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Tirilazad amelioriates extracellular effects of photooxidative stress by sealing the membrane of UVA irradiated human dermal fibroblasts

European Journal of Dermatology. Volume 16, Number 5, 530-6, September-October 2006, Investigative report

DOI : 10.1684/ejd.2006.0026

Summary

Author(s) : Lars Alexander Schneider, Joachim Dissemond, Edith Schwamborn, Meinhard Wlaschek, Peter Brenneisen, Karin Scharffetter-Kochanek , Department of Dermatology and Allergology, University of Ulm, Maienweg 12, D-89081 Ulm, Germany, Department of Dermatology and Allergology, University of Essen, Germany, Department of Biochemistry & Molecular Biology I, University of Duesseldorf, Germany, Department of Dermatology, University of Cologne, Germany, Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany.

Summary : The evaluation of antioxidant medication might provide further tools to protect the skin against the detrimental effects of photooxidative stress. In this context we have previously shown that the lazaroid tirilazad protects fibroblasts effectively against lipid peroxidation (LPO). Now we investigated whether and how tirilazad also influences two typical stress responses after UVA exposure, i.e. IL-6 and collagenase (MMP-1) release. Fibroblasts pre-incubated with tirilazad at a concentration of 30 μM show significantly less IL-6 in the extracellular medium after UVA exposure. Correspondingly, pre-incubation with tirilazad also significantly diminishes the extracellular MMP-1 protein concentration 24h post-irradiation. These effects observed are due to a membrane stabilisation, as tirilazad neither diminishes IL-6 mRNA production nor intracellular IL-6/MMP-1 protein levels after UVA exposure and thus most likely acts by sealing off the cell, delaying the typical leakage of IL-6 and MMP-1.

Keywords : UVA, lazaroid, tirilazad, IL-6, MMP-1, collagenase, fibroblast

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ARTICLE

Auteur(s) : Lars Alexander Schneider^1,4, Joachim Dissemond², Edith Schwamborn^4,5, Meinhard Wlaschek^1,4, Peter Brenneisen^3,4, Karin Scharffetter-Kochanek^1,4

¹Department of Dermatology and Allergology, University of Ulm, Maienweg 12, D-89081 Ulm, Germany
²Department of Dermatology and Allergology, University of Essen, Germany
³Department of Biochemistry & Molecular Biology I, University of Duesseldorf, Germany
⁴Department of Dermatology, University of Cologne, Germany
⁵Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany

accepté le 1 Mai 2006

Ambient UV radiation, to which our skin is daily exposed, consists of over 90% UVA. With its penetrating capacity into the dermis, UVA creates reactive oxygen species (ROS) and photooxidative stress, defined as an imbalance between generation of ROS and existing antioxidative protection [1]. One major result of photooxidative stress in dermal fibroblasts is membrane damage caused by lipid peroxidation (LPO) [2-4]. In biological membranes with a high content of polyunsatured fatty acids, LPO results in destruction of membrane structure and function, disturbed membrane fluidity, protein modifications, perforation of the cell membrane and finally even lysis of cells [5, 6]. This is one pathway via which long term effects of UVA such as photocarcinogenesis, immunosuppression and photoaging are promoted [7, 8]. We have recently shown that the lazaroid tirilazad is able to inhibit UVA mediated LPO in human dermal fibroblasts [9].The effects of tirilazad are permitted by the intercalation of the lipophilic part into the phospholipid-bilayer of cellular membranes. Unspecific physical and chemical attributes are changed and through a loss of fluidity a stabilisation of the membrane occurs. This very effectively terminates the LPO chain reaction [10]. Apart from this stabilising effect tirilazad also acts as an antioxidant by quenching hydroxyl- and peroxyl radicals and keeps the vitamin E content in the cell membranes constant [11]. Despite being a 21-amino-steroid-molecule, tirilazad does not exert the typical genomic effects of mineral- or corticosteroids, like modulation of cytokine production, protein synthesis and immunosuppression [11]. Lazaroids are thus non-hormonal 21-aminosteroids.Since tirilazad diminishes UVA induced LPO and has a further potential as an antioxidant we aimed to evaluate its possible effect on the increase of typical markers for acute photooxidative stress in human dermal fibroblasts. Such a typical photooxidative stress response is the release of inflammatory cytokines like interleukin-6 (IL-6). Another typical effect causing solar scarring and photoaging in the long term is the release of matrix-degrading enzymes like matrix-metalloproteinase-1 (MMP-1) [12, 13]. Both of these acute markers of stress are furthermore linked as UVA and UVB induce MMP-1 production and -enzymatic activity in the skin via autocrine and paracrine loops involving IL-6 [14-16].It has recently been shown that the induction of MMP-1 is partly linked with LPO as well, since LPO induced by UVA radiation leads to activation of the transcription factor NFκB, which then mediates IL-6 induction [17]. In summary, there is a distinct rationale behind the investigation of the possible effects tirilazad might have against the acute UVA stress response, i.e. release of IL-6 respectively MMP-1 into the extracellular space.

