mediates induction of interleukin-8 expression by deoxynivalenol, Shiga toxin 1, and ricin in monocytes. Toxicol Sci,2008,105:322-330.
[57]Kuyama M, Nakanishi G, Arata J, et al. Expression of double-stranded RNA-activated protein kinase in keratinocytes and keratinocytic neoplasia. J Dermatol,2003,30:579-589.
[58]Ahn JH, Park TJ, Jin SH, Kang HY. Human melanocytes express functional Toll-like receptor 4. Exp Dermatol,2008,17:412-417.
[59]Ahn JH, Jin SH, Kang HY. LPS induces melanogenesis through p38 MAPK activation in human melanocytes. Arch Dermatol Res,2008,300:325-329.
[60]Kroll TM, Bommiasamy H, Boissy RE, et al.4-Tertiary butyl phenol exposure sensitizes human melanocytes to dendritic cell-mediated killing:relevance to vitiligo. J Invest Dermatol,2005,124:798-806.
[61]Kang HY, Park TJ, Jin SH. Imiquimod, a Toll-like receptor 7 agonist, inhibits melanogenesis and proliferation of human melanocytes. J Invest Dermatol,2009, 129:243-246.
[62]Dorn A, Ludwig RJ, Bock A, et al. Oligonucleotides suppress IL-8 in skin keratinocytes in vitro and offer anti-inflammatory properties in vivo. J Invest Dermatol,2007,127:846-854.
[63]Kim HS, Lee MS. Role of innate immunity in triggering and tuning of autoimmune diabetes. Curr Mol Med,2009,9(1):30-44.
[64]Li M, Zheng DJ, Field LL, et al. Murine pancreatic beta TC3 cells show greater 2', 5'-oligoadenylate synthetase (2'5'AS) antiviral enzyme activity and apoptosis following IFN-alpha or poly(I:C) treatment than pancreatic alpha TC3 cells. Exp Diabetes Res,2009,2009:631026.
[65]Garcia M, Dogusan Z, Moore F, et al. Regulation and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells. Biochim Biophys Acta.2009, 1793(11):1768-1775.
[66]Wong FS, Wen L. Toll-like receptors and diabetes. Ann N Y Acad Sci,2008,1150: 123-132.
[1]Berardesca E, Maibach H. Racial differences in skin pathophysiology. J Am Acad Dermatol. 1996; 34(4):667-72.
[2]Wesley NO, Maibach HI.Racial (ethnic) differences in skin properties:the objective data. Am J Clin Dermatol.2003;4(12):843-60.
[3]Schallreuter, K.U., Kothari, et al. Regulation of melanogenesis-controversies and new concepts. Exp. Dermatol.2008; 17,395-404.
[4]Gunathilake, R., Schurer, N.Y., Shoo, B.A., et al. H-regulated mechanisms account for pigmenttype differences in epidermal barrier function. J. Invest. Dermatol.2009; 129, 1719-1729.
[5]Hachem, J.P., Man, et. Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity. J. Invest. Dermatol.2005; 125,510-520.
[6]SCHRAERMEYER, U., PETERS, S., THUMANN, G., KOCIOK, N.& HEIMANN, K. Melanin granules of retinalpigment epithelium are connected with the lysosomaldegradation pathway. Exp. Eye Res.1999; 68,237-245.
[7]DELL'ANGELICA, E. C., MULLINS, et al. Lysosome-related organelles. FASEB J.2000; 14,1265-1278.
[8]LE POOLE, I. C., MUTIS, T., et al. A novel antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders. J. Immunol.1993; 151,7284-7292.
[9]Swope VB, Sauder DN, McKenzie RC, et al. Synthesis of interleukin-1 alpha and beta by normal human melanocytes. J Invest Dermatol.1994; 102:749-53.
[10]Kruger-Krasagakes S, Krasagakis K, Garbe C, et al. Production of cytokines by human melanoma cells and melanocytes. Recent Results Cancer Res.1995; 139:155-68.
[11]Lu Y, Zhu WY, Tan C,et al. Melanocytes are potential immunocompetent cells:evidence from recognition of immunological characteristics of cultured human melanocytes. Pigment Cell Res.2002; 15:454-60.
[12]Ahn JH, Park TJ, Jin SH, et al. Human melanocytes express functional Toll-like receptor 4. Exp Dermatol.2008; 17(5):412-7.
[13]Ahn JH, Jin SH, Kang HY. LPS induces melanogenesis through p38 MAPK activation in human melanocytes. Arch Dermatol Res.2008; 300(6):325-9.
[14]Kang HY, Park TJ, Jin SH. Imiquimod, a Toll-like receptor 7 agonist, inhibits melanogenesis and proliferation of human melanocytes. J Invest Dermatol.2009; 129(1):243-6.
[15]Denman CJ, McCracken J, Hariharan V, et al. HSP70i Accelerates Depigmentation in a Mouse Model of Autoimmune Vitiligo. J Invest Dermatol.2008; 13.
[16]TSATMALI, M., GRAHAM, A., SZATKOWSKI, D., et al. Alpha-melanocyte-stimulating hormone modulates nitric oxide production in melanocytes. J. Invest. Dermatol.2000; 114, 520-526.
[17]SASAKI, M., HORIKOSHI, T., UCHIWA, H.& MIYACHI, Y. Up-regulation of tyrosinase gene by nitric oxide in human melanocytes. Pigment Cell Res.2000; 13,248-252.
[18]LUGER, T. A., SCHOLZEN, T.& GRABBE, S. The role of a-melanocyte stimulating hormone in cutaneous biology. J. Invest. Dermatol. Symp. Proc.1997; 2,87-93.
[19]YOSHIDA, M., TAKAHASHI, Y.& SHINTARO, I. Histamine induces melanogenesis and morphologic changes by Protein Kinase A activation via H2 receptors in human normal melanocytes. J. Invest. Dermatol.2000; 114,334-342.