局部温热对HPV感染表皮朗格汉斯细胞功能活性的影响
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摘要
背景
人乳头瘤病毒(Human papillomavirus,HPV)是一组可引发人类上皮细胞发生增殖反应的环状DNA病毒。HPV感染可以引起皮肤粘膜的良性增生性病变甚至发生恶性肿瘤。由于感染局部的免疫缺陷以及缺乏有效的特异性抗病毒制剂和彻底根治的物理方法,使感染迁延不愈。近年来随着加温、测温和控温技术的不断发展和完善,以及有关热疗的生理学和病理生理学的研究逐步深入,热疗得到了迅速发展。有学者将局部温热用于病毒疣的治疗,取得了一定的效果,而且局部温热疗法无创伤,痛苦小,复发率低,患者易于接受,有可能成为一种简捷、常规、有效的治疗手段。
造成HPV持续感染的可能原因是机体不能建立有效的免疫反应及机体的免疫监视功能障碍。朗格汉斯细胞(Langerhans cell,LC)为树突状细胞系统中的一种独特类型,作为皮肤及黏膜的抗原提呈细胞,在摄取、加工和处理抗原的过程中,同时发生形态学变化,并逐渐离开表皮,移行至引流淋巴结,将抗原递呈给T辅助细胞,在协同刺激信号的作用下使之活化,从而启动免疫应答,因此在免疫系统中起着重要作用。HPV进入人体后常形成持续感染,其原因在于局部的免疫抑制和/或不能识别病毒蛋白,导致免疫逃逸。
在HPV感染的皮肤组织中,LC有充足的机会接触HPV抗原,单独的抗原和或淋巴细胞并不能激活机体的免疫功能,只有在抗原提呈细胞的作用下,多方面因素相结合,才能激发机体的免疫应答。LC作为迄今为止所发现的功能最强大、表皮内分布最广泛的抗原提呈细胞,对其在温热治疗病毒疣治疗的发病机制中作用研究就显得非常必要。
本研究中,我们通过检测:①不同温热对LC数量和形态的影响;②不同温热对朗格汉斯细胞游走及成熟功能的影响;③温热对LC信号传导功能的影响。以说明温热对促进LC有效诱发免疫应答中的作用,为病毒疣的温热治疗提供可靠的依据和指导。
材料和方法
一、材料
临床资料:女性尖锐湿疣(Condyloma acuminatum,CA)患者均为我院皮肤科、妇科科2006年1月-2008年2月的门诊患者,符合2000年卫生部防疫司制订的CA诊断标准。均为初发患者,未经过任何治疗。
对照组:对照为同时期我院急诊外伤缝合的非CA患者。
二、方法
(一)取材:
1、征得患者知情同意后,局部消毒麻醉,分别切取疣体/正常组织,每例组织等分为4份。其中一份OTC包埋剂包埋后直接冷冻保存,另3份真皮侧向下,放入少量RPMI-1640培养液,只将疣体底部浸于培养液中分别以37℃、42℃和45℃三个温度照射30分钟后,将疣体取出,OCT包埋剂包埋后冷冻保存。
2、征得患者知情同意后,局部消毒麻醉,切取疣体/正常组织各,每例组织用以上相同方法处理后,将疣体全部浸于RPMI-1640培养液,37℃培养箱孵育12小时后将组织取出,OCT包埋剂包埋后冷冻保存,同时收集培养液中的细胞。
(二)温热对表皮内LC数量和形态的影响
采用免疫组化方法对20例CA患者和20例正常人表皮内LC染色,高倍光学显微镜下观察不同温热处理后LC数目和形态变化。
(三)采用直接免疫荧光方法,用CD1a、CD83单克隆抗体标记LC,利用共聚焦显微镜观察未经过任何处理CA和正常皮肤组织CD1a~+,CD83~+LC的分布。
(四)通过流式细胞分析仪检测CA/正常人不同温热处理后CD1a~+,CD83~+LC细胞百分率。观察不同温热治疗后LC成熟标志—CD83的变化。
(五)采用QRT-PCR方法检测不同温热治疗对LC表面趋化因子受体CCR6和CCR7 mRNA的表达变化。
(六)采用QRT-PCR方法检测不同温热治疗对LC信号传导分子—PI3-KmRNA的表达变化。
(七)统计学方法:采用SPSS13.0版本软件对资料进行统计学分析。测得结果用(?)±s表示,采用区组方差分析、t检验统计方法。P＜0.05有统计学意义。
结果
一、温热治疗后皮肤组织内LC数量和形态的变化
1、温热治疗后CA和正常人表皮内LC的数量变化:
不同温热治疗后,随温度升高,LC数量逐渐减少,经统计学处理,差异具有显著性(P＜0.01);CA组织比正常组织表皮内LC减少更明显,也随温度变化而变化。正常人和CA组织相同温度之间的LC数量经统计学处理,也具有显著性差异(P＜0.01)。