Materials and methods

Chemicals

All chemicals were obtained from Sigma (Deisenhofen, FRG) unless otherwise stated and were of HPLC grade.

Cell culture

Dermal foreskin fibroblasts were cultured after outgrowth from skin biopsies of healthy human donors aged 3-6 years. The fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO-BRL, Eggenstein, FRG) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, FRG), glutamine (2 mM), penicillin (400 U/mL), streptomycin (50 μg/mL), and grown on plastic dishes in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C [18]. Prior to UVA irradiation, confluent fibroblasts were either pre-incubated 24 h in medium with non-toxic concentrations of the lazaroid tirilazad mesylate (30 μmol/L) or mock treated as control fibroblasts.

Fibroblast gels

Collagen type-I from rat tails (Sigma Aldrich) was solved in 0.1% acetic acid over night and mixed with 0.1% NaOH, FCS, DMEM and cultured fibroblasts at a density of 10⁵ cells per dish, layered out as collagen-fibroblast gel and left to contract for 4 days covered by medium. Prior to UVA irradiation, gels were either pre-incubated 24 h with 30 μM/L lazaroid tirilazad mesylate or mock treated as controls. UVA Irradiation was performed as described below.

Tirilazad

The molecular structure of tirilazad is shown in ( figure 1 ). Tirilazad is a member of a new class of synthetic non-hormonal (nonglucocorticoid) 21-aminosteroids and was available and distributed under the brand name Freedox^® by Pharmacia, Kalamazoo,USA before this company was merged with Pfizer. The commercially available Freedox^® solution contained 1.5 mg tirilazad mesylate, equivalent to 1.3 mg tirilazad free base, per ml of the raw solution plus citrate buffer, sodium chloride and water for injection. For our investigations Freedox^® solution was used exclusively. The concentration of 30 μM used in our experiments was selected on the basis of a toxicity analysis in which fibroblasts were incubated with a range of different tirilazad concentrations and exposed to UVA as described below (data not shown). Afterwards the cell survival rate was tested with the MTT test described below. Concentrations up to 50 μM still resulted in a cell survival rate of greater than 80%. However, as we wanted to stay well above this cut-off margin we chose 30 μM as the working concentration of tirilazad for the experiments as it resulted in a cell survival rate of > 90%.

UVA irradiation

Confluent fibroblast monolayer cultures or fibroblast gels were irradiated at a distance of 40 cm using a high-intensity halogen metalloid UVA-source (UVASUN3000) Mutzhas, Munich, FRG). Spectral irradiance was measured routinely with an OL-754 UV/visible light spectroradiometer calibrated against the OL-752-10 spectral irradiance standard certified by the National Institute of Standards and Technology (Optronic Laboratories, Orlando, FL). The irradiation intensity at the surface of the fibroblasts was 60 mW/second. Irradiation fluence was monitored with a UVA ultraviolet-dosimeter. During irradiation, cells/gels were incubated in phosphate-buffered saline (PBS) and maintained at 37 °C in a thermostatically controlled water bath. Following irradiation, PBS was replaced by fresh DMEM with supplements.

IL-6 ELISA

Post UVA exposure fibroblasts were kept in FCS free medium. For extracellular IL-6 measurement cell culture supernatant was harvested at the desired time point, centrifuged for 5 min at 10,000 rpm and used directly as sample in a commercially available IL-6 ELISA kit (Pelikine kit by CLB company Amsterdam, NL) according to the manufacturer’s instructions. For determination of intracellular IL-6 levels cell lysate samples were generated by washing the cells with 0.9% saline, adding 0.3% lysis buffer, scraping the fibroblasts off into 3 mL medium and homogenising by ultrasound. In all cases total protein content of fibroblasts in the relevant culture dish was determined by an assay (Bio-Rad, Munich, FRG) according to Bradford [19]. ELISA results obtained in ng IL-6 per ml sample fluid were then related to total protein and expressed as percent of corresponding control values set to 100%.

