Immunohistochemical analysis of photodamaged skin after treatment with delta 5-aminolevulinic acid

Received on: 14/06/2011
Approved on: 15/12/2011

This study was carried out at the Hospital das
Clínicas da Universidade de São Paulo (USP) -
São Paulo (SP), Brazil.

Financial support: None
Conflicts of interest: None

Abstract

Introduction: Photodynamic therapy destroys target cells by directly harming them, injuring the vascular stroma, and activating the immune system.
Objective: To study photoaged skin''''''''s immune response to photodynamic therapy.
Methods: Thirteen female patients underwent three treatment sessions with delta 5-aminolevulinic acid combined with 630 nm diode light. The immunohistochemical response of the skin''''''''s immune system was evaluated using the markers anti-CD1a, anti-CD4+, anti-CD8+, anti-TNFa, anti-IFNy and anti-IL4 before, 24 hours after the first session, and 21 days after the third session.
Results: At the final evaluation, patients demonstrated a decrease in CD4 and CD8 lymphocytes, an increase in interleukin-4 in the epidermis and dermis, and a reduction and an increase of TNF-a in the epidermis and dermis, respectively. The Langerhans cell and INF-y populations remained unchanged.
Conclusion: Regarding the immune system, photodynamic therapy did not cause changes in Langerhans cells; therefore, there was little local immunosuppression. CD4 and CD8 lymphocyte apoptosis was observed, suggesting that photodynamic therapy can be useful in treating inflammatory diseases. The changes in TNF-a and an increase of IL4 in the dermis and epidermis suggest a humoral type response, which was also evidenced by the absence of INFy.

Keywords: PHOTOCHEMOTHERAPY, AMINOLEVULINIC ACID , SKIN AGING

Photodynamic therapy (PDT) is currently used to treat inflammatory diseases such as psoriasis, acne, sebaceous hyper- plasia, and rosacea – and, more recently, photoaging. In addition, it has applications in the treatment of tumors. 1-7 PDT involves accumulating a specific photosensitizer agent in the target tissue, which is then activated by light. In the presence of oxygen, this process produces the singlet oxygen, which damages the target cells through direct cell destruction, injury to the vascular stro- ma, and activation of the immune system. 1 The role of PDT is to cause a cytotoxic reaction in the tissue with a delayed respon- se (post-PDT), triggering an inflammatory immune response, which is innate and adaptive, to assist in the successful eradica- tion of remaining residual cells. 8 Despite the lesser importance of the immune response compared to other effects in the cell ablation stage, this characteristic seems to be critical in maintai- ning the effects of the therapy over the long term 9 .

High-intensity PDT destroys important immune cells, while low-intensity PDT stimulates the immune system. On the one hand, this process takes place through the activation of monocytes and macrophages, and through the production of inflammatory mediators. On the other hand, it is caused by the massive attraction of neutrophils to the inflammation site, which stimulates the production of specific mediators 8, 9 .

In the treatment of tumors, the main factors that seem to be involved in the induction of that response are the expression of several cytokines and other immunologically important genes. Among the cytokines, the expression of which is repor- tedly modulated by PDT, the most important are IL6, IL10 and TNFa, in addition to IL1ß, IL2, the granulocyte colony stimu- lating factor, the epidermal growth factor, and the modulation of the expression of various genes involved in cellular adhesion and the presentation of antigens 8, 9 .

METHODS

Women (n = 13) aged 50-78 (mean = 64) were prospec- tively studied. The inclusion criteria were females with photo- types I to IV 10 and Glogau photoaging classification Grade I-IV 11 . The exclusion criteria were: use of an abrasive method or keratolytic substance in the past month, systemic diseases and recent surgery, history of keloid formation and/or hypertrophic scarring, skin cancer, personal or family history of melanoma, and use of photosensitizers or immunosuppressive medications. The patients signed a term of free and informed consent, and the study was conducted according to the Declaration of Helsinki 2000 guidelines.

The patients underwent a biopsy in the right preauricu- lar region, which was meticulously measured from the begin- ning to the end of the ear lobe and divided into 3 parts, where each biopsy was performed. The pre-procedure biopsy was car- ried out in the first region; 24 hours after the first session, a con- trol biopsy was carried out in the second region; 21 days later, a control biopsy was carried out in the third region.

Three PDT sessions were administered at 15-day inter- vals. A control picture was taken 24 hours after the first applica- tion and a biopsy was carried out 24 hours after the second application. Twenty-one days after the third session, a photo- graphic control and biopsy were carried out in the third region. The face was first cleansed with alcohol, and 20% 5-delta-ami- nolevulinic acid (ALA) was applied evenly with the Levulan® KerastickTM application stick (Laboratórios Stiefel, Guarulhos, São Paulo, Brazil). Two hours later, the ALA was irradiated with LED (light emitting diode) equipment-based light, with an out- put intensity of 3,100 mW/cm2 , an optical intensity of 100 mW/cm2 , and a 40x80 mm active surface emitting light at the 630 nm wavelength for 10 minutes on each side of the face. The patients were informed about possible side effects, instructed to use sunscreen, prohibited from using other chemical products without prior authorization, instructed not to remove peeling skin, and were required to return on preset dates.

