Results

Influence of IL-6-type cytokines on the mRNA expression of MRP in primary human dermal fibroblasts and NHEK

Primary human dermal fibroblasts (Figure 1) and proliferating normal epidermal keratinocytes (Figure 2) isolated from foreskin were treated with IL-6/soluble human IL-6 receptor (sIL-6R) or OSM for 48 h. Expression of the subtypes 1, 3, 4, and 5 of the MRP family was studied by real-time PCR (TaqMan) or semiquantitative reverse transcription PCR (RT-PCR). As shown in Figure 1, mRNA levels for all four family members were significantly increased in OSM-treated dermal fibroblasts of three individual donors. Particularly, MRP3 and MRP4 were upregulated more than 2-fold. IL-6/sIL-6R had a reproducible stimulatory effect on the transcription of MRP3, whereas effects on MRP1, MRP4, and MRP5 were only marginal.

Figure 1.

Real-time PCR analysis of multidrug resistance-associated proteins (MRP) expression in dermal fibroblasts in response to interleukin-6 (IL-6)/soluble human IL-6 receptor (sIL-6R) or oncostatin M (OSM). Primary human dermal fibroblasts were stimulated with 20 ng per mL IL-6 and 1 g per mL sIL-6R or 20 ng per mL OSM for 48 h. Total RNA was isolated and untreated fibroblasts were used as control. The relative MRP mRNA levels of the subtypes 1, 3, 4, and 5 were normalized to -actin.

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Figure 2.

Real-time PCR, reverse transcription PCR (RT-PCR), and cDNA microarray analysis of multidrug resistance-associated proteins (MRP) expression in proliferating normal human epidermal keratinocytes (NHEK) after incubation with interleukin-6 (IL-6)/soluble human IL-6 receptor (sIL-6R), oncostatin M (OSM), or other inflammatory cytokines. (a) TaqMan real-time PCR analyses of MRP3 expression in proliferating NEHK. Primary keratinocytes were stimulated with 20 ng per mL IL-6 and 1 g per mL sIL-6R or 20 ng per mL OSM for 48 h. Total RNA was isolated and untreated keratinocytes were used as control. The relative MRP3 RNA level was normalized to -actin. (b) RT-PCR analysis of MRP1 and MRP3 expression in keratinocytes after stimulation for 48 h with IL-6 (20 ng per mL)/sIL-6R (1 g per mL) or OSM (20 ng per mL). PCR products of MRP1, MRP3, and -actin as internal standard were separated on agarose gels and stained with ethidium bromide. (c) RT-PCR analysis of MRP1, MRP3, and -actin in proliferating NHEK after incubation with different cytokines for 24 h. Lane 1, unstimulated keratinocytes; lane 2, IL-6 (20 ng per mL)/sIL-6R (1 g per mL); lane 3, IL-1 (2 ng per mL), lane 4: TNF (1 ng per mL), lane 5: TGF (1 ng per mL); lane 6: DNA-Marker pBR322 HaeIII Digest. (d) Analysis of differentially expressed genes in NHEK using microarray analysis. Human keratinocytes were incubated with 20 ng per mL IL-6 and 1 g per mL sIL-6R for 24 h; untreated NHEK were used as control. Generation of ³³P-labeled cDNA probes was achieved by reverse transcription of 10 g total RNA isolated from IL-6/sIL-6R-treated and control keratinocytes. Radiolabeled probes were applied to the GeneFilters microarrays (ID1001, Research Genetics) for hybridization.

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Primary NHEK, on the other hand responded much stronger to IL-6/sIL-6R than to OSM. Although MRP3 mRNA levels demonstrated a 150% increase after incubation with IL-6/sIL6R for 48 h, the mRNA levels remained rather unchanged in response to OSM (Figure 2a). MRP1 mRNA levels had to be analyzed in RT-PCR experiments using established MRP1-specific primers (^{Kool et al, 1997;Baron et al, 2001}) since the Assay-on-Demand primers used in dermal fibroblasts turned out to be unsuitable for the keratinocytes. Similar amounts of template mRNA and the same cycle no. (35) were used for each RT-PCR reaction using primer pairs for MRP1 and MRP3. As shown in Figure 2b, keratinocytes revealed a constitutive expression of MRP1. Stimulation for 48 h with IL-6, in combination with its soluble receptor (sIL-6R), however, also enhanced the expression of this transport protein. For MRP3, an equivalent upregulation was observed as seen before in the real-time PCR (Figure 2a). Other pro- or anti-inflammatory cytokines such as IL-1, tumor necrosis factor (TNF), or transforming growth factor (TGF) had no significant effect on mRNA expression of MRP in NHEK (Figure 2c). Additionally, we could detect higher amounts of MRP1 transcripts using a cDNA microarray focusing on genes expressed in human skin (Dermarray, ID1001). Incubation of NHEK with IL-6/sIL-6R for 24 h confirmed the RT-PCR data and revealed a 1.74-fold upregulation of MRP1 expression (Figure 2d).

