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Influence of 5-Aminolevulinic Acid and Red Light on Collagen Metabolism of Human Dermal Fibroblasts
ORIGINAL ARTICLE
In£uence of 5-Aminolevulinic Acid and Red Light on Collagen Metabolism of Human Dermal Fibroblasts Sigrid Karrer, Anja Kathrin Bosserho¡,n Petra Weiderer, Michael Landthaler, and Rolf-Markus Szeimies Department of Dermatology, University of Regensburg, Regensburg, Germany; nInstitute of Pathology, Molecular Pathology, University of Regensburg, Regensburg, Germany
Patients with localized scleroderma receiving topical photodynamic therapy with 5-aminolevulinic acid show a reduction in skin tightness, suggesting that this therapy reduces skin sclerosis. To investigate potential mechanisms, the e¡ects of 5-aminolevulinic acid and light on collagen metabolism were studied in vitro. Normal and scleroderma ¢broblasts were treated with sublethal doses of 5-aminolevulinic acid and red light and transferred to three-dimensional collagen lattices. Cell supernatants were taken 6^72 h after photodynamic therapy to determine protein levels of the matrix metalloproteinases 1, 2, and 3, and of their inhibitors, tissue inhibitor of metalloproteinase 1 and 2 by enzymelinked immunosorbent assay. Cellular mRNA expression of these proteins and of collagen type I and III was measured by quantitative real-time polymerase chain reaction. A signi¢cant, time-dependent induction of matrix metalloproteinase 1 (up to 2.4-fold after 48 h) and matrix metalloproteinase 3 (up to 4.3-fold after 48 h) protein levels was seen after 5-aminolevulinic acidphotodynamic therapy. Irradiation with ultraviolet A light, used as a positive control, showed a similar induction of matrix metalloproteinase 1 (2.3-fold after 48 h).
The mRNA levels of matrix metalloproteinase 1 and matrix metalloproteinase 3 were signi¢cantly increased 12 h after irradiation, whereas collagen type I mRNA was strongly decreased already 6 h following irradiation. Collagen type III, tissue inhibitor of metalloproteinase 1, and matrix metalloproteinase 2 did not change after photodynamic therapy. Addition of nontoxic concentrations of sodium azide, a singlet-oxygen quencher, signi¢cantly inhibited induction of matrix metalloproteinase 1 by 5-aminolevulinic acid and light. These data show that 5-aminolevulinic acid and light induce matrix metalloproteinase 1 and matrix metalloproteinase 3 expression in normal and scleroderma ¢broblasts in a singlet oxygen-dependent way while reducing collagen type I mRNA expression. Induction of collagen-degrading enzymes together with reduction of collagen production might be responsible for the anti-sclerotic e¡ects of 5-aminolevulinic acid-photodynamic therapy observed in vivo. Key words: collagen type I/collagen type III/extracellular matrix/immunomodulation/ localized scleroderma/matrix metalloproteinase/photodynamic therapy/skin sclerosis/tissue inhibitor of metalloproteinase. J Invest Dermatol 120:325 ^331, 2003
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cells (Aveline, 2001). Photochemically generated singlet oxygen is mainly responsible for cytotoxicity induced by PDT. If targeted cells are not disintegrated, photo-oxidative stress leads to transcription and translation of various stress response and cytokine genes. Tumor necrosis factor and interleukins 1 and 6 have been shown to be induced by PDT, supporting in£ammatory action and immunologic response (Kick et al, 1995). Thus far, however, the exact underlying mechanisms of PDT regarding cellular responses and gene regulation are poorly understood. Recently, ¢ve patients with recalcitrant localized scleroderma have been successfully treated by topical PDT using 5-aminolevulinic acid. After several treatments with low doses of ALA and light a marked softening of the sclerotic plaques was observed in all patients (Karrer et al, 2000). Localized scleroderma is an in£ammatory disorder that manifests itself as excessive sclerosis of the skin. It is generally accepted that dermal ¢broblasts are the key in the pathogenesis of skin sclerosis by synthesizing increased amounts of collagen type I and III, whereas collagen degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3] are decreased (K@h@ri et al, 1988; Petersen et al, 1992; Hunzelmann et al, 1998; Hawk and English, 2001). At present, photochemotherapy using
hotodynamic therapy (PDT) is a therapeutic approach currently under investigation in clinical studies for the treatment of super¢cial skin tumors (Morton et al, 1996; Szeimies et al, 2002) and chronic in£ammatory dermatoses, e.g., psoriasis (Boehncke et al, 1994). Topical PDT using 5-aminolevulinic acid (ALA) is based on the photosensitization of diseased tissue by ALA-induced porphyrins and subsequent irradiation with red light (Kennedy and Pottier, 1992). The excitation of the photosensitizer results in the generation of reactive oxygen species, particularly singlet oxygen. Reactive oxygen species mediate cellular, e.g., lipid peroxidation, and vascular effects resulting in direct or indirect cytotoxic e¡ects on the treated Manuscript received June 20, 2002; revised July 30, 2002; accepted for publication October 1, 2002 Reprint requests to: Sigrid Karrer, MD, Department of Dermatology, University of Regensburg, 93042 Regensburg, Germany. Email: sigrid. [email protected] Abbreviations: ALA, 5-aminolevulinic acid; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; PDT, photodynamic therapy; XTT, 2,3 -bis-(2-methoxy- 4 -nitro-5-sulfenyl)-(2H)-tetrazolium5-carboxanilide assay
0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 325
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photosensitizing psoralen derivatives and ultraviolet (UV) A (PUVA) is one of the most e¡ective and widely applied therapies for localized scleroderma (Morison, 1997). PUVA, however, is associated with an increased risk of squamous cell carcinoma and malignant melanoma (Stern et al, 1997; Stern and Lunder, 1998). Recently, also UVA1 phototherapy has been shown to improve or clear localized scleroderma markedly (Stege et al, 1997). Induction of MMP is supposed to be the mechanism by which UVA light exhibits its anti-sclerotic e¡ects (Herrmann et al, 1993). MMP are a growing family of zinc-dependent endopeptidases that are characterized by their ability to degrade various extracellular matrix components (Woessner, 1991). At present, there are more than 20 human MMP described. The family includes collagenases, gelatinases, stromelysins, metalloelastases, and membrane type metalloproteinases. MMP are expressed by various cell types during the process of development, as well as during certain physiologic and pathologic processes. MMP-1 (interstitial collagenases) degrades extracellular ¢bers comprised of types I, II, III, IX, and XI collagen. MMP-2 (72 kDa gelatinase) degrades types IV, V, and VII collagen. MMP-3 (stromelysin-1) has a broad spectrum of proteolytic activity, including degradation of proteoglycans and ¢bronectin as well as native types III, IV, and V collagen. MMP are regulated by: (i) cytokines, growth factors, and cell^cell and cell^matrix interactions that control gene expression; (ii) activation of their proenzyme form; and (iii) the presence of MMP inhibitors (tissue inhibitors of metallopro-teinases: TIMP) (Raza and Cornelius, 2000). Similar to MMP, TIMP are synthesized by ordinarily resident dermal ¢broblasts. TIMP-1 binds to MMP-1 and MMP-2 and inhibits their activities forming a 1 : 1 stochiometric complex (Gomez et al, 1997). TIMP-2 preferentially binds to a distinct domain of the active site of MMP-2. Any imbalance between the expression of extracellular matrix components and their degrading factors could potentially lead to abnormal accumulation of extracellular matrix as found in scleroderma. Although UV light therapies are e¡ective in the treatment of localized scleroderma by inducing MMP, ALA-PDT has the advantage of not being carcinogenic, as the DNA is not a target for cytotoxicity in PDT (Berg, 1996). The aim of this study was to investigate the mechanisms by which ALA and light might generate its anti-sclerotic e¡ects by analyzing the expression of MMP and their counteracting inhibitors in normal human ¢broblasts and scleroderma ¢broblasts in comparison with UVA light and by investigating the e¡ect of ALA-PDT on collagen type I and type III metabolism in vitro.
MATERIALS AND METHODS Cell culture Primary normal human dermal ¢broblast strains were established by outgrowth from skin biopsies of healthy human donors or from involved skin of patients with localized scleroderma after written informed consent and local Institutional Review Board approval. A total of six cell strains (three scleroderma ¢broblasts and three adult dermal ¢broblasts) were used. The ¢broblasts were maintained in Dulbecco modi¢ed Eagle’s medium supplemented with 10% fetal bovine serum, 1% HEPES (1 mol per liter), 100 U per ml penicillin, and 100 mg streptomycin per ml, and grown as monolayers on plastic Petri dishes in a humidi¢ed atmosphere of a CO2 incubator at 371C. Fibroblast cultures were subcultured by trypsinization and used between the third and tenth passages. Treatment with ALA After the cells in monolayer culture had grown to con£uence medium was removed, serum-free medium containing ALA (75 mmol per liter; Merck AG, Darmstadt, Germany) was added, and cells were allowed to take up ALA for 24 h. The medium containing ALA was removed, the cells were rinsed and then submerged with phosphatebu¡ered saline. Irradiation was performed immediately afterwards. The light dose and ALA dose applied resulted in a 10% cell death (CLD10) compared with the untreated control as determined by a tetrazolium salt assay, sodium 30 -[1-(phenylaminocarbonyl)-3,4 -tetrazolium]-bis(4 -methoxy6 -nitro) benzene sulfonic acid hydrate (XTT test; Sigma Chemie, Deisenhofen, Germany) to measure cell viability. Addition of a singlet oxygen quencher To investigate the role of singlet oxygen, sodium azide (Sigma Chemie), a potent chemical quencher that is speci¢c for singlet oxygen, was added to the cell culture at a concentration of 50 mmol per liter in phosphate-bu¡ered saline. Sodium azide was applied to the cells after they had been allowed to take up ALA for 24 h and immediately prior to the irradiation procedure and removed again immediately after irradiation. The sodium azide dose used was not toxic to the cells as con¢rmed by the XTT test. Supernatants were taken 24 h, 48 h, and 72 h following irradiation. The control group was also treated with 50 mmol per liter sodium azide in phosphate-bu¡ered saline, but received no ALA or light. Irradiation procedure Irradiation of the cells within monolayer culture was performed using an incoherent light source with a 1200 W metal halogen lamp (PDT 1200L, Waldmann-Medizintechnik, VillingenSchwenningen, Germany, emission wavelength lem 580^740 nm), with a light intensity of 40 mW per cm2 and a £uence of 24 J per cm2 (Szeimies et al, 1994). UVA light was used as a positive control (UVA lamp 700, Waldmann-Medizintechnik, emission wavelength lem 340^460 nm, 10 J per cm2, 60 mW per cm2). Fibroblast groups Seven treatment groups were formed: (i) the ¢rst group of ¢broblasts served as a control and received neither sensitizer nor
Table I. Summary of study results
MMP-1 protein MMP-2 protein MMP-3 protein TIMP-1 protein TIMP-2 protein MMP-1 mRNA MMP-3 mRNA TIMP-1 mRNA Collagen type I mRNA Collagen type III mRNA
Normal ¢broblasts (maximal increase or decrease after PDT as compared with the untreated control)
Scleroderma ¢broblasts (maximal increase or decrease after PDT as compared with the untreated control)
* (max. after 48 h) 3 * (max. after 48 h) 3 3 * (max. after 12 h) * (max. after 12 h) 3 + (max. after 24 h) 3
* (max. after 48^72 h) 3 * (max. after 48 h) 3 3 * (max. after 12 h) * (max. after 12 h) 3 + (max. after 24 h) 3
* Signi¢cant increase after PDT as compared with untreated control. + Signi¢cant decrease after PDT as compared with untreated control. 3No signi¢cant change after PDT as compared with untreated control.