MMP-1 and Tissue inhibitor of MMP-1 (TIMP-1) ELISA with Cell Supernatant

Post UVA exposure fibroblasts in cell cultures were kept in FCS-free medium for 24 h as FCS is known to alter MMP-1 expression. For determination of extracellular MMP-1/TIMP-1 (tissue inhibitor of metalloproteinases-1) levels, supernatant was then harvested, centrifuged for 5 min at 10,000 rpm and used as sample in commercially available ELISA systems. MMP-1 and TIMP-1“sandwich” ELISA assays were performed according to the manufacturer’s protocols (Amersham). Again, in all cases total protein content of fibroblasts in the relevant culture dish was determined by an assay (Bio-Rad, Munich, FRG) according to Bradford [19]. ELISA results obtained in ng MMP-1/TIMP-1 protein per mL sample fluid were then related to total protein and expressed as percent of corresponding control values set to 100%.The MMP-1 ELISA system measures total MMP-1 as well as pro MMP-1 and active MMP-1.

MMP-1 ELISA with lysed fibroblasts from collagen gels

In case of the collagen gels cell lysate was used as sample for the MMP-1 ELISA. This lysate was generated by taking off the supernatant, washing once in cold PBS and sonicating the gel with 200 μL PBS on ice. After centrifugation the clear lysate supernatant was then used as samples. The ELISA procedure was the same as described above. ELISA results were here also related to the total protein content of fibroblasts in the gel dish.

IL-6 mRNA Northern blot analysis

At two different time points after UVA exposure total RNA was isolated from cultured fibroblasts using the Trizol method. Then equal amounts of RNA were fractionated by size on an agarose gel. After diffusion transfer to nitrocellulose filters and vacuum baking (Schleicher & Schuell, Dassel, Germany), hybridizations were performed and studied by Northern blot analysis using a cDNA probe for IL-6 and 18S ribosomal RNA (as a control). Specific bands were normalised against the 18S ribosomal bands and subsequent densitometry, i.e. grey value measurement with Adobe Photoshop 5.5 Software was performed.

Cell Viability assay (MTT-test)

The viability of the fibroblasts was monitored 24h after tirilazad supplementation, ± exposure to UVA. This was done by means of the MTT test as described by Moosmann [20]. We used non-cytotoxic concentrations and set the cut off for tirilazad pre-incubation plus UVA exposure to a minimum of 90% cell survival.

Statistical analysis

Statistical analysis was performed on an exploratory basis only using GraphPad Prism version 3.0a (GraphPad Software, San Diego California, USA). Values of p < 0.05 were considered to be significant. Each of the different subgroups was described by mean and standard deviation. Multi group comparisons of unpaired observations were subjected to an ANOVA analysis.

Results

Tirilazad reduces extracellular IL-6 concentration post UVA exposure

In order to evaluate the effect of tirilazad against UVA induced release of IL-6 human dermal fibroblasts were incubated for 24 h with 30 μmol/L tirilazad and exposed to a single dose of 200 kJ/m² UVA. The non-toxicity of this experimental setting was controlled by the MTT test as described in materials and methods. The cell culture supernatant was harvested at time points 2-24 h post-irradiation and the IL-6 concentration in it was determined and related to the total protein content of the culture dish. ( Figure 2 ) shows the results from n = 3 independent experiments. For each time point the IL-6 concentration in the supernatant is expressed in % of the value for the corresponding mock-irradiated (± tirilazad pre-incubated) control fibroblasts of the same time-point. Fibroblasts without tirilazad pre-incubation produced markedly increased extracellular IL-6 levels with a peak at 12 h post-irradiation reaching then a level of 700% compared to mock treated control fibroblasts. In contrast tirilazad pre-incubated fibroblasts showed a clearly diminished increase of IL-6 that also peaked at 12 h post-irradiation but reached only values of 280% of mock treated control fibroblasts. This difference between the tirilazad treated and untreated fibroblasts was significant in an exploratory ANOVA analysis at time points from 3, 6 and 12 h post-irradiation.