The pre-procedure, 24 hours post-procedure and 21 days post-procedure histological samples of the patients'''''''' skin were embedded in paraffin wax and submitted to immunohis- tochemical reaction analysis using the avidin-biotin peroxidase complex method12 . This method involves incubating primary antibodies at the following dilutions: 1/20 CD1, 1/400 CD4, and 1/50 CD8 (DAKO). The other antibodies used were: 1/30 IFNg (RD Systems – Code MAB285), 1/20 IL4 (RD System – Code AF-204-NA), and 1/20 TNF (RD System – Code AF- 210-NA).

The immunohistochemistry analysis was conducted using a Zeiss light microscope with a 20x objective lens and 10x eyepiece. The quantitative evaluation (calculation of area, length, and particle count) was performed using the Image Analyzer System (Kontron Eletronic 300, Zeiss). All samples were evaluated by two independent researchers. The histo- morphometric quantitative data regarding the number of CD1 positive cells and the semi-quantitative data from the inflamma- tory response – from the CD4 and CD8 positive cells, and from the IL4, IFNg, and TNFa positive reactions – were analyzed using descriptive statistics (mean, standard deviation, minimum and maximum, and median). The statistical tests were performed using the SigmaStat software (Jandel Cientific, CA, USA), with a significance level of p < 0.05.

RESULTS

Thirteen patients with facial photodamage received treatment with PDT and topical ALA. According to Glogau''''''''s classification, 11 six patients (46.15%) (patients 1, 4, 9, 10, 11, and 12) had grade III photoaging, five (38.46%) (patients 2, 3, 5, 6, and 13) had grade IV, and two (15.38%) (patients 7 and 8) had grade II. Two patients (15.38%) (patients 2 and 5) were classi- fied as Fitspatrick phototype I, five (38.46%) (patients 1, 3, 6, 9, and 12) presented phototype II, five (38.46%) (patients 4, 7, 10, 11, and 13) had phototype III, and one (7.69%) (patient 8) pre- sented phototype IV.

The histological, immunohistochemical, and morpho- metric analyses'''''''' results of the immune system were evaluated in only 12 patients due to technical problems with the biopsy. The immunohistochemistry analysis of the histological sections for anti-CD8 revealed that CD8 positive mononuclear inflammatory cells were present around blood vessels and annexes in the dermis. (Figures 1A, 1B). The morphometric analysis was semi- quantitative, and the individual results of each patient were stu- died.

Comparing the three time points evaluated in the same treatment (before, 24 hours after, and 21 days after the third ses- sion) using the Kruskal-Wallis nonparametric statistical test of variance analysis, a significant difference was observed (H = 10.185, p = 0.006). To better specify the differences, the Dunn''''''''s multiple comparison method was applied, revealing a highly significant reduction of CD8 cells after treatment (Q = 2.68, p < 0.05) (Graph 1).

Regarding the CD4 antibody (used to demonstrate lymphocytes), the analysis of histological samples submitted to immunohistochemistry reaction to anti-CD4 showed that CD4 positive mononuclear inflammatory cells were present around blood vessels and annexes in the dermis. The morphometric analysis was semi-quantitative.

Comparing the three time points studied for the same treatment regarding CD4+ cells in the dermis, the Kruskal- Wallis nonparametric statistical test of variance analysis found no significant differences (H = 4.821, p = 0.090).

Regarding the antibody CD1 (which indicates the pre- sence of Langehrans cells), Langerhans cells were present in the skin 24 hours after the PDT procedure and after the full treat- ment course had been completed (Figures 2A, B).

The quantitative morphometric analysis was used to evaluate the number of CD1+ cells per area of epidermis. The mean values and standard deviations were: CD1before = 0.000458 ± 0.000151; CD124h = 0.000429 ± 0.000255; CD1final = 0.000934 ± 0.000810.

Comparing the three time points studied for the same treatment (before, 24 hours after, and 21 days after) for the pre- sence of CD1+ cells in the epidermis using the Kruskal-Wallis nonparametric statistical test of variance analysis, no significant differences were observed (H = 1.977, p = 0.372).

Regarding the antibody IL4 (used to demonstrate the intensity of interleukin 4), it was verified that PDT treatment promotes the presence of IL4 in the epidermis (Figure 3) and in the dermis (Figure 4).

