Effects Of Localized Hyperthermia On Function Of Langerhans Cells In HPV-infected Skin

BackgroundHuman 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 methodsMaterialsClinical 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.Methods1.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%CO₂) 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 treatmentStandard 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 MicroscopyTo 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 LCsTo 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) assayThe 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.Result1.Quantitative and morphological changes of LCs of skin tissue after hyperthermia treatmentâ‘ Quantitative changes of epidermal LCs after hyperthermia treatmentThe 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).â‘¡Morphological changes of epidermal LCs after hyperthermia treatmentThe 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 LCsImmunofluorescence 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 LCsFlow 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.Conclusion1.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.