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irradiation; (ii) group 2 received ALA only; (iii) group 3 was irradiated only with red light; (iv) group 4 received ALA and red light; (v) group 5 was treated with UVA light only (positive control); (vi) group 6 received sodium azide only; and (vii) group 7 was treated with ALA and light with the addition of sodium azide prior to irradiation. Collagen lattice cultures Collagen type I from rat tail tendon was prepared and used for lattice preparation according to the following conditions: 0.45 ml of a sterile solution of type I collagen was mixed with 1.4 ml of Dulbecco modi¢ed Eagle’s medium with fetal bovine serum, and transferred into a 35 mm diameter Petri dish. Immediately after irradiation viable ¢broblasts (2.5 106 viable cells per dish) were placed in the collagen lattices and incubated for several time periods between 6 h and 72 h at 371C. Once embedded in the gel the ¢broblasts contract the collagen to form a very dense matrix. The circular shape retained during contraction and the diameter of the contracting ¢broblast populated collagen lattice was measured at each time-point and the conditioned medium surrounding the lattices was harvested from n ¼ 3 individual ¢broblast populated collagen lattices for further analysis by enzyme-linked immunosorbent assay (ELISA). Then cell viability was determined using the XTT test and cells were snap frozen in liquid nitrogen and stored at ^ 801C until required for RNA extraction and quantitative reverse transcriptase^polymerase chain reaction (reverse transcriptase^PCR).
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Protein levels of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 were measured by ELISA in cell supernatants 6, 12, 24, 48, and 72 h following treatment with ALA and light of scleroderma ¢broblasts and of normal human dermal ¢broblasts. In addition to the MMP-1 ELISA, an MMP-1 activity assay was performed. A time-dependent signi¢cant increase of protein levels of MMP-1 after PDT was detected (Fig 1A,B). MMP-1 (active and total MMP-1) gradually increased in a time-dependent manner with a maximal induction (up to 3.7-fold as compared with the control) at 48 h following PDT, the active form of MMP-1 presumably being the biologically relevant measure. There was no statistically signi¢cant di¡erence regarding the induction of MMP-1 between scleroderma and normal ¢broblasts. Irradiation of ¢broblasts with sublethal doses of UVA
Cell survival as determined by a tetrazolium salt assay (XTT) Viability of cells after ALA and/or light treatment was assessed by means of the XTT assay (Roehm et al, 1991) (Roche Diagnostics, Mannheim, Germany). All ELISA results were referred to the number of viable cells as measured by the XTT test. ELISA for MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 Human MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 ELISA kits and the monoclonal antibodies against these molecules were obtained from Amersham Pharmacia Biotech (Freiburg, Germany). For time-course experiments supernatants were collected 0, 6, 12, 24, 48, and 72 h after irradiation. The concentration of the enzymes was determined using the above-mentioned ELISA kits. To di¡erentiate between active and other forms of MMP-1 an activity assay system for MMP-1 was additionally used (Biotraks MMP-1 activity assay; Amersham Pharmacia Biotech). Reverse transcriptase^PCR analysis Total RNA was isolated from cells using the NucleoSpins RNA II kit from Macherey-Nagel (Dˇren, Germany). Total RNA was stored at 801C until use in reverse transcriptase^PCR analysis. A LightCycler assisted PCR approach was used to measure MMP-1, MMP-3, TIMP-1, collagen type I, and collagen type III mRNA. The LightCycler PCR and detection system (Roche Diagnostics, Mannheim, Germany) was used for ampli¢cation and online quanti¢cation. Speci¢c oligonucleotide primers for MMP-1, MMP-3,TIMP-1, collagen type I [a1(I) collagen], and collagen type III [a1(III) collagen] were used in PCR reactions with total cDNA from cells. b-actin served as an internal standard. Quanti¢cation was performed by online monitoring for identi¢cation of the exact time point at which the logarithmic linear phase could be distinguished from the background (crossing point). The cycle numbers of the logarithmic linear phase were plotted against the logarithm of the concentration of template DNA. Each quantitative PCR was performed at least in duplicate. Statistics Independent experiments were performed at least in triplicate. Statistical signi¢cance was evaluated in paired analyses using the Student’s paired t test or the U test (nonparametric), depending on the data distribution. Data values are expressed as mean7SEM. Statistical signi¢cance was de¢ned as a pp0.05.
RESULTS Time-dependent e¡ects of ALA and light on the MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 protein levels Constitutive MMP-1 production in the collected medium as measured by ELISA was 2.44 -fold higher for untreated normal ¢broblasts as compared with untreated scleroderma ¢broblasts (scleroderma ¢broblasts: 1.29770.24 ng per ml; normal ¢broblasts: 3.16570.49 ng per ml; per 250,000 cells, po0.05). Also constitutive MMP-3 production was 2.1-fold higher for normal ¢broblasts as compared with scleroderma ¢broblasts (po0.05).
Figure 1. Time-dependent regulation of protein levels of MMP-1. Results of the MMP-1 ELISA from cell culture supernatants of normal ¢broblasts (A) and of scleroderma ¢broblasts (B) treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and examined at di¡erent time points (6^72 h) following irradiation are shown. The x-fold change of protein levels of MMP-1 (black bars) and active MMP-1 as measured by an activity assay (dark gray bars) in cell supernatants of ¢broblasts after treatment with ALA and light are shown as compared with the untreated control (the control is set ¼ 1 at each measured time point, controls not shown). Fibroblasts were also irradiated with sublethal doses of UVA light (10 J per cm2) as a positive regulator of MMP-1 production in this cell type. The x-fold change of total MMP-1 after irradiation with UVA light is illustrated by the light gray bars. Fibroblasts treated with red light only or ALA only did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from triplicate determinations of at least three independent experiments for each case. nA signi¢cant di¡erence compared with the untreated control (po0.05).
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Figure 2. Time-dependent regulation of MMP-3 protein levels. Results of the MMP-3 ELISA from cell culture supernatants of normal ¢broblasts (black bars) and scleroderma ¢broblasts (dark gray bars) treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and examined at di¡erent time points (6^72 h) following irradiation are shown. Data represent fold increase of MMP-3 protein levels over the untreated control (control is set ¼ 1, controls not shown). Fibroblasts treated with light or ALA alone did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from triplicate determinations of at least three independent experiments for each case. There was no statistically signi¢cant di¡erence between normal and scleroderma ¢broblasts. nA signi¢cant di¡erence compared with the untreated control (po0.05).
light (10 J per cm2) used in this study as a positive regulator of MMP-1 production in this cell type, led also to a signi¢cant time-dependent increase of MMP-1 production (Fig 1A,B). There was no signi¢cant in£uence of red light alone or ALA alone on the expression of MMP-1 as compared with the untreated controls. MMP-3 protein levels showed a very similar behavior after PDT as compared with MMP-1. Also, MMP-3 increased after PDT in a time-dependent manner with a maximal induction (about 4.3fold as compared with the control) at 48 h after treatment with ALA and light (Fig 2). Red light alone or ALA alone had no in£uence on the expression of MMP-3 as compared with the untreated controls. MMP-2, TIMP-1, and TIMP-2 protein levels remained unaltered by light, ALA and, in contrast to MMP-1 and MMP-3, also after ALA-PDT (data not shown). Time-dependent e¡ects of ALA and light on MMP-1, MMP-3, TIMP-1, and collagen type I and type III mRNA levels Total RNA was isolated 0, 4, 12, and 24 h after irradiation of scleroderma and normal ¢broblasts and subjected to quantitative real-time PCR using the LightCycler. MMP-1 and MMP-3 mRNA expression was signi¢cantly increased as compared with the untreated controls and to the cells treated with ALA alone or with light alone in a time-dependent manner in response to the treatment with 75 mmol per ml ALA and red light in both normal and scleroderma ¢broblasts (Fig 3A,B). An increase occurred as soon as 4 h after irradiation and was signi¢cant 12 h after treatment. At 24 h post-PDT mRNA levels were already returning to the levels of the untreated controls. Control ¢broblasts receiving no ALA and light were often beyond detection limits. Collagen type I mRNA, measured 12, 24, and 72 h after PDT by real-time PCR, signi¢cantly decreased in normal and in scleroderma ¢broblasts after PDT showing a maximal decrease 24 h following PDT (Fig 4). TIMP-1 mRNA and collagen type III mRNA levels did not change up to 72 h following PDT (data not shown).
Figure 3. (MMP-1 (A) and MMP-3 (B) mRNA levels after quantitative real-time PCR using the LightCycler. Normal human ¢broblasts were treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and subjected to reverse transcriptase^PCR at di¡erent time points following irradiation (gray bars). Change of MMP-1 and MMP3 mRNA as compared with the untreated control (black bars) is expressed in arbitrary units, as quantitative mRNA levels of ¢broblasts prior to PDT were often beyond detection limits. Fibroblasts treated with light or ALA alone did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from duplicate determinations of three independent experiments for each case. Scleroderma ¢broblasts showed the same results as compared with normal ¢broblasts and are therefore not shown. nA signi¢cant di¡erence compared with the untreated control (po0.05).