2、不同温热治疗后表皮内LC的形态变化
不同温热治疗后,LC减少、变短或消失,具有温度相关性。CA组织较正常组织变化更明显。
二、我们采用免疫荧光方法,用CD1a、CD83单克隆抗体双标记正常皮肤组织和疣组织,发现多数LC位于表皮,而且只表达CD1a,不表达CD83。
三、不同温热条件对LC游走的影响:正常皮肤组织和疣组织经不同温度处理,并孵育12小时后,培养液中有较多的游走细胞。本研究采用双色色标记法,检测了游走至培养液中CD1a,CD83阳性细胞的百分数。经37℃,42℃和45℃处理后游走至培养液中LC的比例不同。随温度升高,CD1a~+,CD83~+LC百分比逐渐增加。
四、为进一步证明温热对LC成熟功能的影响,我们采用荧光实时定量PCR的方法检测趋化因子受体CCR6和CCR7 mRNA水平变化。
正常皮肤组织、疣体组织经不同温热条件处理后,游走至培养液中LC趋化因子受体mRNA表达比率不同;同一组织利用不同温度处理后,CCR6mRNA的表达量随温度增加逐渐减少,而CCR7mRNA的表达量随温度增加逐渐增加。
五、温热对LC信号传导功能的影响
本研究采用RT-PCR方法检测游走纯化的LC发现PI3Kp110αmRNA表达量随温度升高而逐渐增加。而未经过处理的疣组织的表达量低于正常皮肤组织。
结论
1、局部温热治疗后皮损部位LC较正常组织LC数量和结构均发生了明显改变,推测温热能促进LC离开表皮,通过真皮向局部淋巴结迁移,促进局部细胞免疫应答反应。
2、局部温热治疗后游走至培养液中的LC明显增多,而且随温度升高CD83~+LC逐渐增多,CCR7 mRNA表达量也逐渐增多,说明温热能够促进LC成熟,温热能够促进LC的迁移和增强抗原提呈能力,LC通过真皮向局部淋巴结迁移,在局部细胞免疫应答中发挥作用。温热能够有效治疗HPV感染皮肤病,是协助增强了机体免疫系统的作用。
3、温热可能通过抑制HPV感染皮肤LC PI3-K的活性,调控信号传导通路。
Background
Human papilloma virus(HPV) is a group of circular-DNA viruses that can induce proliferation of epithelial cells in humans.HPV infection can cause benign proliferation of the skin and mucous membrane,or even malignancy.Once infected by this group of viruses,recovery becomes difficult due to local immunodeficiency of the HPV-infected parts,lack of effective antiviral agents specific to this infection and lack of effective physical methods to permanently get rid of the infection.The rapid development of new techniques in heat production,temperature measurement and temperature control in recent years has made studies in physiology and pathophysiology of thermotherapy possible,hence the rapid evolution of this field of Medicine in recent years Some scholars reported good results of the application of local hyperthermia in the treatment of warts.Local hyperthermia may become a simple, routine and effective therapeutic tool because it's wound-free,painless,patient-friendly and is associated with low recurrences.