The semi-quantitative morphometric analysis was used to evaluate the intensity of the immunohistochemical reaction in the epidermis and dermis separately. Comparing the intensi- ty of IL4 in the epidermis for the same patient for the three time points studied (before, 24 hours after the procedure, and 21 days after the treatment) using the Kruskal-Wallis nonparametric sta- tistical test of variance analysis, a significant difference was observed (H = 16.090, p < 0.001). To better specify the diffe- rences, the Dunn''''''''s multiple comparison method was applied, which revealed a highly significant increase in IL4 in the epider- mis after treatment (Q = 4.00, p < 0.05) (Graph 2).

Comparing the intensity of IL4 in the dermis for the same patient for the three time points studied (before, 24 hours after the procedure, and 21 days after the treatment) using the Kruskal-Wallis nonparametric statistical test of variance analysis, a significant difference was observed (H = 13.77, p = 0.001). To better specify the differences, the Dunn''''''''s multiple comparison method was applied, which revealed a highly significant in IL4 in the dermis after treatment (Q = 3.71, p < 0.05) (Graph 3).

Regarding the TNFa antibody, it was observed that PDT treatment reduces the amount of TNFa in the epidermis and increases its intensity in the dermis (Figure 5).

The semi-quantitative morphometric evaluation assessed the intensity of the immunohistochemical reaction in the epidermis and dermis separately. Comparing the intensity of TNFa in the dermis for the same patient for the three time points stu- died (before, 24 hours after the procedure, and 21 days after the treatment) using the Kruskal-Wallis nonparametric statistical test of variance analysis, significant differences were observed (H = 12.895, p = 0.002). To better specify the differences, the Dunn''''''''s multiple comparison method was applied, which revea- led a highly significant reduction in the intensity of TNFa in the epidermis after treatment (Q = 3.57, p <0.05) Graph 4 shows the results, represented by the mean values of each case, depicted in a box plot graph.

Comparing the intensity of TNFa in the dermis for the same patient for the three time points studied (before, 24 hours after the procedure, and 21 days after the treatment) using the Kruskal-Wallis nonparametric statistical test of variance analysis, significant differences were observed (H = 11.78, p = 0.003). To better specify the differences, the Dunn''''''''s multiple comparison method was applied, revealing a highly significant increase in the intensity of TNFa in the dermis after treatment (Q = 3.61, p <0. 05). Graph 5 shows the results, represented by the mean values of each case, depicted in a box plot graph.

Regarding IFN¿ antibody, there was no positive reac- tion in either the dermis or the epidermis at the three time points studied (Figure 5A and B).

The analysis of the Hematoxylin-Eosin. stained histolo- gical sections revealed that mononuclear inflammatory cells were present around vessels and annexes in the dermis. Comparing the intensity of the inflammatory infiltrate in the dermis for the same patient at the three time points studied (before, 24 hours after the procedure, and 21 days after the treat- ment) using the Kruskal-Wallis nonparametric statistical test of variance analysis, no significant differences were observed (H = 1.11, p = 0.572).

DISCUSSION

From the histological point of view, after 5-15 minutes of PDT, vasodilation and perivascular acute inflammation with neutrophils, eosinophils, and mastocytes takes place – neverthe- less, the tumor does not change 1, 13. One to three days after, vacuolar degeneration of the basal layer occurs with foci of epi- dermal necrosis, acute perivascular inflammation, and dispersed neutrophils and invasion of neutrophils in the epidermis. There is prominent vasodilation and edema in the dermis. Acute neu- trophilic infiltrate can be seen in the sebaceous glands, which can present degeneration of their nuclei. During this period, the tumor still does not present alterations. On the third day, there is hydropic degeneration of the basal layer and intracellular edema of keratinocytes, and the nuclei becomes hyperchroma- tic. The inflammatory infiltrate then begins to exhibit acute and subacute characteristics. On the fourth day, keratinocyte necro- sis begins to result in reticular degeneration, and tumoral necro- sis and the regeneration of sebaceous glands starts. On the seventh day, the regeneration of the skin takes place, and is com- pleted 4-6 weeks after PDT-ALA. Specific staining indicate reparation of the subepidermal zone with collagen fibers paral- lel to the epidermis, reducing the pre-existent elastotic mass 13 .

The progression of histological alterations observed in this study was similar to those reported in the literature: edema and vacuolization of the basal layer were observed after 24 hours, with the complete restructuring of the epidermis and formation of new collagen observed 21 days after the procedu- re – nonetheless, there was no significant alteration in the inflammatory infiltrate. The acute inflammation that is formed after PDT was not observed in this study, suggesting that this may occur more frequently in the treatment of tumors, where both the time of exposure to ALA and the systemic administra- tion are longer; the destruction affect of the topical therapy occurs with the formation of singlet oxygen. The immunologic markers were chosen in order to define a tumoral and cellular immunity profile for the treatment.