E¡ect of sodium azide on MMP-1 induction Addition of sodium azide (50 mmol per liter) prior to irradiation led to a signi¢cant inhibition of MMP-1 increase 24, 48, and 72 h after treatment by ALA plus red light in normal and in scleroderma ¢broblasts (Fig 5A,B). Thus, the induction of MMP-1 by ALA and light could be abrogated by the addition of a singlet oxygen quencher prior to illumination. The addition of 50 mmol per liter sodium azide without ALA and light did not in£uence cell viability as measured by the XTT test nor did it result in any change of MMP-1 levels as compared with the untreated controls. DISCUSSION
In this study we present experimental evidence that ALA and light induced MMP-1 and MMP-3 synthesis in human dermal ¢broblasts cultivated within a three-dimensional collagen gel
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Figure 4. Collagen type I mRNA levels after quantitative real-time PCR using the LightCycler. Normal human ¢broblasts (black bars) and scleroderma ¢broblasts (dark gray bars) were treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and subjected to reverse transcriptase^PCR at di¡erent time points (12^72 h) following irradiation. Data represent the fold decrease of collagen type I mRNA over the untreated control (control is set ¼ 1, controls are represented by the black bars). Collagen mRNA levels were reduced signi¢cantly already 12 h following PDT showing a maximal reduction at 24 h after PDT. Fibroblasts treated with light or ALA alone did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from duplicate determinations of three independent experiments for each case. nA signi¢cant di¡erence as compared with the untreated control (po0.05).
while reducing collagen type I mRNA expression. ALA and light a¡ected the regulation of MMP at various levels, including the pretranslational level with increased amounts of MMP-1 and MMP-3 mRNA at 12 h following irradiation. ELISA showed an induction of MMP-1 and MMP-3 protein levels with a maximum at 48 h following irradiation, thus indicating that the PDT-induced speci¢c mRNA of distinct MMP are translated and actively secreted into the supernatants. There was no signi¢cant di¡erence between normal human ¢broblasts and scleroderma ¢broblasts, indicating that these responses are not speci¢c for ¢broblasts derived from patients with localized scleroderma, but occur also in normal human ¢broblasts in vitro. As scleroderma ¢broblasts exhibited more than 2-fold lower constitutive levels of MMP-1 and MMP-3 as compared with normal ¢broblasts, sublethal PDT enhanced MMP levels of scleroderma ¢broblasts to the physiologic level of normal ¢broblasts. The ¢broblast-populated collagen lattice model was used to study the behavior of normal and scleroderma ¢broblasts and their interactions with the extracellular matrix, because dermal ¢broblasts embedded in this collagen gel function in a more in vivo-like environment than do monolayer cultures (Bell et al, 1983; Mauch et al, 1988; Fertin et al, 1991; Serpier et al, 1992). Once embedded in the collagen lattice, ¢broblasts reorganize and contract the collagen ¢bers to form a very dense matrix resembling authentic connective tissue. There is a di¡erence, however, between the behavior of ¢broblasts in this rapidly remodeling culture and the behavior of ¢broblasts in dermis, so that this dynamic model cannot exactly mimic normal dermis. As MMP and TIMP production is in£uenced by cell/extracellular matrix interactions and mechanical forces (Stephens et al, 2001; Scott et al, 1998), the three-dimensional extracellular matrix environment was nevertheless considered a valuable model to study collagen metabolism of ¢broblasts after PDT. Remodeling of the extracellular matrix plays a pivotal role during many biologic and pathologic processes, as diverse as embryonal development, wound healing, tumor invasion, and
Figure 5. E¡ect of the singlet oxygen quencher sodium azide on MMP-1 induction after incubation of normal ¢broblasts (A) and scleroderma ¢broblasts (B) with ALA (75 mmol per liter) and addition of sodium azide (50 mmol per liter) prior to irradiation with red light (40 mW per cm2, 24 J per cm2). Untreated controls are set ¼ 1 (data not shown), dark gray bars show MMP-1 induction after PDT and light gray bars show MMP-1 protein levels after PDT plus addition of sodium azide during irradiation with red light. Addition of sodium azide to otherwise untreated ¢broblasts did not alter cell viability nor MMP-1 expression as compared with the untreated controls. PDT-induced MMP-1 increase was signi¢cantly inhibited by sodium azide, indicating that singlet oxygen is an important mediator of MMP-1 expression. nA signi¢cant di¡erence between ¢broblasts treated with PDT and those treated with PDT and sodium azide (po0.05).
¢brosis in scleroderma. The e¡ect of solar irradiation (315^800 nm) on the steady-state levels of the mRNA of type I and III collagens and their degrading enzymes (MMP-1 and MMP-3) were measured in human dermal ¢broblasts cultured in a threedimensional collagen gel (Le-Tallec et al, 1998). Exposure to low levels of solar irradiation (315^800 nm, 0^10 J per cm2 in the UVA doses) caused a transient decrease in type I procollagen mRNA, an increase in MMP mRNA, and no change in type III procollagen mRNA steady-state levels. These results are similar to our results with ALA and light inducing MMP-1 and MMP-3 and reducing collagen type I mRNA expression while not altering expression of collagen type III mRNA in human dermal ¢broblasts, embedded in a collagen lattice. The activity of MMP-1 is regulated by a family of TIMP, in particular TIMP-1. ALA and light upregulated MMP-1 and MMP-3 without altering TIMP-1 and TIMP-2 mRNA as well
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as secreted proteins. Obviously, the biologic e¡ect, i.e., increased matrix deposition or increased degradation, will depend on the local balance between collagen, MMP, and TIMP. The ratio of MMP-1 and TIMP-1 was clearly in favor of MMP-1 in our experiments, thus favoring collagen degradation, which is the desired e¡ect when treating skin sclerosis. Scleroderma, although uncommon, is a good model for the study of sclerosis. Previous evidence suggests that there are activated ¢broblasts in scleroderma that display abundant transcripts of collagen, whereas MMP synthesis is reduced, so the net balance in the tissue is clearly in favor of collagen deposition (K@h@ri et al, 1988; Takeda et al, 1994). UVA irradiation, in the form of bath and oral PUVA, as well as high- and low-dose UVA1 therapy, have been successfully used for the treatment of localized scleroderma (Morison, 1997; Stege et al, 1997). UVA irradiation is postulated to increase the activity of metalloproteinases, and modulate expression of cytokines that participate in connective tissue remodeling. UVA irradiation has been shown to induce MMP-1, MMP-2, and MMP-3 in cultured human ¢broblasts up to 5-fold in a co-ordinate manner, whereas TIMP-1 synthesis was not altered (Herrmann et al, 1993). Kawaguchi et al (1996) were able to demonstrate an increase of MMP-1 mRNA up to 2.3 -fold following UVA, but did not stimulate MMP-2 or TIMP-2 mRNA expression. Photosensitization of human dermal ¢broblasts with uroporphyrin followed by long-wave UV irradiation resulted in an increase of MMP-1 and MMP-3 with singlet oxygen being the major intermediate in the upregulation of MMP (Herrmann et al, 1996). As compared with UV light, red light (580^740 nm) used for PDT penetrates deeper into the dermis of the skin, making ¢broblasts a well accessible target. To compare the biologic e¡ects of UVA and of ALA-PDT in our experimental setting, ¢broblasts were also irradiated with UVA light. UVA light as well as treatment with ALA and red light resulted in a signi¢cant increase of MMP-1 protein levels. There was no signi¢cant di¡erence between the e¡ects of UVA and of ALA-PDT in vitro, suggesting that similar mechanisms (induction of interstitial collagenase) might be responsible for the anti-sclerotic e¡ects of both therapies. This theory is supported by the fact, that biologic e¡ects of PDT as well as of UVA are mediated by singlet oxygen (Bensasson et al, 1993; Morita et al, 1997). The role of singlet oxygen can be assessed by addition of a singlet oxygen quencher (Agarwal et al, 1991). In our experiments addition of nontoxic doses of the singlet oxygen quencher sodium azide during irradiation inhibited the induction of MMP-1 by ALA and light suggesting that singlet oxygen is involved in the induction of MMP-1 by ALA-PDT. Respectively, Schar¡etter-Kochanek et al (1993) and Wlaschek et al (1995) showed that singlet oxygen, generated in a dark reaction by thermodissociation of an endoperoxide, as well as UVA irradiation induced MMP-1 in human skin ¢broblasts. This e¡ect could be abolished in the presence of nontoxic doses of sodium azide. Although some biologic e¡ects of PDT as well as of UVA light are mediated by reactive oxygen species, in particular singlet oxygenRand this may be a reason for some similarities observed regarding the e¡ects on collagen metabolismRit has to be stressed that there are important di¡erences regarding the cellular responses and biologic e¡ects of UVA light and PDT. UVA produces mainly oxidative DNA damage, especially of guanine and causes DNA strand breakage, nuclear base damage, and mutations (Wang et al, 2001). Chronic exposure to UVA can promote photodamage such as premature aging (photoaging) and skin cancer (Young, 1990; Wang et al, 2001). There are no experimental nor clinical data on the e¡ects of chronic ALA-PDT on photoaging, so that it cannot be excluded that ALA-PDT might enhance photoaging. In contrast to UVA light, however, PDT using ALA is not supposed to be carcinogenic (Berg, 1996). ALA induced porphyrins, in particular protoporphyrin IX, are synthesized in the mitochondria and accumulate preferentially in plasma membranes suggesting that the DNA is not a target for cytotoxicity in PDT (Steinbach et al, 1995). ALA-PDT has even been shown to
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have an anti-carcinogenic potential by delaying UV photocarcinogenesis in hairless mice (Stender et al, 1997). Weekly systemic suberythemogenic PDT with ALA has also been able to delay the appearance of UV-induced skin tumors in mice without increasing mortality or the incidence of large tumors (Sharfaei et al, 2001). Some data suggest that reactive oxygen species are also able to decrease collagen production (Tanaka et al, 1993). Skin collagen, synthesized by ¢broblasts, comprises 80^85% of type I collagen and 10^15% of type III collagen, type I collagen being the major constituent of the interstitial connective tissue. Degradation of mature collagen ¢brils in the extracellular space requires the interstitial collagenase (MMP-1), which cleaves the interstitial collagens at a de¢ned position releasing two fragments of one and three-quarter lengths that are available to other proteases. Mempel et al (2000) found that UVA1 phototherapy resulted in a decrease of mature collagen type I and collagen type III in skin samples from irradiated patients with atopic eczema. Schar¡etter et al (1991) demonstrated that collagen type I mRNA levels remained unaltered following irradiation of human ¢broblasts with UVA light. In this study, ALA and light were able reduce collagen type I production in human dermal ¢broblasts in vitro, whereas collagen type III mRNA did not change quantitatively. At the same time MMP-1 and MMP-3, which are important for collagen degradation, were induced by ALA-PDT, whereas their counteracting inhibitors TIMP-1 and TIMP-2 were not a¡ected. Thus, an increased synthesis of MMP contributing to the dissolution of dermal collagen in conjunction with a reduced collagen synthesis probably explains the anti-sclerotic e¡ects of ALA-PDT observed in patients with localized scleroderma; however, the effects of ALA and light on MMP and collagen synthesis appear to be transient in vitro (Figs 1A^5B). This might probably correlate with the clinical observation that multiple treatments are necessary to achieve a signi¢cant improvement of sclerotic lesions in patients with localized scleroderma (Karrer et al, 2000). In addition to the e¡ects on collagen metabolism shown here, also other e¡ects of PDT on in£ammatory or immune cells may be contributing to the long-term remissions found with multiple treatments. For PDT, the occurrence of apoptosis in T lymphocytes has been documented (Hryhorenko et al, 1998; Hunt et al, 1999). Thus, activated T cells may be also valuable targets for the treatment of T cell-mediated diseases, such as psoriasis, and perhaps also in£ammatory states of localized scleroderma, with PDT. The e¡ects of low-dose ALA-PDT on T lymphocytes still remain to be investigated. This work was supported by a grant from the German scleroderma research group (Deutsche Stiftung fˇr Sklerodermie). Scleroderma ¢broblasts were kindly provided by Prof. Dr med. Rˇdiger Hein, Department of Dermatology, University of Munich.