The possible reason for persistent HPV infection is inability of the body to establish effective immune reaction and functional impairment of body's immune surveillance. Langerhans cells(LCs) are a unique set of epidermal dendritic cells(DCs).They are antigen-presenting cells of skin and mucous membrane.LCs are very important in the functioning of the immune system as they trap and process antigens in the skin.As they do so,they simultaneously undergo morphological changes,gradually leaving the epidermis,migrate to the draining lymph node(LN),and handle the antigen to T helper cells.LCs are activated by assistant irritation signals prompting immune response.The reason for persistent HPV infection is local immune suppression and/or inability to identify viral protein,resulting in immune escape.
An immune response is brought about by a combination of many factors.Neither antigens nor lymphocytes can act independently in stimulating body's immune function. In HPV-infected skin tissues,LCs have a great opportunity to contact HPV antigens.As antigen-presenting cells,LCs have an important role to play in the initiation of an immune response.So far,LCs are the most powerful and the most extensive antigen-presenting cells of the epidermis.It is,therefore,extremely important that researches on the role of LCs in the mechanism of hyperthermia in wart treatment be done.
This research aims at illustrating the role of hyperthermia in promoting LCs to provoke an immune response.It aims at establishing a reliable foundation and guidance in the treatment of warts.To achieve this objective,our research puts more emphasis on:
1.How different temperatures in hyperthermia affect the quantity and morphology of LCs,
2.How different temperatures in hyperthermia affect the migration and maturation of LCs.
3.Whether different temperatures in hyperthermia affect the immune recognition (or immune surveillance) of HPV through Langerhans Cell Phosphoinositide 3-Kinase Activation.
Materials and methods
Materials
Clinical data:All patients with clinically diagnosed condyloma acuminatum (CA) were recruited into the study from the outpatient clinic of China Medical University from Jan.2006 to Feb.2008.Only patients with the first visit were included in the study.Patients with any documented systemic disease(s) were excluded from the study.
Control group:patients without CA,undergoing surgical treatment in our hospital,were chosen as control group.
Methods
1.skin tissue
(1) human skin was obtained as resected material of surgery with informed consent of the patient.After removal of subcutaneous fat,sterile biopsies were cut and divided into 4 equal portions and 3 were respectively placed(the remaining piece were used for routine histological examination),dermal side down,in culture media in tissue culture plate and exposed to hyperthermia at 37℃,42℃and 45℃respectively for 30min.The skin biopsies were taken out and embedded in OCT compound,frozen in liquid nitrogen and stored at -70℃.
(2) Another ten pieces of resected CA and 10 normal tissues were similarly treated using the protocol as mentioned above.While after exposure to hyperthermia at 37℃,42℃,45℃respectively for 30min,the specimen and unexposed specimen were completely immersed in the media,and were subsequently incubated at 37℃for 12h (with 5%CO_2) The specimen were then taken and embedded in OCT compound, frozen in liquid nitrogen and stored at -70℃.Meanwhile the cells that migrated into the medium were recovered for further analysis(see below Method 4,5 and 6).
2.Quantitative and morphological changes of LCs of skin tissue after hyperthermia treatment
Standard Immunohistochemical staining methods were performed on cryostat sections of CA/normal skin(n=10) using antibodies for CD1a and examined under a light microscope.
3.Confocal Microscopy
To evaluate the initial status of LCs,we performed direct immunofluorescence (DIF) with FITC-labeled CD1a and PE-labeled CD83.Normal skin tissue and HPV-infected skin tissue from two donors were routinely prepared for DIF staining.
4.Flow cytometric analysis of migrating LCs
To nalysis the migrating LC after hyperthermia treatment,the cells were collected and stained with mouse anti-human FITC-labeled CD1a and PE-labeled CD83 monoclonal antibodies(mAbs).After staining,cells were analyzed with flow cytometry.
5.RNA extraction and Real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR) assay
The RT-PCR was performed to detect the mRNA variation of CCR6 and CCR7 after hyperthermia treatment in CA/normal skin.
6.The qRT-PCR was performed to detect the mRNA variation of PI3K P110α,P85αafter hyperthermia treatment in CA/normal skin.
7.Statistical analysis:
Data were analyzed with SPSS 13.0 software.The differences between groups were analyzed by repeated measures analysis of variance(ANOVA),and independent t tests.A two-tailed P-value of＜0.05 was considered statistically significant.