Analysis of keratinocyte–fibroblast interaction in a two-chamber cell cultivation system

Expression of MRP was measured by RT-PCR in NHEK cocultured with dermal fibroblasts using the transwell system (TW) or in an NHEK from a monoculture (MC) (Figure 3). Incubation of cells with IL-6/sIL-6R for 24 h revealed the upregulation of MRP1 and -3 in both experimental setups, i.e. keratinocytes of the keratinocyte–fibroblast coculture and NHEK monoculture. Stimulation with IL-1 for 24 h, however, showed the upregulation of MRP1 and -3 only in those keratinocytes cocultured with fibroblasts in a TW (Figure 3), but not in keratinocytes taken from the NHEK monoculture.

Figure 3.

Coculture of dermal fibroblasts and normal human epidermal keratinocytes (NHEK) using the transwell system (TW) (left panel) in contrast to NHEK monoculture (MC) (right panel).Lane 1, control; lane 2, incubation with 20 ng per mL interleukin-6 (IL-6) and 1 g per mL soluble human IL-6 receptor (sIL-6R) for 24 h; lane 3, incubation with 2 ng per mL IL-1; lane 4, DNA-Marker pBR322 HaeIII Digest.

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Effect of MAP kinase and protein kinase B (PKB/Akt) inhibitors on MRP expression in dermal fibroblasts

Both IL-6/sIL-6R as well as OSM are strong activators of the Jak/STAT and the mitogen-activated protein kinase (MAPK) pathway (^{Heinrich et al, 1998, 2003}). Numerous studies have identified the Jak-mediated phosphorylation, dimerization, and nuclear translocation of STAT3 as a key component for transcription of a majority of IL-6-type cytokine-stimulated genes. But previous studies on regulatory mechanisms driving MDR genes have suggested a significant contribution of the MAPK pathway (^{Sukhai and Piquette-Miller, 2000}).

The increase in mRNA levels of the various MRP in primary dermal fibroblasts is a long-term effect visible after 24–48 h incubation with either OSM or IL-6/sIL-6R. We therefore first investigated the time kinetics of the different signaling pathways known to be activated by IL-6-type cytokines (^{Heinrich et al, 2003}). As expected, STAT3 as well as STAT1 are strongly tyrosine phosphorylated in response to OSM as well as to IL-6/sIL-6R. Interestingly, after the initial peak between 10 min and 1 h, an enhanced level of tyrosine phosphorylated STAT remained detectable at least up to 24 h (Figure 4a, first and third panel). The effect was more pronounced for STAT3 than for STAT1. In contrast, the increased phosphorylation status of the MAPK Erk1/2 and p38 as well as of PKB/Akt was only transient with the same initial peak between 10 min and 1 h, but then a rapid return to basal levels (Figure 4a, fifth, seventh, and ninth panel). In contrast to IL-6, OSM turned out to be a much more potent activator of p38 and PKB/Akt (Figure 4a).

Figure 4.

Signaling pathways initiated by IL-6 and OSM in dermal fibroblasts and their impact on the enhanced transcription of MRP. (a) Analysis of signaling pathways initiated in dermal fibroblasts in response to oncostatin M (OSM) (left panel) or interleukin-6 (IL-6)/soluble human IL-6 receptor (sIL-6R) (right panel). Human dermal fibroblasts were stimulated with 20 ng per mL OSM or 20 ng per mL IL-6 and 1 g per mL sIL-6R for the indicated time points. Total cell lysates were separated by SDS-PAGE. Western blots were developed with antisera specific for the indicated proteins. (b) Effect of inhibition of Erk1/2 on multidrug resistance-associated proteins (MRP) transcription. Real-time PCR analysis of MRP1, 3, 4, and 5 expression in cytokine-stimulated dermal fibroblasts pretreated with the MEK-1 inhibitor U0126: Human dermal fibroblasts were stimulated for 48 h with 20 ng per mL OSM or pretreated for 45 min with 5 M U0126 followed by cytokine stimulation. The inhibitor as well as the cytokine were refreshed once after 24 h incubation. Total RNA was isolated, and untreated fibroblasts were used as control. The relative MRP RNA levels of the different subtypes were normalized to -actin. (c) Effect of inhibition of phosphaditylinositol 3-kinase (PI3 kinase) on MRP4 transcription. Real-time PCR analysis of MRP4 expression in OSM-stimulated dermal fibroblasts pre-treated with the PI3-kinase inhibitor LY294002: Human dermal fibroblasts were stimulated for 48 h with OSM or pre-treated for 45 min with 5 M LY294002 followed by cytokine stimulation. The inhibitor as well as the cytokine were refreshed once after 24 h incubation. Total RNA was isolated and untreated fibroblasts were used as control. The relative MRP4 mRNA level was normalized to 18S rRNA.