REFERENCES Agarwal R, Athar M, Urban SA, Bickers DR, Mukhtar H: Involvement of singlet oxygen in chloroaluminum phthalocyanine tetrasulfonate-mediated photoenhancement of lipid peroxidation in rat epidermal microsomes. Cancer Lett 56:125^129, 1991 Aveline BM: Primary processes in photosensitization mechanisms. In: CalzavaraPinton P, Szeimies RM, Ortel B (eds). Photodynamic Therapy and Fluorescence Diagnosis in Dermatology. Amsterdam: Elsevier Science, 2001: pp 17^37 Bell E, Sher S, Hull B, et al: The reconstitution of living skin. J Invest Dermatol 81:2^ 10, 1983 Bensasson RV, Land EJ,Truscott TG: Excited States and Free Radicals in Biology and Medicine. Oxford: Oxford University Press, 1993 Berg K: Mechanism of cell damage in photodynamic therapy. In: H˛nigsmann H, Jori G, Young AR (eds). The Fundamental Bases of Phototherapy. Milano: OEMF Spa, 1996: pp 181^207 Boehncke WH, Sterry W, Kaufmann R: Treatment of psoriasis by topical photodynamic therapy with polychromatic light. Lancet 343:801, 1994 Fertin C, Nicolas JF, Gillery P, Kalis B, Banchereau J, Maquart FX: Interleukin- 4 stimulates collagen synthesis by normal and scleroderma ¢broblasts in dermal equivalents. Cell Mol Biol 37:823^829, 1991
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Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP: Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 74:111^122, 1997 Hawk A, English FC: Localized and systemic scleroderma. Semin Cutan Med Surg 20:27^37, 2001 Herrmann G, Wlaschek M, Lange TS, Prenzel K, Goerz G, Schar¡etter-Kochanek K: UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMP) in cultured human ¢broblasts. Exp Dermatol 2:92^97, 1993 Herrmann G, Wlaschek M, Bolsen K, Prenzel K, Goerz G, Schar¡etter-Kochanek K: Photosensitization of uroporphyrin augments the ultraviolet A-induced synthesis of matrix metalloproteinases in human dermal ¢broblasts. J Invest Dermatol 107:398^403, 1996 Hryhorenko EA, Rittenhouse-Diakun K, Harvey N, Morgan J, Steward CC, Osero¡ AR: Analysis and characterization of protoporphyrin IX accumulation in activated peripheral blood lymphocytes by four color £ow cytometry. Photochem Photobiol 67:565^572, 1998 Hunt DW, Jiang H, Granville DJ, Chan AH, Leong S, Levy JG: Consequences of the photodynamic treatment of resting and activated peripheral T lymphocytes. Immunopharmacology 41:31^44, 1999 Hunzelmann N, Schar¡etter Kochanek K, Hager C, Krieg T: Management of localised scleroderma. Semin Cutan Med Surg 17:34^40, 1998 K@h@ri VM, Sandberg M, Kalimo H, Vuorio T, Vuorio E: Identi¢cation of ¢broblasts responsible for increased collagen production in localized scleroderma by in situ hybridization. J Invest Dermatol 90:664^670, 1988 Karrer S, Abels C, Landthaler M, Szeimies RM: Topical photodynamic therapy for localized scleroderma. Acta Dermatol Venereol 80:26^27, 2000 Kawaguchi Y, Tanaka H, Okada T, Konishi H,Takahashi M, Ito M, Asai J: The e¡ects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal ¢broblasts. Arch Dermatol Res 288:39^44, 1996 Kennedy JC, Pottier RH: Endogenous protoporphyrin IX, a clinical useful photosensitizer for photodynamic therapy. J Photochem Photobiol B Biol 14:275^292, 1992 Kick G, Messer G, Goetz A, Plewig G, Kind P: Photodynamic therapy induces expression of interleukin 6 by activation of AP-1 but not NF-kappa B DNA binding. Cancer Res 55:2373^2376, 1995 Le-Tallec L, Zmimowska CK, Adolphe M: E¡ects of simulated solar radiation on type I and type III collagens, collagenase (MMP-1) and stromelysin (MMP-3) gene expression in human dermal ¢broblasts cultured in collagen gels. J Photochem Photobiol B Biol 42:226^232, 1998 Mauch C, Hatamochi A, Schar¡etter K, Krieg T: Regulation of collagen synthesis in ¢broblasts within a three-dimensional collagen gel. Exp Cell Res 178:493^503, 1988 Mempel M, Schmidt T, Boeck K, et al: Changes in collagen I and collagen III metabolism in patients with generalized atopic eczema undergoing medium-dose ultraviolet A1 phototherapy. Br J Dermatol 142:473^480, 2000 Morison WL: Psoralen UVA therapy for linear and generalized morphea. J Am Acad Dermatol 37:657^659, 1997 Morita A, Werfel T, Stege H, et al: Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultraviolet-A radiation phototherapy. J Exp Med 186:1763^1768, 1997 Morton CA, Whitehurst C, Moseley H, McColl JH, Moore JV, Mackie RM: Comparison of photodynamic therapy with cryotherapy in the treatment of Bowen’s disease. Br J Dermatol 135:766^771, 1996 Petersen MJ, Hansen C, Craig S: Ultraviolet A irradiation stimulates collagenase production in cultured human ¢broblasts. J Invest Dermatol 99:440^444, 1992 Raza SL, Cornelius LA: Matrix metalloproteinases: pro- and anti-angiogenic activities. J Invest Dermatol Symp Proc 5:47^54, 2000
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Roehm NW, Rodgers GH, Hat¢eld SM, Glasebrook AL: An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods 142:257^265, 1991 Schar¡etter K, Wlaschek M, Hogg A, et al: UVA irradiation induced collagenases in human dermal ¢broblasts in vitro and in vivo. Arch Dermatol Res 283:506^511, 1991 Schar¡etter-Kochanek K, Wlaschek M, Briviba K, Sies H: Singlet oxygen induces collagenase expression in human skin ¢broblasts. FEBS Lett 331:304^306, 1993 Scott KA, Wood EJ, Karran EH: A matrix metalloproteinase inhibitor which prevents ¢broblast-mediated collagen lattice contraction. FEBS Lett 441:137^140, 1998 Serpier H, Gillery P, Polette M, et al: Modulation of the organization of the extracellular matrix and the production of collagen by interferon gamma in threedimensional cultures of normal and sclerodermic ¢broblasts. Pathol Biol 40:865^870, 1992 Sharfaei S, Viau G, Lui H, Bou¡ard D, Bissonnette R: Systemic photodynamic therapy with aminolevulinic acid delays the appearance of ultraviolet-induced skin tumours in mice. Br J Dermatol 144:1207^1214, 2001 Stege H, Berneburg M, Humke S, et al: High-dose UVA1 radiation therapy for localized scleroderma. J Am Acad Dermatol 36:938^944, 1997 Steinbach P, Weingandt H, Baumgartner R, Kriegmaier M, Hofst@dter F, Knˇchel R: Cellular £uorescence of the endogenous photosensitizer protoporphyrin IX following exposure to 5-aminolevulinic acid. Photochem Photobiol 62:887^ 895, 1995 Stender IM, Bech-Thomsen N, Poulsen T, Wulf HC: Photodynamic therapy with topical d-aminolevulinic acid delays UV photocarcinogenesis in hairless mice. Photochem Photobiol 66:493^496, 1997 Stephens P, Davies KJ, Occleston N, et al: Skin and oral ¢broblasts exhibit phenotypic di¡erences in extracellular matrix reorganization and matrix metalloproteinases activity. Br J Dermatol 144:229^237, 2001 Stern RS, Lunder EJ: Risk of squamous cell carcinoma and methoxsalen (psoralen) and UV-A radiation (PUVA). A meta-analysis. Arch Dermatol 134:1582^1585, 1998 Stern RS, Nichols KT, V@kev@ LH: Malignant melanoma in patients treated for psoriasis with methoxsalen (psoralen) and ultraviolet A radiation (PUVA). N Engl J Med 336:1041^1045, 1997 Szeimies RM, Hein R, B@umler W, Heine A, Landthaler M: A possible new incoherent lamp for photodynamic treatment of super¢cial skin lesions. Acta Derm Venereol (Stockh) 74:117^119, 1994 Szeimes RM, Karrer S, Radakovic-Fijan S, et al: Photodynamic therapy using topical 5-aminolevulinate compared with cryotherapy for actinic keratosis: A prospective, randomized study. Am Acad Dermatol 47:258^262, 2002 Takeda K, Hatamochi A, Ueki H, Nakata M, Oishi Y: Decreased collagenase expression in cultured systemic sclerosis ¢broblasts. J Invest Dermatol 103:359^363, 1994 Tanaka H, Okada T, Konishi H, Tsuji T: The e¡ect of reactive oxygen species on the biosynthesis of collagen and glycosaminoglycans in cultured human dermal ¢broblasts. Arch Dermatol Res 285:352^355, 1993 Wang SQ, Setlow R, Berwick M, Polsky D, Marghoob AA, Kopf AW, Bart RS: Ultraviolet A and melanoma: a review. J Am Acad Dermatol 44:837^846, 2001 Wlaschek M, Briviba K, Stricklin GP, Sies H, Schar¡etter-Kochanek K: Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase. J Invest Dermatol 104:194^198, 1995 Woessner JF: Matrix metalloproteinases and their inhibitors in connective tissue remodelling. FASEB J 5:2145^2154, 1991 Young AR: Cumulative e¡ects of ultraviolet radiation on the skin; cancer and photoaging. Semin Dermatol 9:21^31, 1990
In£uence of 5-Aminolevulinic Acid and Red Light on Collagen Metabolism of Human Dermal Fibroblasts Sigrid Karrer, Anja Kathrin Bosserho¡,n Petra Weiderer, Michael Landthaler, and Rolf-Markus Szeimies Department of Dermatology, University of Regensburg, Regensburg, Germany; nInstitute of Pathology, Molecular Pathology, University of Regensburg, Regensburg, Germany
Patients with localized scleroderma receiving topical photodynamic therapy with 5-aminolevulinic acid show a reduction in skin tightness, suggesting that this therapy reduces skin sclerosis. To investigate potential mechanisms, the e¡ects of 5-aminolevulinic acid and light on collagen metabolism were studied in vitro. Normal and scleroderma ¢broblasts were treated with sublethal doses of 5-aminolevulinic acid and red light and transferred to three-dimensional collagen lattices. Cell supernatants were taken 6^72 h after photodynamic therapy to determine protein levels of the matrix metalloproteinases 1, 2, and 3, and of their inhibitors, tissue inhibitor of metalloproteinase 1 and 2 by enzymelinked immunosorbent assay. Cellular mRNA expression of these proteins and of collagen type I and III was measured by quantitative real-time polymerase chain reaction. A signi¢cant, time-dependent induction of matrix metalloproteinase 1 (up to 2.4-fold after 48 h) and matrix metalloproteinase 3 (up to 4.3-fold after 48 h) protein levels was seen after 5-aminolevulinic acidphotodynamic therapy. Irradiation with ultraviolet A light, used as a positive control, showed a similar induction of matrix metalloproteinase 1 (2.3-fold after 48 h).