Result
1.Quantitative and morphological changes of LCs of skin tissue after hyperthermia treatment
①Quantitative changes of epidermal LCs after hyperthermia treatment
The number of LCs in the normal skin tissue and HPV-infected skin tissue decreased with an increase in temperature.At any given temperature,the differences in the number of epidermal LCs between normal skin tissue and HPV-infected skin tissue have obvious statistical significance(p＜0.01).The differences in the number of epidermal LCs in the same skin tissue,at different temperatures,have obvious statistical significance(again,p＜0.01).
②Morphological changes of epidermal LCs after hyperthermia treatment
The dendrites were shortened,decreased in number or even disappeared with increased temperatures in normal skin tissue and HPV-infected skin tissue.This was more obvious in HPV-infected skin tissue.
2.Confocal Microscopic Studies of LCs
Immunofluorescence labeling of HPV-infected skin tissues and normal skin tissues with CD1a and CD83 mAbs revealed that most LCs were located in the epidermis.Double staining of skin tissues with CD1a and CD83 mAbs showed that the LCs of the epidermis in both normal and HPV-infected skin tissues only expressed CD1a.None of them expressed CD83.
3.Flow cytometric analysis of migrating LCs
Flow cytometric assessments of mAb reactivity against LC was undertaken by measuring the%of reactivity and mean fluorescent intensity(MFI) of the two populations of LC at 37℃,42℃and 45℃.
The normal tissues and HPV-infected tissues were,respectively,treated at different temperature,and then incubated for 12h in media at 37℃.There were many migratory LCs in the media.At different temperatures,the migratory LCs,as expressed by CD1a and CD83,were found to be different.The percentages of CD1a~+,CD83~+ positive cells were increased with increased temperatures in normal skin and HPV-infected skin.But the percentages of migratory LCs in HPV infected skin were greater than those in normal skin.
4.To confirm the observation mentioned above,qRT-PCR assay was performed to detect the expressions of different chemokine receptors at mRNA level.
The real-time quantitative detection of qRT-PCR for mRNA of CCR6,CCR7,in freshly purified LCs inémigrés from normal skin tissues and HPV-infected tissues.The expression of CCR6 mRNA was decreased with increased temperatures.In contrast,the expression of CCR7 mRNA was increased with increased temperatures.Again,the decrement of CCR6 mRNA expression at each given hyperthermia point was more prominent inémigrés from CA than those from normal skin,meanwhile the increase of CCR7 expression at each given hyperthermia point was more prominent inémigrés from CA than those from normal skin.
5.QRT-PCR assay was performed to detect the expressions of phosphatidylinositol 3-kinase(PI3-K) at mRNA level in inémigrés from normal skin tissues and HPV-infected tissues after hyperthermia treatment.
The expression of PI3K p110αmRNA was increased with increased temperatures. In contrast,the expression of PI3K p110αmRNA was decreased in HPV-infected tissues than those normal skin tissues having no hyperthermia treatment.
Conclusion
1.Our results show that after local hyperthermia there were more significant changes in the number and structure of LCs in HPV infected tissue than in normal tissue.These data have led to the hypothesis that hyperthermia can promote LCs leaving epidermis to draining lymph nodes(DLN) to induce a specific immune response.
2.The percentages of CD83~+ positive cells were increased with an increase in temperature in the normal skin and HPV infected skin,and the expressions of CCR7at mRNA level were increased with increased temperature.Taken together these data illustrate that hyperthermia can promote migration of LCs and enhance antigen-presenting ability.LCs migrate to regional lymph nodes through dermis and exert their effects in local cellular immune responses.Its ability to help build up the immune system explains why hyperthermia can be used in the treatment of HPV-related skin diseases.
3.Hyperthermia can down-regulate PI3-K at mRNA level in LCs inémigrés at mRNA level inémigrés from normal skin tissue and HPV-infected tissue.Human papillomavirus possiblely can escape immune recognition through LC PI3-K activation. This finding suggests that hyperthermia may enhance immunity to anti-HPV immune response.Hyperthermia may serves as an effective clinical option to treat HPV-infected skin diseases.