Tirilazad does not reduce IL-6 mRNA post UVA irradiation

In order to investigate whether the decreased IL-6 level in the extracellular space of tirilazad pre-incubated fibroblasts would result from an inhibition of IL-6 production by tirilazad on the transcriptional level, we next looked at IL-6 mRNA production post-irradiation with and without tirilazad pre-incubation. For this approach RNA was isolated from fibroblasts irradiated with 200 kJ/m² UVA with and without tirilazad pre-incubation at the two time points 3 h and 6 h post-irradiation, i.e. in the time frame of a steep-but-not-yet-peaking IL-6 increase in the supernatant. Mock-irradiated fibroblasts were used as controls. ( Figure 3A ) shows one representative northern blot and ( figure 3B ) the results of the densitometric analysis of n = 3 blots. As a reference control 18S RNA was used in the blots. The 18S RNA bands of all probes gave similar grey values in the densitometric analysis indicating a comparable loading of the samples (data not shown). As to the IL-6 mRNA levels, a stronger signal was observed at 3 h in all irradiated and at 6 h only in the irradiated and tirilazad pre-incubated fibroblasts compared to the mock-irradiated control fibroblasts. Furthermore it is evident that at both time points the pre-incubated tirilazad revealed increased IL-6 mRNA levels compared to the fibroblasts that were simply irradiated. Finally tirilazad pre-incubation without UVA exposure did not increase the IL-6 RNA level compared to mock irradiation. Densitometry analysis revealed then that the actual increase in specific IL-6 mRNA signal at 3 h reached 130% of the mock treated control fibroblasts (= 100%) for fibroblasts without and 151% for fibroblasts with tirilazad pre-incubation. The 6h value for tirilazad pre-incubated fibroblasts was 130% of control fibroblasts whilst irradiated fibroblasts without tirilazad had a signal back in the range of mock irradiated controls (= 100%). Statistically the difference in signal strength for IL-6 mRNA between tirilazad pre-incubation and wihtout tirilazad exposure was not significant. However, the IL-6 production on mRNA level is visibly enhanced by tirilazad pre-incubation, which indicates that the decrease in extracellular IL-6 concentration, which we observed with tirilazad, can only result from an effect on a different step in IL-6 regulation than mRNA levels.

Tirilazad leads to IL-6 accumulation in the cell preventing leakage

As the decreased IL-6 concentration in the fibroblast supernatant that tirilazad achieves, did not result from an inhibition of IL-6 genome transcription it could be due to a “tyre-fitting”-effect of tirilazad in the cell membrane keeping the newly produced IL-6 from leaking out. This hypothesis was investigated further. For that purpose we irradiated fibroblasts with 200 kJ/m² again with and without pre-incubation with 30 μmol/L tirilazad and analysed this time the amount of IL-6 in the fibroblasts 3 h and 24 h post-irradiation. The results were calculated in relation to the total protein content of the fibroblasts. ( Figure 4 ) summarises the results of n = 3 independent experiments. We found, 3 h post-irradiation, in tirilazad pre-incubated and irradiated fibroblasts, an excessively high level of IL-6 of 2700% the value of mock-treated control fibroblasts, whilst lysate from UVA exposed fibroblasts without tirilazad showed IL-6 levels of “only” 745% of control fibroblasts. The huge difference in IL-6 levels found between these two groups of fibroblasts reached statistical significance in an exploratory multiple group comparison analysis with ANOVA.

Comparing these excessive levels with the values present in the fibroblasts 24 h post-irradiation it becomes evident that in both tirilazad pre-incubated and control fibroblasts, the IL-6 content in the cells had gone down significantly to 200% in tirilazad pre-incubated fibroblasts and 150% in just irradiated fibroblasts, again in relation to mock treated controls. This decline in intracellular IL-6 levels from 3 to 24 h post-irradiation going along with the observed increase of IL-6 in the supernatant over the same period, suggests that the accumulated IL-6 has been leaking out between the two time points examined.

Tirilazad inhibits the UVA induced extracellular MMP-1 increase in fibroblasts post UVA exposure resulting in a lower MMP-1/TIMP-1 ratio

Having demonstrated a significant effect of tirilazad on IL-6 secretion, we next addressed the question whether this substance might also be able to decrease the extracellular MMP-1 concentration following UVA irradiation of fibroblasts. Fibroblasts were therefore pre-incubated again with 30 μmol/L tirilazad for 24 h and exposed to 200 kJ/m²UVA. Mock-irradiated and pre-incubated fibroblasts served as controls. The concentrations of MMP-1 and its tissue inhibitor TIMP-1 in the supernatant were determined 24 h post-irradiation. This time point is known to represent the maximum increase of MMP-1 activity and protein in vivo post irradiation [13]. Figure 5 shows the results of n = 3 independent experiments. The basic level of MMP-1/TIMP-1 in mock treated control fibroblasts was again set to 100%. After UVA exposure untreated and irradiated fibroblasts revealed an increase to 580% of MMP-1 in the supernatant, whilst TIMP-1 levels were left unchanged. The tirilazad pre-incubated fibroblasts revealed a reduced 200% MMP-1 induction in relation to controls with a TIMP-1 level decreasing to 70% of controls. The basic 1:1 ratio of MMP-1/TIMP-1 in mock treated controls was calculated to increase to 4.61:1 after UVA exposure without tirilazad pre-incubation and 2.87:1 in the presence of tirilazad. The difference in the increase of MMP-1 between simply irradiated and tirilazad pre-incubated and irradiated fibroblasts reaches statistical significance in an exploratory multiple group comparison analysis (ANOVA).