The mRNA levels of matrix metalloproteinase 1 and matrix metalloproteinase 3 were signi¢cantly increased 12 h after irradiation, whereas collagen type I mRNA was strongly decreased already 6 h following irradiation. Collagen type III, tissue inhibitor of metalloproteinase 1, and matrix metalloproteinase 2 did not change after photodynamic therapy. Addition of nontoxic concentrations of sodium azide, a singlet-oxygen quencher, signi¢cantly inhibited induction of matrix metalloproteinase 1 by 5-aminolevulinic acid and light. These data show that 5-aminolevulinic acid and light induce matrix metalloproteinase 1 and matrix metalloproteinase 3 expression in normal and scleroderma ¢broblasts in a singlet oxygen-dependent way while reducing collagen type I mRNA expression. Induction of collagen-degrading enzymes together with reduction of collagen production might be responsible for the anti-sclerotic e¡ects of 5-aminolevulinic acid-photodynamic therapy observed in vivo. Key words: collagen type I/collagen type III/extracellular matrix/immunomodulation/ localized scleroderma/matrix metalloproteinase/photodynamic therapy/skin sclerosis/tissue inhibitor of metalloproteinase. J Invest Dermatol 120:325 ^331, 2003
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cells (Aveline, 2001). Photochemically generated singlet oxygen is mainly responsible for cytotoxicity induced by PDT. If targeted cells are not disintegrated, photo-oxidative stress leads to transcription and translation of various stress response and cytokine genes. Tumor necrosis factor and interleukins 1 and 6 have been shown to be induced by PDT, supporting in£ammatory action and immunologic response (Kick et al, 1995). Thus far, however, the exact underlying mechanisms of PDT regarding cellular responses and gene regulation are poorly understood. Recently, ¢ve patients with recalcitrant localized scleroderma have been successfully treated by topical PDT using 5-aminolevulinic acid. After several treatments with low doses of ALA and light a marked softening of the sclerotic plaques was observed in all patients (Karrer et al, 2000). Localized scleroderma is an in£ammatory disorder that manifests itself as excessive sclerosis of the skin. It is generally accepted that dermal ¢broblasts are the key in the pathogenesis of skin sclerosis by synthesizing increased amounts of collagen type I and III, whereas collagen degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3] are decreased (K@h@ri et al, 1988; Petersen et al, 1992; Hunzelmann et al, 1998; Hawk and English, 2001). At present, photochemotherapy using
hotodynamic therapy (PDT) is a therapeutic approach currently under investigation in clinical studies for the treatment of super¢cial skin tumors (Morton et al, 1996; Szeimies et al, 2002) and chronic in£ammatory dermatoses, e.g., psoriasis (Boehncke et al, 1994). Topical PDT using 5-aminolevulinic acid (ALA) is based on the photosensitization of diseased tissue by ALA-induced porphyrins and subsequent irradiation with red light (Kennedy and Pottier, 1992). The excitation of the photosensitizer results in the generation of reactive oxygen species, particularly singlet oxygen. Reactive oxygen species mediate cellular, e.g., lipid peroxidation, and vascular effects resulting in direct or indirect cytotoxic e¡ects on the treated Manuscript received June 20, 2002; revised July 30, 2002; accepted for publication October 1, 2002 Reprint requests to: Sigrid Karrer, MD, Department of Dermatology, University of Regensburg, 93042 Regensburg, Germany. Email: sigrid. [email protected] Abbreviations: ALA, 5-aminolevulinic acid; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; PDT, photodynamic therapy; XTT, 2,3 -bis-(2-methoxy- 4 -nitro-5-sulfenyl)-(2H)-tetrazolium5-carboxanilide assay
0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 325
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photosensitizing psoralen derivatives and ultraviolet (UV) A (PUVA) is one of the most e¡ective and widely applied therapies for localized scleroderma (Morison, 1997). PUVA, however, is associated with an increased risk of squamous cell carcinoma and malignant melanoma (Stern et al, 1997; Stern and Lunder, 1998). Recently, also UVA1 phototherapy has been shown to improve or clear localized scleroderma markedly (Stege et al, 1997). Induction of MMP is supposed to be the mechanism by which UVA light exhibits its anti-sclerotic e¡ects (Herrmann et al, 1993). MMP are a growing family of zinc-dependent endopeptidases that are characterized by their ability to degrade various extracellular matrix components (Woessner, 1991). At present, there are more than 20 human MMP described. The family includes collagenases, gelatinases, stromelysins, metalloelastases, and membrane type metalloproteinases. MMP are expressed by various cell types during the process of development, as well as during certain physiologic and pathologic processes. MMP-1 (interstitial collagenases) degrades extracellular ¢bers comprised of types I, II, III, IX, and XI collagen. MMP-2 (72 kDa gelatinase) degrades types IV, V, and VII collagen. MMP-3 (stromelysin-1) has a broad spectrum of proteolytic activity, including degradation of proteoglycans and ¢bronectin as well as native types III, IV, and V collagen. MMP are regulated by: (i) cytokines, growth factors, and cell^cell and cell^matrix interactions that control gene expression; (ii) activation of their proenzyme form; and (iii) the presence of MMP inhibitors (tissue inhibitors of metallopro-teinases: TIMP) (Raza and Cornelius, 2000). Similar to MMP, TIMP are synthesized by ordinarily resident dermal ¢broblasts. TIMP-1 binds to MMP-1 and MMP-2 and inhibits their activities forming a 1 : 1 stochiometric complex (Gomez et al, 1997). TIMP-2 preferentially binds to a distinct domain of the active site of MMP-2. Any imbalance between the expression of extracellular matrix components and their degrading factors could potentially lead to abnormal accumulation of extracellular matrix as found in scleroderma. Although UV light therapies are e¡ective in the treatment of localized scleroderma by inducing MMP, ALA-PDT has the advantage of not being carcinogenic, as the DNA is not a target for cytotoxicity in PDT (Berg, 1996). The aim of this study was to investigate the mechanisms by which ALA and light might generate its anti-sclerotic e¡ects by analyzing the expression of MMP and their counteracting inhibitors in normal human ¢broblasts and scleroderma ¢broblasts in comparison with UVA light and by investigating the e¡ect of ALA-PDT on collagen type I and type III metabolism in vitro.
MATERIALS AND METHODS Cell culture Primary normal human dermal ¢broblast strains were established by outgrowth from skin biopsies of healthy human donors or from involved skin of patients with localized scleroderma after written informed consent and local Institutional Review Board approval. A total of six cell strains (three scleroderma ¢broblasts and three adult dermal ¢broblasts) were used. The ¢broblasts were maintained in Dulbecco modi¢ed Eagle’s medium supplemented with 10% fetal bovine serum, 1% HEPES (1 mol per liter), 100 U per ml penicillin, and 100 mg streptomycin per ml, and grown as monolayers on plastic Petri dishes in a humidi¢ed atmosphere of a CO2 incubator at 371C. Fibroblast cultures were subcultured by trypsinization and used between the third and tenth passages. Treatment with ALA After the cells in monolayer culture had grown to con£uence medium was removed, serum-free medium containing ALA (75 mmol per liter; Merck AG, Darmstadt, Germany) was added, and cells were allowed to take up ALA for 24 h. The medium containing ALA was removed, the cells were rinsed and then submerged with phosphatebu¡ered saline. Irradiation was performed immediately afterwards. The light dose and ALA dose applied resulted in a 10% cell death (CLD10) compared with the untreated control as determined by a tetrazolium salt assay, sodium 30 -[1-(phenylaminocarbonyl)-3,4 -tetrazolium]-bis(4 -methoxy6 -nitro) benzene sulfonic acid hydrate (XTT test; Sigma Chemie, Deisenhofen, Germany) to measure cell viability. Addition of a singlet oxygen quencher To investigate the role of singlet oxygen, sodium azide (Sigma Chemie), a potent chemical quencher that is speci¢c for singlet oxygen, was added to the cell culture at a concentration of 50 mmol per liter in phosphate-bu¡ered saline. Sodium azide was applied to the cells after they had been allowed to take up ALA for 24 h and immediately prior to the irradiation procedure and removed again immediately after irradiation. The sodium azide dose used was not toxic to the cells as con¢rmed by the XTT test. Supernatants were taken 24 h, 48 h, and 72 h following irradiation. The control group was also treated with 50 mmol per liter sodium azide in phosphate-bu¡ered saline, but received no ALA or light. Irradiation procedure Irradiation of the cells within monolayer culture was performed using an incoherent light source with a 1200 W metal halogen lamp (PDT 1200L, Waldmann-Medizintechnik, VillingenSchwenningen, Germany, emission wavelength lem 580^740 nm), with a light intensity of 40 mW per cm2 and a £uence of 24 J per cm2 (Szeimies et al, 1994). UVA light was used as a positive control (UVA lamp 700, Waldmann-Medizintechnik, emission wavelength lem 340^460 nm, 10 J per cm2, 60 mW per cm2). Fibroblast groups Seven treatment groups were formed: (i) the ¢rst group of ¢broblasts served as a control and received neither sensitizer nor
Table I. Summary of study results
MMP-1 protein MMP-2 protein MMP-3 protein TIMP-1 protein TIMP-2 protein MMP-1 mRNA MMP-3 mRNA TIMP-1 mRNA Collagen type I mRNA Collagen type III mRNA
Normal ¢broblasts (maximal increase or decrease after PDT as compared with the untreated control)
Scleroderma ¢broblasts (maximal increase or decrease after PDT as compared with the untreated control)
* (max. after 48 h) 3 * (max. after 48 h) 3 3 * (max. after 12 h) * (max. after 12 h) 3 + (max. after 24 h) 3
* (max. after 48^72 h) 3 * (max. after 48 h) 3 3 * (max. after 12 h) * (max. after 12 h) 3 + (max. after 24 h) 3
* Signi¢cant increase after PDT as compared with untreated control. + Signi¢cant decrease after PDT as compared with untreated control. 3No signi¢cant change after PDT as compared with untreated control.