人乳头瘤病毒(Human papillomavirus,HPV)是一组可引发人类上皮细胞发生增殖反应的环状DNA病毒。HPV感染可以引起皮肤粘膜的良性增生性病变甚至发生恶性肿瘤。由于感染局部的免疫缺陷以及缺乏有效的特异性抗病毒制剂和彻底根治的物理方法,使感染迁延不愈。近年来随着加温、测温和控温技术的不断发展和完善,以及有关热疗的生理学和病理生理学的研究逐步深入,热疗得到了迅速发展。有学者将局部温热用于病毒疣的治疗,取得了一定的效果,而且局部温热疗法无创伤,痛苦小,复发率低,患者易于接受,有可能成为一种简捷、常规、有效的治疗手段。
造成HPV持续感染的可能原因是机体不能建立有效的免疫反应及机体的免疫监视功能障碍。朗格汉斯细胞(Langerhans cell,LC)为树突状细胞系统中的一种独特类型,作为皮肤及黏膜的抗原提呈细胞,在摄取、加工和处理抗原的过程中,同时发生形态学变化,并逐渐离开表皮,移行至引流淋巴结,将抗原递呈给T辅助细胞,在协同刺激信号的作用下使之活化,从而启动免疫应答,因此在免疫系统中起着重要作用。HPV进入人体后常形成持续感染,其原因在于局部的免疫抑制和/或不能识别病毒蛋白,导致免疫逃逸。
在HPV感染的皮肤组织中,LC有充足的机会接触HPV抗原,单独的抗原和或淋巴细胞并不能激活机体的免疫功能,只有在抗原提呈细胞的作用下,多方面因素相结合,才能激发机体的免疫应答。LC作为迄今为止所发现的功能最强大、表皮内分布最广泛的抗原提呈细胞,对其在温热治疗病毒疣治疗的发病机制中作用研究就显得非常必要。
本研究中,我们通过检测:①不同温热对LC数量和形态的影响;②不同温热对朗格汉斯细胞游走及成熟功能的影响;③温热对LC信号传导功能的影响。以说明温热对促进LC有效诱发免疫应答中的作用,为病毒疣的温热治疗提供可靠的依据和指导。
材料和方法
一、材料
临床资料:女性尖锐湿疣(Condyloma acuminatum,CA)患者均为我院皮肤科、妇科科2006年1月-2008年2月的门诊患者,符合2000年卫生部防疫司制订的CA诊断标准。均为初发患者,未经过任何治疗。
对照组:对照为同时期我院急诊外伤缝合的非CA患者。
二、方法
(一)取材:
1、征得患者知情同意后,局部消毒麻醉,分别切取疣体/正常组织,每例组织等分为4份。其中一份OTC包埋剂包埋后直接冷冻保存,另3份真皮侧向下,放入少量RPMI-1640培养液,只将疣体底部浸于培养液中分别以37℃、42℃和45℃三个温度照射30分钟后,将疣体取出,OCT包埋剂包埋后冷冻保存。
2、征得患者知情同意后,局部消毒麻醉,切取疣体/正常组织各,每例组织用以上相同方法处理后,将疣体全部浸于RPMI-1640培养液,37℃培养箱孵育12小时后将组织取出,OCT包埋剂包埋后冷冻保存,同时收集培养液中的细胞。
(二)温热对表皮内LC数量和形态的影响
采用免疫组化方法对20例CA患者和20例正常人表皮内LC染色,高倍光学显微镜下观察不同温热处理后LC数目和形态变化。
(三)采用直接免疫荧光方法,用CD1a、CD83单克隆抗体标记LC,利用共聚焦显微镜观察未经过任何处理CA和正常皮肤组织CD1a~+,CD83~+LC的分布。
(四)通过流式细胞分析仪检测CA/正常人不同温热处理后CD1a~+,CD83~+LC细胞百分率。观察不同温热治疗后LC成熟标志—CD83的变化。
(五)采用QRT-PCR方法检测不同温热治疗对LC表面趋化因子受体CCR6和CCR7 mRNA的表达变化。
(六)采用QRT-PCR方法检测不同温热治疗对LC信号传导分子—PI3-KmRNA的表达变化。
(七)统计学方法:采用SPSS13.0版本软件对资料进行统计学分析。测得结果用(?)±s表示,采用区组方差分析、t检验统计方法。P＜0.05有统计学意义。
结果
一、温热治疗后皮肤组织内LC数量和形态的变化
1、温热治疗后CA和正常人表皮内LC的数量变化:
不同温热治疗后,随温度升高,LC数量逐渐减少,经统计学处理,差异具有显著性(P＜0.01);CA组织比正常组织表皮内LC减少更明显,也随温度变化而变化。正常人和CA组织相同温度之间的LC数量经统计学处理,也具有显著性差异(P＜0.01)。
2、不同温热治疗后表皮内LC的形态变化
不同温热治疗后,LC减少、变短或消失,具有温度相关性。CA组织较正常组织变化更明显。
