PDT is a two-step therapeutic technique in which the topical or systemic delivery of photosensitizing drugs is followed by irradiation with light, and requires simultaneous presence of photosensitizer, light and oxygen inside the tissue. Although systemic photosensitizers are widely used for the treatment of a variety of internal cancers, topical photosensitizers are preferred in dermatology to avoid prolonged photosensitivity and the side effects like nausea and vomiting caused by a systemic photosensitizer.
5-ALA is the most popular topical photosensitizer. It is a small molecule (167.6 daltons) which diffuses most readily into cutaneous tissue from a topical delivery system. After penetration into the skin, ALA is selectively accumulated at a greater extent, and/or retained longer, by hyperproliferative tumoral and inflammatory cell populations and by sebaceous glands. This explains the therapeutic effects of PDT for tumors, inflammatory dermatoses and acne vulgaris. However, keratinocytes, the major cell type in skin, are also suggested to be a target of PDT5
ALA is not a photosensitizer by itself. However, it is metabolized to photosensitive protoporphyrin IX (PpIX) through the intrinsic cellular heme biosynthetic pathway. PpIX has a high absorption peak corresponding to the Soret band at about 405 nm and other absorption maxima - the Q-bands - at approximately 510, 545, 580 and 630 nm. Although the Q-bands are 10-20-fold smaller than the peak in the Soret band, most clinical studies have used lights corresponding to Q-bands as they allow for deeper penetration into the skin, for example 630 nm red light penetrates 5~10 mm into the skin6
. Recently, IPL, which emits a wide range of wavelengths, has been used to take advantage of the properties of various wavelengths. We used IPL, in the present study, with an emission spectrum (555~950 nm) that corresponds to Q bands.
Biologic effects after PDT can be modulated by different combinations of light and drug doses. At higher doses of one or both components of PDT, the disruption of cell membranes and organelles cause necrosis, which contributes to the formation of an inflammatory state. At intermediate combinations of light and drug, cells may undergo apoptosis. With low light and drug doses, cell viability may be maintained while other traits (signaling activity, cytokine formation, receptor expression) may be altered7
. The apoptosis-inducing action and sublethal effects of PDT typically occur within a relatively narrow range of photosensitizer concentrations. Immunomodulatory PDT utilizes apoptotic and sublethal doses and is used to treat a variety of inflammatory dermatoses. In vivo
studies have shown that PDT was effective in alleviating contact hypersensitivity (CHS) and retention of allogenic skin grafts8,9
, and also was effective in reducing disease severity when applied in different models of autoimmunity10
IL-10 is one of the regulatory cytokines that inhibit cytokine production in activated T lymphocytes and antigen-presenting cells. Epidermal keratinocytes are a major source of IL-10 in skin and its production by keratinocytes is upregulated after UV irradiation, leading to local and systemic immunosuppression. IL-10 was markedly induced in the skin of mice exposed to PDT using porfirmer and a 630 nm argon dye laser at doses that strongly inhibited the CHS. This suggests that enhanced IL-10 expression might play a role in the suppression of cell-mediated responses seen following PDT4
In this study, IL-10 protein increased in both the IPL treatment and PDT groups, with larger increases noted in the IPL treatment group. Compared to an approximately 9-fold increase of IL-10 in the skin of mice exposed to a systemic PDT regime using porfirmer and 630 nm argon dye, we observed only a 2.85-fold increase, at maximum, after PDT. This difference in magnitude of IL-10 induction may be attributable to the fact that different photosensitizers and light sources were used, and that other cells besides keratinocytes would have participated in vivo
. In addition, IL-10 was more markedly increase only after IPL treatment, which may suggest the possibility that mechanisms other than IL-10 are involved in the anti-inflammatory effect of PDT. Gollnick et al11
also suggested the implication of different mechanisms, as the ability of PDT to induce CHS suppression was sustained in IL-10 knockout mice, and after administration of anti-IL-10 antibodies.
is known to be a potent stimulus for fibrosis, which stimulates fibroblast proliferation and dramatically increases the production of collagen and other extracellular matrix proteins. It also inhibits degradation of these matrix proteins by reducing metalloproteinase synthesis. In addition, TGF-β1
is a pivotal immunosuppressive cytokine which promotes inflammation resolution. It is also intimately involved in the development of immune tolerance. Being produced by regulatory T cells, it suppresses Th1 and Th2 immune responses and is able to convert CD4+ T lymphocytes to CD25+ regulatory T cells. Besides, TGF-β1
is the most potent known inhibitor of keratinocyte proliferation, and causes practical growth arrest12
In this study, TGF-β1 slightly increased in both the IPL treatment and PDT groups, with slight larger increases in the latter group. The increase of TGF-β1 after PDT may be implicated in the beneficial effect of PDT for psoriasis and in extended engraftment of allogenic skin grafts with PDT.
TNF-α evokes two types of responses in cells: proinflammatory effects and induction of apoptotic cell death. It is an important mediator of cutaneous inflammation and is produced by keratinocytes after stimulation, such as with UV light. In previous studies, TNF-α gene transcription was found to increase in the keratinocytes of mice treated with phthalocyanine and light13
, and the expression of TNF-α protein was reported to increase up to 2.3-fold in cultured keratinocytes treated with sublethal doses of ALA and red light5
In this study, however, TNF-α mRNA and protein showed no marked change in the PDT group. The fact our findings disagree with these previous studies may be attributable to the different conditions, such as different cell types, light sources and energy, reported here.
There are some limitations of our study. Because this study was an in vitro study, the concentration of ALA and the energy of IPL used in the study cannot be applied in vivo. Moreover, normal keratinocytes and abnormal keratinocytes in diseased states may respond differently to HaCaT cells. In an in vivo condition, many other cell types, like fibroblasts, may also respond to PDT, and various cytokines other than those investigated in this study may also participate in the reaction. Thus, the actual biologic responses are difficult to predict.
In conclusion, we observed the increased expression of IL-10 and TGF-β1 in HaCaT cells after PDT. The induction of IL-10 may contribute to the anti-inflammatory effect, which explains the therapeutic benefit of PDT for inflammatory dermatoses. The induction of TGF-β1 may be related to its therapeutic effect for psoriasis. The finding that IL-10 induction was more marked after IPL treatment than after PDT suggests that some mechanism other than IL-10 induction participates in the anti-inflammatory effect of PDT. We believe that these findings may contribute to a better understanding of the immunologic effects of PDT, and that further studies, including in vivo procedures, are required.