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irradiation; (ii) group 2 received ALA only; (iii) group 3 was irradiated only with red light; (iv) group 4 received ALA and red light; (v) group 5 was treated with UVA light only (positive control); (vi) group 6 received sodium azide only; and (vii) group 7 was treated with ALA and light with the addition of sodium azide prior to irradiation. Collagen lattice cultures Collagen type I from rat tail tendon was prepared and used for lattice preparation according to the following conditions: 0.45 ml of a sterile solution of type I collagen was mixed with 1.4 ml of Dulbecco modi¢ed Eagle’s medium with fetal bovine serum, and transferred into a 35 mm diameter Petri dish. Immediately after irradiation viable ¢broblasts (2.5 106 viable cells per dish) were placed in the collagen lattices and incubated for several time periods between 6 h and 72 h at 371C. Once embedded in the gel the ¢broblasts contract the collagen to form a very dense matrix. The circular shape retained during contraction and the diameter of the contracting ¢broblast populated collagen lattice was measured at each time-point and the conditioned medium surrounding the lattices was harvested from n ¼ 3 individual ¢broblast populated collagen lattices for further analysis by enzyme-linked immunosorbent assay (ELISA). Then cell viability was determined using the XTT test and cells were snap frozen in liquid nitrogen and stored at ^ 801C until required for RNA extraction and quantitative reverse transcriptase^polymerase chain reaction (reverse transcriptase^PCR).
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Protein levels of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 were measured by ELISA in cell supernatants 6, 12, 24, 48, and 72 h following treatment with ALA and light of scleroderma ¢broblasts and of normal human dermal ¢broblasts. In addition to the MMP-1 ELISA, an MMP-1 activity assay was performed. A time-dependent signi¢cant increase of protein levels of MMP-1 after PDT was detected (Fig 1A,B). MMP-1 (active and total MMP-1) gradually increased in a time-dependent manner with a maximal induction (up to 3.7-fold as compared with the control) at 48 h following PDT, the active form of MMP-1 presumably being the biologically relevant measure. There was no statistically signi¢cant di¡erence regarding the induction of MMP-1 between scleroderma and normal ¢broblasts. Irradiation of ¢broblasts with sublethal doses of UVA
Cell survival as determined by a tetrazolium salt assay (XTT) Viability of cells after ALA and/or light treatment was assessed by means of the XTT assay (Roehm et al, 1991) (Roche Diagnostics, Mannheim, Germany). All ELISA results were referred to the number of viable cells as measured by the XTT test. ELISA for MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 Human MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 ELISA kits and the monoclonal antibodies against these molecules were obtained from Amersham Pharmacia Biotech (Freiburg, Germany). For time-course experiments supernatants were collected 0, 6, 12, 24, 48, and 72 h after irradiation. The concentration of the enzymes was determined using the above-mentioned ELISA kits. To di¡erentiate between active and other forms of MMP-1 an activity assay system for MMP-1 was additionally used (Biotraks MMP-1 activity assay; Amersham Pharmacia Biotech). Reverse transcriptase^PCR analysis Total RNA was isolated from cells using the NucleoSpins RNA II kit from Macherey-Nagel (Dˇren, Germany). Total RNA was stored at 801C until use in reverse transcriptase^PCR analysis. A LightCycler assisted PCR approach was used to measure MMP-1, MMP-3, TIMP-1, collagen type I, and collagen type III mRNA. The LightCycler PCR and detection system (Roche Diagnostics, Mannheim, Germany) was used for ampli¢cation and online quanti¢cation. Speci¢c oligonucleotide primers for MMP-1, MMP-3,TIMP-1, collagen type I [a1(I) collagen], and collagen type III [a1(III) collagen] were used in PCR reactions with total cDNA from cells. b-actin served as an internal standard. Quanti¢cation was performed by online monitoring for identi¢cation of the exact time point at which the logarithmic linear phase could be distinguished from the background (crossing point). The cycle numbers of the logarithmic linear phase were plotted against the logarithm of the concentration of template DNA. Each quantitative PCR was performed at least in duplicate. Statistics Independent experiments were performed at least in triplicate. Statistical signi¢cance was evaluated in paired analyses using the Student’s paired t test or the U test (nonparametric), depending on the data distribution. Data values are expressed as mean7SEM. Statistical signi¢cance was de¢ned as a pp0.05.
RESULTS Time-dependent e¡ects of ALA and light on the MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 protein levels Constitutive MMP-1 production in the collected medium as measured by ELISA was 2.44 -fold higher for untreated normal ¢broblasts as compared with untreated scleroderma ¢broblasts (scleroderma ¢broblasts: 1.29770.24 ng per ml; normal ¢broblasts: 3.16570.49 ng per ml; per 250,000 cells, po0.05). Also constitutive MMP-3 production was 2.1-fold higher for normal ¢broblasts as compared with scleroderma ¢broblasts (po0.05).
Figure 1. Time-dependent regulation of protein levels of MMP-1. Results of the MMP-1 ELISA from cell culture supernatants of normal ¢broblasts (A) and of scleroderma ¢broblasts (B) treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and examined at di¡erent time points (6^72 h) following irradiation are shown. The x-fold change of protein levels of MMP-1 (black bars) and active MMP-1 as measured by an activity assay (dark gray bars) in cell supernatants of ¢broblasts after treatment with ALA and light are shown as compared with the untreated control (the control is set ¼ 1 at each measured time point, controls not shown). Fibroblasts were also irradiated with sublethal doses of UVA light (10 J per cm2) as a positive regulator of MMP-1 production in this cell type. The x-fold change of total MMP-1 after irradiation with UVA light is illustrated by the light gray bars. Fibroblasts treated with red light only or ALA only did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from triplicate determinations of at least three independent experiments for each case. nA signi¢cant di¡erence compared with the untreated control (po0.05).
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Figure 2. Time-dependent regulation of MMP-3 protein levels. Results of the MMP-3 ELISA from cell culture supernatants of normal ¢broblasts (black bars) and scleroderma ¢broblasts (dark gray bars) treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and examined at di¡erent time points (6^72 h) following irradiation are shown. Data represent fold increase of MMP-3 protein levels over the untreated control (control is set ¼ 1, controls not shown). Fibroblasts treated with light or ALA alone did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from triplicate determinations of at least three independent experiments for each case. There was no statistically signi¢cant di¡erence between normal and scleroderma ¢broblasts. nA signi¢cant di¡erence compared with the untreated control (po0.05).
light (10 J per cm2) used in this study as a positive regulator of MMP-1 production in this cell type, led also to a signi¢cant time-dependent increase of MMP-1 production (Fig 1A,B). There was no signi¢cant in£uence of red light alone or ALA alone on the expression of MMP-1 as compared with the untreated controls. MMP-3 protein levels showed a very similar behavior after PDT as compared with MMP-1. Also, MMP-3 increased after PDT in a time-dependent manner with a maximal induction (about 4.3fold as compared with the control) at 48 h after treatment with ALA and light (Fig 2). Red light alone or ALA alone had no in£uence on the expression of MMP-3 as compared with the untreated controls. MMP-2, TIMP-1, and TIMP-2 protein levels remained unaltered by light, ALA and, in contrast to MMP-1 and MMP-3, also after ALA-PDT (data not shown). Time-dependent e¡ects of ALA and light on MMP-1, MMP-3, TIMP-1, and collagen type I and type III mRNA levels Total RNA was isolated 0, 4, 12, and 24 h after irradiation of scleroderma and normal ¢broblasts and subjected to quantitative real-time PCR using the LightCycler. MMP-1 and MMP-3 mRNA expression was signi¢cantly increased as compared with the untreated controls and to the cells treated with ALA alone or with light alone in a time-dependent manner in response to the treatment with 75 mmol per ml ALA and red light in both normal and scleroderma ¢broblasts (Fig 3A,B). An increase occurred as soon as 4 h after irradiation and was signi¢cant 12 h after treatment. At 24 h post-PDT mRNA levels were already returning to the levels of the untreated controls. Control ¢broblasts receiving no ALA and light were often beyond detection limits. Collagen type I mRNA, measured 12, 24, and 72 h after PDT by real-time PCR, signi¢cantly decreased in normal and in scleroderma ¢broblasts after PDT showing a maximal decrease 24 h following PDT (Fig 4). TIMP-1 mRNA and collagen type III mRNA levels did not change up to 72 h following PDT (data not shown).
Figure 3. (MMP-1 (A) and MMP-3 (B) mRNA levels after quantitative real-time PCR using the LightCycler. Normal human ¢broblasts were treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and subjected to reverse transcriptase^PCR at di¡erent time points following irradiation (gray bars). Change of MMP-1 and MMP3 mRNA as compared with the untreated control (black bars) is expressed in arbitrary units, as quantitative mRNA levels of ¢broblasts prior to PDT were often beyond detection limits. Fibroblasts treated with light or ALA alone did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from duplicate determinations of three independent experiments for each case. Scleroderma ¢broblasts showed the same results as compared with normal ¢broblasts and are therefore not shown. nA signi¢cant di¡erence compared with the untreated control (po0.05).