二、我们采用免疫荧光方法,用CD1a、CD83单克隆抗体双标记正常皮肤组织和疣组织,发现多数LC位于表皮,而且只表达CD1a,不表达CD83。
三、不同温热条件对LC游走的影响:正常皮肤组织和疣组织经不同温度处理,并孵育12小时后,培养液中有较多的游走细胞。本研究采用双色色标记法,检测了游走至培养液中CD1a,CD83阳性细胞的百分数。经37℃,42℃和45℃处理后游走至培养液中LC的比例不同。随温度升高,CD1a~+,CD83~+LC百分比逐渐增加。
四、为进一步证明温热对LC成熟功能的影响,我们采用荧光实时定量PCR的方法检测趋化因子受体CCR6和CCR7 mRNA水平变化。
正常皮肤组织、疣体组织经不同温热条件处理后,游走至培养液中LC趋化因子受体mRNA表达比率不同;同一组织利用不同温度处理后,CCR6mRNA的表达量随温度增加逐渐减少,而CCR7mRNA的表达量随温度增加逐渐增加。
五、温热对LC信号传导功能的影响
本研究采用RT-PCR方法检测游走纯化的LC发现PI3Kp110αmRNA表达量随温度升高而逐渐增加。而未经过处理的疣组织的表达量低于正常皮肤组织。
结论
1、局部温热治疗后皮损部位LC较正常组织LC数量和结构均发生了明显改变,推测温热能促进LC离开表皮,通过真皮向局部淋巴结迁移,促进局部细胞免疫应答反应。
2、局部温热治疗后游走至培养液中的LC明显增多,而且随温度升高CD83~+LC逐渐增多,CCR7 mRNA表达量也逐渐增多,说明温热能够促进LC成熟,温热能够促进LC的迁移和增强抗原提呈能力,LC通过真皮向局部淋巴结迁移,在局部细胞免疫应答中发挥作用。温热能够有效治疗HPV感染皮肤病,是协助增强了机体免疫系统的作用。
3、温热可能通过抑制HPV感染皮肤LC PI3-K的活性,调控信号传导通路。
Background
Human papilloma virus(HPV) is a group of circular-DNA viruses that can induce proliferation of epithelial cells in humans.HPV infection can cause benign proliferation of the skin and mucous membrane,or even malignancy.Once infected by this group of viruses,recovery becomes difficult due to local immunodeficiency of the HPV-infected parts,lack of effective antiviral agents specific to this infection and lack of effective physical methods to permanently get rid of the infection.The rapid development of new techniques in heat production,temperature measurement and temperature control in recent years has made studies in physiology and pathophysiology of thermotherapy possible,hence the rapid evolution of this field of Medicine in recent years Some scholars reported good results of the application of local hyperthermia in the treatment of warts.Local hyperthermia may become a simple, routine and effective therapeutic tool because it's wound-free,painless,patient-friendly and is associated with low recurrences.