Tirilazad does not diminish the intracellular MMP-1 protein concentration after UVA irradiation

Since the decreased MMP-1 levels in the extracellular space that tirilazad achieved could again, as with IL-6, be due to a membrane sealing effect or due to a reduced intracellular production of IL-6, we next addressed this final question. For this purpose we opted for a model of fibroblasts seeded in collagen gels, i.e. closer to an in vivo setting. Fibroblasts seeded in collagen gels were pre-incubated with 30 μmol/L tirilazad for 24 h and then exposed to 200 kJ/m²UVA. Mock-irradiated fibroblast-gels with and without tirilazad pre-incubation served as controls. The concentration of MMP-1 protein was then determined in the lysed fibroblasts from the gels 24 h post-irradiation, i.e. at the same time point as when we found a decreased extracellular level under the influence of tirilazad before. The results from n = 4 independent experiments are shown in ( figure 6 ). As is evident, intracellular MMP-1 levels increase in irradiated fibroblast-gels with and without pre-incubation of tirilazad. The mean values found were 127% of mock irradiated controls without and 147% with tirilazad pre-incubation. Tirilazad thus clearly achieves no diminished MMP-1 protein production within the cell but rather again, similarly as for IL-6, leads to intracellular accumulation of MMP-1.

Discussion

Tirilazad represents a new synthetic group of 21-aminosteroids called lazaroids, derived from the molecular structure of glucocorticosteroids. Lazaroids have been subject to both in-vivo and in-vitro studies in the context of LPO [21]. To our knowledge tirilazad has not been evaluated as to its potential in the field of photodermatology. In a first study we demonstrated the protective effects of tirilazad against UVA induced LPO in human dermal fibroblasts in-vitro [9]. The mechanism behind this effect is so far believed to be a combination of radical scavenging activity and an alteration of membrane fluidity [10, 22]. Lazaroids intercalate with their lipophilic areas into the phospholipid bilayer [23]. This intercalation results in an unspecific change of physico-chemical properties of membranes, as observed by a lower fluidity and static stabilisation. As lazaroids are not able to inhibit LPO in organisms without intact cell-membranes, their intercalation into the membrane seems to be one central mode of action and means of protecting membrane function [21]. As tirilazad is in addition a quencher of hydroxyl-, phenoxyl- and lipid peroxyl radicals and furthermore helps to keep the vitamin E content in the cell membrane constant it also functions as an antioxidant [10, 11]. Therefore we set out to explore its possible protective capacity against acute cellular responses to photo-oxidative stress. Our study focussed on 2 exemplary markers IL-6 and MMP-1. We chose an in-vitro system with cultured human fibroblasts in cell culture dishes and for the last experimental series, in collagen gels. Great care was taken to eliminate influential factors affecting the cellular reaction to UVA in addition to the substance tirilazad which we investigated as supplement as much as possible. Therefore the fibroblasts were rinsed before and kept in PBS during irradiation. It is important to be aware of this fact as for example antibiotics used in the culture medium can influence LPO during UV exposure [24].

The IL-6 release after UVA exposure of fibroblasts is an interesting parameter because it is linked with LPO mediated NFκB-activation and thus a direct connection to the membrane protective potential of tirilazad might be seen [17]. Furthermore IL-6 is known to be a key cytokine involved in autocrine loops activating MMP-1 [14] and thus also linked with collagen matrix degradation, a hallmark of photoaged skin [12, 25].