E¡ect of sodium azide on MMP-1 induction Addition of sodium azide (50 mmol per liter) prior to irradiation led to a signi¢cant inhibition of MMP-1 increase 24, 48, and 72 h after treatment by ALA plus red light in normal and in scleroderma ¢broblasts (Fig 5A,B). Thus, the induction of MMP-1 by ALA and light could be abrogated by the addition of a singlet oxygen quencher prior to illumination. The addition of 50 mmol per liter sodium azide without ALA and light did not in£uence cell viability as measured by the XTT test nor did it result in any change of MMP-1 levels as compared with the untreated controls. DISCUSSION
In this study we present experimental evidence that ALA and light induced MMP-1 and MMP-3 synthesis in human dermal ¢broblasts cultivated within a three-dimensional collagen gel
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Figure 4. Collagen type I mRNA levels after quantitative real-time PCR using the LightCycler. Normal human ¢broblasts (black bars) and scleroderma ¢broblasts (dark gray bars) were treated with ALA (75 mmol per liter) and light (40 mW per cm2, 24 J per cm2) and subjected to reverse transcriptase^PCR at di¡erent time points (12^72 h) following irradiation. Data represent the fold decrease of collagen type I mRNA over the untreated control (control is set ¼ 1, controls are represented by the black bars). Collagen mRNA levels were reduced signi¢cantly already 12 h following PDT showing a maximal reduction at 24 h after PDT. Fibroblasts treated with light or ALA alone did not show statistically signi¢cant changes as compared with the untreated controls and are therefore not shown. Data are mean7SEM from duplicate determinations of three independent experiments for each case. nA signi¢cant di¡erence as compared with the untreated control (po0.05).
while reducing collagen type I mRNA expression. ALA and light a¡ected the regulation of MMP at various levels, including the pretranslational level with increased amounts of MMP-1 and MMP-3 mRNA at 12 h following irradiation. ELISA showed an induction of MMP-1 and MMP-3 protein levels with a maximum at 48 h following irradiation, thus indicating that the PDT-induced speci¢c mRNA of distinct MMP are translated and actively secreted into the supernatants. There was no signi¢cant di¡erence between normal human ¢broblasts and scleroderma ¢broblasts, indicating that these responses are not speci¢c for ¢broblasts derived from patients with localized scleroderma, but occur also in normal human ¢broblasts in vitro. As scleroderma ¢broblasts exhibited more than 2-fold lower constitutive levels of MMP-1 and MMP-3 as compared with normal ¢broblasts, sublethal PDT enhanced MMP levels of scleroderma ¢broblasts to the physiologic level of normal ¢broblasts. The ¢broblast-populated collagen lattice model was used to study the behavior of normal and scleroderma ¢broblasts and their interactions with the extracellular matrix, because dermal ¢broblasts embedded in this collagen gel function in a more in vivo-like environment than do monolayer cultures (Bell et al, 1983; Mauch et al, 1988; Fertin et al, 1991; Serpier et al, 1992). Once embedded in the collagen lattice, ¢broblasts reorganize and contract the collagen ¢bers to form a very dense matrix resembling authentic connective tissue. There is a di¡erence, however, between the behavior of ¢broblasts in this rapidly remodeling culture and the behavior of ¢broblasts in dermis, so that this dynamic model cannot exactly mimic normal dermis. As MMP and TIMP production is in£uenced by cell/extracellular matrix interactions and mechanical forces (Stephens et al, 2001; Scott et al, 1998), the three-dimensional extracellular matrix environment was nevertheless considered a valuable model to study collagen metabolism of ¢broblasts after PDT. Remodeling of the extracellular matrix plays a pivotal role during many biologic and pathologic processes, as diverse as embryonal development, wound healing, tumor invasion, and
Figure 5. E¡ect of the singlet oxygen quencher sodium azide on MMP-1 induction after incubation of normal ¢broblasts (A) and scleroderma ¢broblasts (B) with ALA (75 mmol per liter) and addition of sodium azide (50 mmol per liter) prior to irradiation with red light (40 mW per cm2, 24 J per cm2). Untreated controls are set ¼ 1 (data not shown), dark gray bars show MMP-1 induction after PDT and light gray bars show MMP-1 protein levels after PDT plus addition of sodium azide during irradiation with red light. Addition of sodium azide to otherwise untreated ¢broblasts did not alter cell viability nor MMP-1 expression as compared with the untreated controls. PDT-induced MMP-1 increase was signi¢cantly inhibited by sodium azide, indicating that singlet oxygen is an important mediator of MMP-1 expression. nA signi¢cant di¡erence between ¢broblasts treated with PDT and those treated with PDT and sodium azide (po0.05).
¢brosis in scleroderma. The e¡ect of solar irradiation (315^800 nm) on the steady-state levels of the mRNA of type I and III collagens and their degrading enzymes (MMP-1 and MMP-3) were measured in human dermal ¢broblasts cultured in a threedimensional collagen gel (Le-Tallec et al, 1998). Exposure to low levels of solar irradiation (315^800 nm, 0^10 J per cm2 in the UVA doses) caused a transient decrease in type I procollagen mRNA, an increase in MMP mRNA, and no change in type III procollagen mRNA steady-state levels. These results are similar to our results with ALA and light inducing MMP-1 and MMP-3 and reducing collagen type I mRNA expression while not altering expression of collagen type III mRNA in human dermal ¢broblasts, embedded in a collagen lattice. The activity of MMP-1 is regulated by a family of TIMP, in particular TIMP-1. ALA and light upregulated MMP-1 and MMP-3 without altering TIMP-1 and TIMP-2 mRNA as well
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as secreted proteins. Obviously, the biologic e¡ect, i.e., increased matrix deposition or increased degradation, will depend on the local balance between collagen, MMP, and TIMP. The ratio of MMP-1 and TIMP-1 was clearly in favor of MMP-1 in our experiments, thus favoring collagen degradation, which is the desired e¡ect when treating skin sclerosis. Scleroderma, although uncommon, is a good model for the study of sclerosis. Previous evidence suggests that there are activated ¢broblasts in scleroderma that display abundant transcripts of collagen, whereas MMP synthesis is reduced, so the net balance in the tissue is clearly in favor of collagen deposition (K@h@ri et al, 1988; Takeda et al, 1994). UVA irradiation, in the form of bath and oral PUVA, as well as high- and low-dose UVA1 therapy, have been successfully used for the treatment of localized scleroderma (Morison, 1997; Stege et al, 1997). UVA irradiation is postulated to increase the activity of metalloproteinases, and modulate expression of cytokines that participate in connective tissue remodeling. UVA irradiation has been shown to induce MMP-1, MMP-2, and MMP-3 in cultured human ¢broblasts up to 5-fold in a co-ordinate manner, whereas TIMP-1 synthesis was not altered (Herrmann et al, 1993). Kawaguchi et al (1996) were able to demonstrate an increase of MMP-1 mRNA up to 2.3 -fold following UVA, but did not stimulate MMP-2 or TIMP-2 mRNA expression. Photosensitization of human dermal ¢broblasts with uroporphyrin followed by long-wave UV irradiation resulted in an increase of MMP-1 and MMP-3 with singlet oxygen being the major intermediate in the upregulation of MMP (Herrmann et al, 1996). As compared with UV light, red light (580^740 nm) used for PDT penetrates deeper into the dermis of the skin, making ¢broblasts a well accessible target. To compare the biologic e¡ects of UVA and of ALA-PDT in our experimental setting, ¢broblasts were also irradiated with UVA light. UVA light as well as treatment with ALA and red light resulted in a signi¢cant increase of MMP-1 protein levels. There was no signi¢cant di¡erence between the e¡ects of UVA and of ALA-PDT in vitro, suggesting that similar mechanisms (induction of interstitial collagenase) might be responsible for the anti-sclerotic e¡ects of both therapies. This theory is supported by the fact, that biologic e¡ects of PDT as well as of UVA are mediated by singlet oxygen (Bensasson et al, 1993; Morita et al, 1997). The role of singlet oxygen can be assessed by addition of a singlet oxygen quencher (Agarwal et al, 1991). In our experiments addition of nontoxic doses of the singlet oxygen quencher sodium azide during irradiation inhibited the induction of MMP-1 by ALA and light suggesting that singlet oxygen is involved in the induction of MMP-1 by ALA-PDT. Respectively, Schar¡etter-Kochanek et al (1993) and Wlaschek et al (1995) showed that singlet oxygen, generated in a dark reaction by thermodissociation of an endoperoxide, as well as UVA irradiation induced MMP-1 in human skin ¢broblasts. This e¡ect could be abolished in the presence of nontoxic doses of sodium azide. Although some biologic e¡ects of PDT as well as of UVA light are mediated by reactive oxygen species, in particular singlet oxygenRand this may be a reason for some similarities observed regarding the e¡ects on collagen metabolismRit has to be stressed that there are important di¡erences regarding the cellular responses and biologic e¡ects of UVA light and PDT. UVA produces mainly oxidative DNA damage, especially of guanine and causes DNA strand breakage, nuclear base damage, and mutations (Wang et al, 2001). Chronic exposure to UVA can promote photodamage such as premature aging (photoaging) and skin cancer (Young, 1990; Wang et al, 2001). There are no experimental nor clinical data on the e¡ects of chronic ALA-PDT on photoaging, so that it cannot be excluded that ALA-PDT might enhance photoaging. In contrast to UVA light, however, PDT using ALA is not supposed to be carcinogenic (Berg, 1996). ALA induced porphyrins, in particular protoporphyrin IX, are synthesized in the mitochondria and accumulate preferentially in plasma membranes suggesting that the DNA is not a target for cytotoxicity in PDT (Steinbach et al, 1995). ALA-PDT has even been shown to
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have an anti-carcinogenic potential by delaying UV photocarcinogenesis in hairless mice (Stender et al, 1997). Weekly systemic suberythemogenic PDT with ALA has also been able to delay the appearance of UV-induced skin tumors in mice without increasing mortality or the incidence of large tumors (Sharfaei et al, 2001). Some data suggest that reactive oxygen species are also able to decrease collagen production (Tanaka et al, 1993). Skin collagen, synthesized by ¢broblasts, comprises 80^85% of type I collagen and 10^15% of type III collagen, type I collagen being the major constituent of the interstitial connective tissue. Degradation of mature collagen ¢brils in the extracellular space requires the interstitial collagenase (MMP-1), which cleaves the interstitial collagens at a de¢ned position releasing two fragments of one and three-quarter lengths that are available to other proteases. Mempel et al (2000) found that UVA1 phototherapy resulted in a decrease of mature collagen type I and collagen type III in skin samples from irradiated patients with atopic eczema. Schar¡etter et al (1991) demonstrated that collagen type I mRNA levels remained unaltered following irradiation of human ¢broblasts with UVA light. In this study, ALA and light were able reduce collagen type I production in human dermal ¢broblasts in vitro, whereas collagen type III mRNA did not change quantitatively. At the same time MMP-1 and MMP-3, which are important for collagen degradation, were induced by ALA-PDT, whereas their counteracting inhibitors TIMP-1 and TIMP-2 were not a¡ected. Thus, an increased synthesis of MMP contributing to the dissolution of dermal collagen in conjunction with a reduced collagen synthesis probably explains the anti-sclerotic e¡ects of ALA-PDT observed in patients with localized scleroderma; however, the effects of ALA and light on MMP and collagen synthesis appear to be transient in vitro (Figs 1A^5B). This might probably correlate with the clinical observation that multiple treatments are necessary to achieve a signi¢cant improvement of sclerotic lesions in patients with localized scleroderma (Karrer et al, 2000). In addition to the e¡ects on collagen metabolism shown here, also other e¡ects of PDT on in£ammatory or immune cells may be contributing to the long-term remissions found with multiple treatments. For PDT, the occurrence of apoptosis in T lymphocytes has been documented (Hryhorenko et al, 1998; Hunt et al, 1999). Thus, activated T cells may be also valuable targets for the treatment of T cell-mediated diseases, such as psoriasis, and perhaps also in£ammatory states of localized scleroderma, with PDT. The e¡ects of low-dose ALA-PDT on T lymphocytes still remain to be investigated. This work was supported by a grant from the German scleroderma research group (Deutsche Stiftung fˇr Sklerodermie). Scleroderma ¢broblasts were kindly provided by Prof. Dr med. Rˇdiger Hein, Department of Dermatology, University of Munich.