The possible reason for persistent HPV infection is inability of the body to establish effective immune reaction and functional impairment of body's immune surveillance. Langerhans cells(LCs) are a unique set of epidermal dendritic cells(DCs).They are antigen-presenting cells of skin and mucous membrane.LCs are very important in the functioning of the immune system as they trap and process antigens in the skin.As they do so,they simultaneously undergo morphological changes,gradually leaving the epidermis,migrate to the draining lymph node(LN),and handle the antigen to T helper cells.LCs are activated by assistant irritation signals prompting immune response.The reason for persistent HPV infection is local immune suppression and/or inability to identify viral protein,resulting in immune escape.
An immune response is brought about by a combination of many factors.Neither antigens nor lymphocytes can act independently in stimulating body's immune function. In HPV-infected skin tissues,LCs have a great opportunity to contact HPV antigens.As antigen-presenting cells,LCs have an important role to play in the initiation of an immune response.So far,LCs are the most powerful and the most extensive antigen-presenting cells of the epidermis.It is,therefore,extremely important that researches on the role of LCs in the mechanism of hyperthermia in wart treatment be done.
This research aims at illustrating the role of hyperthermia in promoting LCs to provoke an immune response.It aims at establishing a reliable foundation and guidance in the treatment of warts.To achieve this objective,our research puts more emphasis on:
1.How different temperatures in hyperthermia affect the quantity and morphology of LCs,
2.How different temperatures in hyperthermia affect the migration and maturation of LCs.
3.Whether different temperatures in hyperthermia affect the immune recognition (or immune surveillance) of HPV through Langerhans Cell Phosphoinositide 3-Kinase Activation.
Materials and methods
Materials
Clinical data:All patients with clinically diagnosed condyloma acuminatum (CA) were recruited into the study from the outpatient clinic of China Medical University from Jan.2006 to Feb.2008.Only patients with the first visit were included in the study.Patients with any documented systemic disease(s) were excluded from the study.
Control group:patients without CA,undergoing surgical treatment in our hospital,were chosen as control group.
Methods
1.skin tissue
(1) human skin was obtained as resected material of surgery with informed consent of the patient.After removal of subcutaneous fat,sterile biopsies were cut and divided into 4 equal portions and 3 were respectively placed(the remaining piece were used for routine histological examination),dermal side down,in culture media in tissue culture plate and exposed to hyperthermia at 37℃,42℃and 45℃respectively for 30min.The skin biopsies were taken out and embedded in OCT compound,frozen in liquid nitrogen and stored at -70℃.
(2) Another ten pieces of resected CA and 10 normal tissues were similarly treated using the protocol as mentioned above.While after exposure to hyperthermia at 37℃,42℃,45℃respectively for 30min,the specimen and unexposed specimen were completely immersed in the media,and were subsequently incubated at 37℃for 12h (with 5%CO_2) The specimen were then taken and embedded in OCT compound, frozen in liquid nitrogen and stored at -70℃.Meanwhile the cells that migrated into the medium were recovered for further analysis(see below Method 4,5 and 6).
2.Quantitative and morphological changes of LCs of skin tissue after hyperthermia treatment
Standard Immunohistochemical staining methods were performed on cryostat sections of CA/normal skin(n=10) using antibodies for CD1a and examined under a light microscope.
3.Confocal Microscopy
To evaluate the initial status of LCs,we performed direct immunofluorescence (DIF) with FITC-labeled CD1a and PE-labeled CD83.Normal skin tissue and HPV-infected skin tissue from two donors were routinely prepared for DIF staining.
4.Flow cytometric analysis of migrating LCs
To nalysis the migrating LC after hyperthermia treatment,the cells were collected and stained with mouse anti-human FITC-labeled CD1a and PE-labeled CD83 monoclonal antibodies(mAbs).After staining,cells were analyzed with flow cytometry.
5.RNA extraction and Real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR) assay
The RT-PCR was performed to detect the mRNA variation of CCR6 and CCR7 after hyperthermia treatment in CA/normal skin.
6.The qRT-PCR was performed to detect the mRNA variation of PI3K P110α,P85αafter hyperthermia treatment in CA/normal skin.
7.Statistical analysis:
Data were analyzed with SPSS 13.0 software.The differences between groups were analyzed by repeated measures analysis of variance(ANOVA),and independent t tests.A two-tailed P-value of＜0.05 was considered statistically significant.