The study of Langerhans cells'''''''' morphology and beha- vior after thermal injury, exposure to UV and trauma caused by adhesive tapes shows that the first disappear within two days and return after 11-15 days 14-16 . Although the majority of patients presented a decrease in the number of CD1, the changes were not significant in the present study. This result is in line with those cited in the literature, in which the change in number of Langerhans cells is a function of the procedure''''''''s degree of abla- tion.

The decrease in the CD8 count 24 hours after the first session was not significant, however it became significant at 21 days after the third session. This reduction was not obtained by Abdel-Hady and others 17 , who found an increase in CD8 in treated areas that presented a positive response compared to treated areas that yielded negative results. On the other hand, Hryhorenko and colleagues 18,19 reported that PpIX accumulates in the activated lymphocytes and Gad (2001) cited Bissonete20 reported apoptosis in those cells after PDT. Grebenova and col- leagues 21 reported a 75% reduction in lymphocytes after PDT. The decrease in the CD4 cell count was not significant.

This reduction in the CD4 and CD8 cell counts suggests the apoptosis of lymphocytes following PDT, which is also reported by Grebenova 21 and Bissonette 20. Notwithstanding, these studies involve treating tumors rather than photoaging; there are no reports in the literature describing alterations in CD4 and CD8 cells in the treatment of tumors. The increase in CD8 reported by Abdel-Hady 17 may be related to the control of the cellular response in cervical intraepithelial neoplasia; the tumor loses control when the treatment is successful.

TNFa, an important cutaneous inflammation mediator, is expressed in all inflammatory processes of the skin, but parti- cularly in two types of reactions: pro-inflammatory effects and induction of death by apoptosis. Keratinocytes and dermal fibroblasts synthesize large amounts of TNFa. In the present study, the expression of TNFa suggested a significant decrease in that cytokine in the epidermis and a significant increase in the dermis. The increase in the dermis could explain the fibroblasts'''''''' stimulus, as evidenced by the increase in the collagen and elas- tic fiber fraction. Nevertheless, its reduction in the epidermis can only be justified by the increase in IL4 in the epidermis. The increase in IL4 in the dermis may have not been sufficient to inhibit fibroblasts, since this IL does not act only in the dermis, but rather in the epidermis. The CD4 and CD8 cells'''''''' apoptosis could also be related to the increase of TNFa in the dermis.

The IL4 acts on the growth and differentiation of Th2 cells. It inhibits IL1, IL6, and TNFa, and the expression of ICAM1. IL4 expression promotes the development of the Th2 response, which is related to the B cells'''''''' responses. This study demonstrated an increase in IL4 in the epidermis and dermis, suggesting a Th2-type response.

In the present study, there was no expression of IFN¿. The production of IFN¿ is restricted to the "natural killer" cell, the CD8 lymphocytes, a subset of CD4s that produces type I cytokines (Th1 cells). It modulates the immune response, acti- vates the macrophages'''''''' properties, increases the capacity of anti- gen presenting cells, and induces adhesion molecules such as ICAM1 and VCAM114-16 . An absence of IFN expression can be related to a decrease in CD4 and CD8 lymphocytes and an increase in IL4, which determines Th2-type responses (while IFN¿ determines Th1-type responses).

Further investigation of ALA-PDT-based photoaging treatment is required to better understand the modulation of the immune system.

CONCLUSIONS

Treating photoaged skin with the topical application of 20% ALA-PDT Levulan® KerastickTM (LED equipment, 630 nm wavelength, output intensity 3100 mw/cm2, optical inten- sity 100 mW/cm2, 40x80 mm active surface, 2 x 10 minutes on each side of the face) using three 20-minute sessions with 15- day intervals presented the following outcomes:

1. The Langerhans cells did not change, suggesting that the procedure caused little local immunosuppression.
2. There was a significant decrease in the CD8 popula- tion count and an insignificant decrease in the CD4 population count, suggesting the apoptosis of lymphocytes.
3. There were significant differences in the expression of TNFa, which was reduced in the epidermis and increased in the dermis; the increase in the dermis has the potential to sti- mulate fibroblasts.
4. There was a significant increase in the expression of interleukin 4 in the epidermis and dermis.
5. The IFN¿ did not present alterations in this protocol, which could be related to the lymphocytes'''''''' apoptosis.
6. The positive expression of IL4 and the negative expression of IFN suggests a Th2-type response.
7. The study''''''''s parameters – three sessions, 15-day inter- val, prior exposure time of 2 hours, 20% concentration ALA and the use of 630 nm red light – effectively treated photoaging. In addition, it was effective in stimulating the synthesis of collagen and elastic fibers, and in acting on the immune system.

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