Our experiments from ( figure 2 ) show that tirilazad leads to significantly diminished IL-6 levels in the surrounding extracellular fluid after a single non-toxic dose of UVA in human dermal fibroblasts. The time pattern of increase is not altered though. Similarly the extracellular release of MMP-1 is diminished by tirilazad pre-incubation as well, as we can show in ( figure 5 ). The underlying mechanism for the reduction of UVA mediated IL-6 and the increase of MMP-1 in the extracellular space that tirilazad achieves remain to be discussed. The ability to quench ROS and to maintain levels of vitamin E in the cell membrane that tirilazad ensures, might be an explanation [11]. However, assuming this is the protective mechanism one would consequently expect an inhibition of IL-6 and MMP-1 production in the fibroblasts themselves after UVA irradiation. This was investigated in detail for IL-6. Looking at the levels of IL-6 mRNA 3 and 6h after irradiation, i.e. at time points when the extracellular increase of IL-6 was already notable but had not yet reached its peak and could thus still be potentiated by a sustained intracellular IL-6 synthesis, we found no beneficial influence of tirilazad as shown in ( figure 3A ) and 3B. In fact what these experiments further reveal is that the specific level mRNA levels for IL-6 increased in the first 3-5 h post-UVA exposure, as specific IL-6 mRNA levels found in our fibroblasts 6h after irradiation had again reached normal control levels of mock-irradiated fibroblasts. In the next step shown in ( figure 4 ), we then compared the intracellular IL-6 protein levels of irradiated fibroblasts with and without tirilazad pre-incubation. This comparison revealed three important further facts: 1^st: Tirilazad pre-incubated fibroblasts accumulate very high levels of intracellular IL-6 already in the first 3 h. 2^nd: These levels are much higher than the ones found in fibroblasts without pre-incubation of tirilazad. 3^rd: at 24 h after irradiation the intracellular levels of fibroblasts with and without tirilazad pre-incubation have greatly decreased and do not differ significantly any more. These data thus confirmed that tirilazad has neither an inhibitory effect on IL-6 mRNA transcription nor does it inhibit the later steps of IL-6 protein assembly as otherwise the extensively high intracellular IL-6 peak found in tirilazad pre-incubated fibroblasts would be unexplainable. In summary our data instead strongly suggest that the stress response following UVA irradiation and the NFκB-dependent pathway triggered by it, via which IL-6 and other cytokines like IL-1 are induced, is not inhibited by tirilazad [26-28]. However, we can here only show this definitely for IL-6.

With these findings the only other likely possibility for the observed significantly ameliorated IL-6 release after UVA irradiation lies in the known membrane stabilising effect of tirilazad, which could account for a delay in release of IL-6 despite high intracellular levels. In fact the data from ( figure 2 ) and ( figure 4 ) taken together strongly support this mechanism of action for tirilazad: The cell membrane is sealed off by the physico-chemical properties of tirilazad described [11] and this results in the IL-6 content in the extracellular space increasing more slowly and reaching a reduced peak in tirilazad pre-incubated fibroblasts compared to unexposed fibroblasts. The IL-6 which cannot get out immediately, accumulates in the meantime and results in the observed excessively high intracellular concentration 3h after UVA exposure. In the following hours IL-6 then leaks out of the cells in the presence of tirilazad more slowly than in its absence, and 24 h later the intracellular levels are still slightly higher in tirilazad pre-incubated fibroblasts than in fibroblasts without contact with this substance.

Such an ameliorating effect on extracellular IL-6 as observed in our experiments with tirilazad could in theory result in a decreased stimulation of MMP-1 production and subsequent release of the cell via the known MMP-1 inducing autocrine loops [14].

Therefore we also investigated finally, whether tirilazad per-incubation is of any benefit for intracellular MMP-1 levels post UVA irradiation. Our final experiments from ( figure 6 ), taking an in-vitro model of human dermis, show that no beneficial influence of tirilazad on the intracellular MMP-1 protein level 24 h post-irradiation can be detected, which fits with the previous data and again supports the view that this 21-aminosteroid is in fact mainly active via its physico-chemical membrane stabilisation rather than its antioxidant function. The antioxidant function which we previously found with respect to decrease of lipid peroxidation does therefore not play a major role in terms of a mode of protective action against acute photooxidative stress responses.

As to the possible potential of tirilazad as a substance for systemic or topical photoprotection it is, important to point out that in-vivo even the time-delay of an otherwise immediate IL-6 or MMP-1 increase after UVA exposure will enhance the situation for the stressed tissue, as it gives more time for dilution of mediators in the constant tissue perfusion stream. In-vitro experiments, such as we have used in this study, eliminate this important factor of tissue perfusion. Tirilazad thus might well be worth exploring further as a photoprotective substance.

Acknowledgements

We are grateful for the support by the Köln Fortune Program/Faculty of Medicine, University of Cologne, by the German Research Foundation DFG SCHA 411/10-1, 10-2 and by the European Union through the MAUVE project (QLK4-1999-01084).

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