Result
1.Quantitative and morphological changes of LCs of skin tissue after hyperthermia treatment
①Quantitative changes of epidermal LCs after hyperthermia treatment
The number of LCs in the normal skin tissue and HPV-infected skin tissue decreased with an increase in temperature.At any given temperature,the differences in the number of epidermal LCs between normal skin tissue and HPV-infected skin tissue have obvious statistical significance(p＜0.01).The differences in the number of epidermal LCs in the same skin tissue,at different temperatures,have obvious statistical significance(again,p＜0.01).
②Morphological changes of epidermal LCs after hyperthermia treatment
The dendrites were shortened,decreased in number or even disappeared with increased temperatures in normal skin tissue and HPV-infected skin tissue.This was more obvious in HPV-infected skin tissue.
2.Confocal Microscopic Studies of LCs
Immunofluorescence labeling of HPV-infected skin tissues and normal skin tissues with CD1a and CD83 mAbs revealed that most LCs were located in the epidermis.Double staining of skin tissues with CD1a and CD83 mAbs showed that the LCs of the epidermis in both normal and HPV-infected skin tissues only expressed CD1a.None of them expressed CD83.
3.Flow cytometric analysis of migrating LCs
Flow cytometric assessments of mAb reactivity against LC was undertaken by measuring the%of reactivity and mean fluorescent intensity(MFI) of the two populations of LC at 37℃,42℃and 45℃.
The normal tissues and HPV-infected tissues were,respectively,treated at different temperature,and then incubated for 12h in media at 37℃.There were many migratory LCs in the media.At different temperatures,the migratory LCs,as expressed by CD1a and CD83,were found to be different.The percentages of CD1a~+,CD83~+ positive cells were increased with increased temperatures in normal skin and HPV-infected skin.But the percentages of migratory LCs in HPV infected skin were greater than those in normal skin.
4.To confirm the observation mentioned above,qRT-PCR assay was performed to detect the expressions of different chemokine receptors at mRNA level.
The real-time quantitative detection of qRT-PCR for mRNA of CCR6,CCR7,in freshly purified LCs inémigrés from normal skin tissues and HPV-infected tissues.The expression of CCR6 mRNA was decreased with increased temperatures.In contrast,the expression of CCR7 mRNA was increased with increased temperatures.Again,the decrement of CCR6 mRNA expression at each given hyperthermia point was more prominent inémigrés from CA than those from normal skin,meanwhile the increase of CCR7 expression at each given hyperthermia point was more prominent inémigrés from CA than those from normal skin.
5.QRT-PCR assay was performed to detect the expressions of phosphatidylinositol 3-kinase(PI3-K) at mRNA level in inémigrés from normal skin tissues and HPV-infected tissues after hyperthermia treatment.
The expression of PI3K p110αmRNA was increased with increased temperatures. In contrast,the expression of PI3K p110αmRNA was decreased in HPV-infected tissues than those normal skin tissues having no hyperthermia treatment.
Conclusion
1.Our results show that after local hyperthermia there were more significant changes in the number and structure of LCs in HPV infected tissue than in normal tissue.These data have led to the hypothesis that hyperthermia can promote LCs leaving epidermis to draining lymph nodes(DLN) to induce a specific immune response.
2.The percentages of CD83~+ positive cells were increased with an increase in temperature in the normal skin and HPV infected skin,and the expressions of CCR7at mRNA level were increased with increased temperature.Taken together these data illustrate that hyperthermia can promote migration of LCs and enhance antigen-presenting ability.LCs migrate to regional lymph nodes through dermis and exert their effects in local cellular immune responses.Its ability to help build up the immune system explains why hyperthermia can be used in the treatment of HPV-related skin diseases.
3.Hyperthermia can down-regulate PI3-K at mRNA level in LCs inémigrés at mRNA level inémigrés from normal skin tissue and HPV-infected tissue.Human papillomavirus possiblely can escape immune recognition through LC PI3-K activation. This finding suggests that hyperthermia may enhance immunity to anti-HPV immune response.Hyperthermia may serves as an effective clinical option to treat HPV-infected skin diseases.
引文
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