REFERENCES Agarwal R, Athar M, Urban SA, Bickers DR, Mukhtar H: Involvement of singlet oxygen in chloroaluminum phthalocyanine tetrasulfonate-mediated photoenhancement of lipid peroxidation in rat epidermal microsomes. Cancer Lett 56:125^129, 1991 Aveline BM: Primary processes in photosensitization mechanisms. In: CalzavaraPinton P, Szeimies RM, Ortel B (eds). Photodynamic Therapy and Fluorescence Diagnosis in Dermatology. Amsterdam: Elsevier Science, 2001: pp 17^37 Bell E, Sher S, Hull B, et al: The reconstitution of living skin. J Invest Dermatol 81:2^ 10, 1983 Bensasson RV, Land EJ,Truscott TG: Excited States and Free Radicals in Biology and Medicine. Oxford: Oxford University Press, 1993 Berg K: Mechanism of cell damage in photodynamic therapy. In: H˛nigsmann H, Jori G, Young AR (eds). The Fundamental Bases of Phototherapy. Milano: OEMF Spa, 1996: pp 181^207 Boehncke WH, Sterry W, Kaufmann R: Treatment of psoriasis by topical photodynamic therapy with polychromatic light. Lancet 343:801, 1994 Fertin C, Nicolas JF, Gillery P, Kalis B, Banchereau J, Maquart FX: Interleukin- 4 stimulates collagen synthesis by normal and scleroderma ¢broblasts in dermal equivalents. Cell Mol Biol 37:823^829, 1991
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Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP: Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 74:111^122, 1997 Hawk A, English FC: Localized and systemic scleroderma. Semin Cutan Med Surg 20:27^37, 2001 Herrmann G, Wlaschek M, Lange TS, Prenzel K, Goerz G, Schar¡etter-Kochanek K: UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMP) in cultured human ¢broblasts. Exp Dermatol 2:92^97, 1993 Herrmann G, Wlaschek M, Bolsen K, Prenzel K, Goerz G, Schar¡etter-Kochanek K: Photosensitization of uroporphyrin augments the ultraviolet A-induced synthesis of matrix metalloproteinases in human dermal ¢broblasts. J Invest Dermatol 107:398^403, 1996 Hryhorenko EA, Rittenhouse-Diakun K, Harvey N, Morgan J, Steward CC, Osero¡ AR: Analysis and characterization of protoporphyrin IX accumulation in activated peripheral blood lymphocytes by four color £ow cytometry. Photochem Photobiol 67:565^572, 1998 Hunt DW, Jiang H, Granville DJ, Chan AH, Leong S, Levy JG: Consequences of the photodynamic treatment of resting and activated peripheral T lymphocytes. Immunopharmacology 41:31^44, 1999 Hunzelmann N, Schar¡etter Kochanek K, Hager C, Krieg T: Management of localised scleroderma. Semin Cutan Med Surg 17:34^40, 1998 K@h@ri VM, Sandberg M, Kalimo H, Vuorio T, Vuorio E: Identi¢cation of ¢broblasts responsible for increased collagen production in localized scleroderma by in situ hybridization. J Invest Dermatol 90:664^670, 1988 Karrer S, Abels C, Landthaler M, Szeimies RM: Topical photodynamic therapy for localized scleroderma. Acta Dermatol Venereol 80:26^27, 2000 Kawaguchi Y, Tanaka H, Okada T, Konishi H,Takahashi M, Ito M, Asai J: The e¡ects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal ¢broblasts. Arch Dermatol Res 288:39^44, 1996 Kennedy JC, Pottier RH: Endogenous protoporphyrin IX, a clinical useful photosensitizer for photodynamic therapy. J Photochem Photobiol B Biol 14:275^292, 1992 Kick G, Messer G, Goetz A, Plewig G, Kind P: Photodynamic therapy induces expression of interleukin 6 by activation of AP-1 but not NF-kappa B DNA binding. Cancer Res 55:2373^2376, 1995 Le-Tallec L, Zmimowska CK, Adolphe M: E¡ects of simulated solar radiation on type I and type III collagens, collagenase (MMP-1) and stromelysin (MMP-3) gene expression in human dermal ¢broblasts cultured in collagen gels. J Photochem Photobiol B Biol 42:226^232, 1998 Mauch C, Hatamochi A, Schar¡etter K, Krieg T: Regulation of collagen synthesis in ¢broblasts within a three-dimensional collagen gel. Exp Cell Res 178:493^503, 1988 Mempel M, Schmidt T, Boeck K, et al: Changes in collagen I and collagen III metabolism in patients with generalized atopic eczema undergoing medium-dose ultraviolet A1 phototherapy. Br J Dermatol 142:473^480, 2000 Morison WL: Psoralen UVA therapy for linear and generalized morphea. J Am Acad Dermatol 37:657^659, 1997 Morita A, Werfel T, Stege H, et al: Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultraviolet-A radiation phototherapy. J Exp Med 186:1763^1768, 1997 Morton CA, Whitehurst C, Moseley H, McColl JH, Moore JV, Mackie RM: Comparison of photodynamic therapy with cryotherapy in the treatment of Bowen’s disease. Br J Dermatol 135:766^771, 1996 Petersen MJ, Hansen C, Craig S: Ultraviolet A irradiation stimulates collagenase production in cultured human ¢broblasts. J Invest Dermatol 99:440^444, 1992 Raza SL, Cornelius LA: Matrix metalloproteinases: pro- and anti-angiogenic activities. J Invest Dermatol Symp Proc 5:47^54, 2000
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331
Roehm NW, Rodgers GH, Hat¢eld SM, Glasebrook AL: An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods 142:257^265, 1991 Schar¡etter K, Wlaschek M, Hogg A, et al: UVA irradiation induced collagenases in human dermal ¢broblasts in vitro and in vivo. Arch Dermatol Res 283:506^511, 1991 Schar¡etter-Kochanek K, Wlaschek M, Briviba K, Sies H: Singlet oxygen induces collagenase expression in human skin ¢broblasts. FEBS Lett 331:304^306, 1993 Scott KA, Wood EJ, Karran EH: A matrix metalloproteinase inhibitor which prevents ¢broblast-mediated collagen lattice contraction. FEBS Lett 441:137^140, 1998 Serpier H, Gillery P, Polette M, et al: Modulation of the organization of the extracellular matrix and the production of collagen by interferon gamma in threedimensional cultures of normal and sclerodermic ¢broblasts. Pathol Biol 40:865^870, 1992 Sharfaei S, Viau G, Lui H, Bou¡ard D, Bissonnette R: Systemic photodynamic therapy with aminolevulinic acid delays the appearance of ultraviolet-induced skin tumours in mice. Br J Dermatol 144:1207^1214, 2001 Stege H, Berneburg M, Humke S, et al: High-dose UVA1 radiation therapy for localized scleroderma. J Am Acad Dermatol 36:938^944, 1997 Steinbach P, Weingandt H, Baumgartner R, Kriegmaier M, Hofst@dter F, Knˇchel R: Cellular £uorescence of the endogenous photosensitizer protoporphyrin IX following exposure to 5-aminolevulinic acid. Photochem Photobiol 62:887^ 895, 1995 Stender IM, Bech-Thomsen N, Poulsen T, Wulf HC: Photodynamic therapy with topical d-aminolevulinic acid delays UV photocarcinogenesis in hairless mice. Photochem Photobiol 66:493^496, 1997 Stephens P, Davies KJ, Occleston N, et al: Skin and oral ¢broblasts exhibit phenotypic di¡erences in extracellular matrix reorganization and matrix metalloproteinases activity. Br J Dermatol 144:229^237, 2001 Stern RS, Lunder EJ: Risk of squamous cell carcinoma and methoxsalen (psoralen) and UV-A radiation (PUVA). A meta-analysis. Arch Dermatol 134:1582^1585, 1998 Stern RS, Nichols KT, V@kev@ LH: Malignant melanoma in patients treated for psoriasis with methoxsalen (psoralen) and ultraviolet A radiation (PUVA). N Engl J Med 336:1041^1045, 1997 Szeimies RM, Hein R, B@umler W, Heine A, Landthaler M: A possible new incoherent lamp for photodynamic treatment of super¢cial skin lesions. Acta Derm Venereol (Stockh) 74:117^119, 1994 Szeimes RM, Karrer S, Radakovic-Fijan S, et al: Photodynamic therapy using topical 5-aminolevulinate compared with cryotherapy for actinic keratosis: A prospective, randomized study. Am Acad Dermatol 47:258^262, 2002 Takeda K, Hatamochi A, Ueki H, Nakata M, Oishi Y: Decreased collagenase expression in cultured systemic sclerosis ¢broblasts. J Invest Dermatol 103:359^363, 1994 Tanaka H, Okada T, Konishi H, Tsuji T: The e¡ect of reactive oxygen species on the biosynthesis of collagen and glycosaminoglycans in cultured human dermal ¢broblasts. Arch Dermatol Res 285:352^355, 1993 Wang SQ, Setlow R, Berwick M, Polsky D, Marghoob AA, Kopf AW, Bart RS: Ultraviolet A and melanoma: a review. J Am Acad Dermatol 44:837^846, 2001 Wlaschek M, Briviba K, Stricklin GP, Sies H, Schar¡etter-Kochanek K: Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase. J Invest Dermatol 104:194^198, 1995 Woessner JF: Matrix metalloproteinases and their inhibitors in connective tissue remodelling. FASEB J 5:2145^2154, 1991 Young AR: Cumulative e¡ects of ultraviolet radiation on the skin; cancer and photoaging. Semin Dermatol 9:21^31, 1990