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In order to analyze whether the activation of MAPK is important to modify MRP gene transcription, dermal fibroblasts were pre-incubated with the MEK-1 inhibitor (U0126) 45 min before stimulation with OSM for 48 h. The inhibitor and the cytokine were refreshed once during the incubation period. Real-time PCR analysis revealed that pre-treatment of dermal fibroblasts with 5 M of U0126 had no inhibitory effect on the enhanced transcription of MRP1, MRP3, or MRP4 under the influence of OSM (Figure 4b).

Western blot studies verified that U0126 was suppressing the phosphorylation of Erk1/2, as expected, but also the phosphorylation of the stress-activated kinase p38. This unspecific activity of the inhibitor might be because of the long incubation time of 48 h. Tyrosine phosphorylation of STAT3, however, as well as serine phosphorylation of PKB/Akt were not influenced (data not shown).

We then examined whether inhibition of the phosphaditylinositol 3-kinase (PI3-kinase) pathway by the specific inhibitor LY294002 altered OSM-induced MRP expression. Interestingly, the significant increase in MRP4 expression mediated through 48 h stimulation with OSM was strongly impaired in dermal fibroblasts treated with OSM in the presence of LY294002 (Figure 4c). Unfortunately, MRP1 and MRP3 mRNA could not be evaluated, since LY294002 itself already had a strong stimulatory effect on transcription of these two transporters (data not shown).

LY294002 remained specific even after 48 h incubation and inhibited only the phosphorylation of PKB/Akt, but not of Erk1/2, p38, or STAT3 (not shown).

Immunohistochemistry

The immunohistochemical staining of skin samples clearly demonstrated the expression of MRP1 in the cell membrane of epidermal keratinocytes (Figure 5; MRP1 expression indicated by arrow). In contrast to normal human skin (Figure 5a), expression of MRP1 was more pronounced in skin samples of patients with lichen planus (Figure 5b) and especially in psoriasis vulgaris (Figure 5c).

Figure 5.

Immunohistological staining of human skin specimen using a monoclonal antibody specific for the detection of multidrug resistance-associated proteins (MRP1). (a) Staining of normal human skin, (b) skin sample taken from a patient with lichen planus, (c) skin sample from a psoriasis plaque. indicates enhanced expression of MRP1 in the plasma membrane of the epidermal keratinocytes.

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Functional activity of MRP in NHEK and primary dermal fibroblasts

To further corroborate the efficient translation of the increased mRNA for the various MRP, the efflux activity of human keratinocytes as well as dermal fibroblasts was determined fluorimetrically using the calcein-acetoxymethylester (calcein-AM) efflux assay (Figure 6a, b. This chemical diffuses passively into cells, remains non-fluorescent in its ester form, and can efficiently be exported by MRP. Cytoplasmic and mitochondrial esterases within the cells convert calcein-AM into a hydrophilic fluorescent dye (calcein) that is trapped inside the cytoplasm. Therefore, a low level of fluorescence indicates a low intracellular retention of calcein, reflecting a high functional activity of efflux transport proteins. Untreated NHEK and fibroblasts were compared with cells incubated with IL-6 and sIL-6R for 72 h. After 3 d of incubation with the cytokine, accumulation of calcein-AM was allowed to proceed for the indicated times. Cells were washed and the remaining incorporated fluorescence was measured. In accordance with the PCR data (Figure 1 and Figure 2), enhanced expression of MRP transporters in the plasma membrane of NHEK as well as in dermal fibroblasts correlated with a decrease in fluorescence after IL-6/sIL-6R treatment (Figure 6a, b).

Figure 6.

Measurement of multidrug resistance-associated proteins (MRP) activity. The net accumulation of calcein-acetoxymethylester (calcein-AM) (a, b), and all-trans retinoic acid (RA) (c) was determined either fluorimetrically (calcein-AM) or radiometrically (all-trans [20-methyl-³H] RA) in normal human epidermal keratinocytes (NHEK) (a, c) or dermal fibroblasts (b). Cells were pre-treated with 20 ng per mL IL-6 and 1 g per mL sIL-6R for 72 h, and untreated cells served as control. (a, b) Accumulation of calcein-AM was allowed to proceed for 15, 20, 30, or 60 min and was performed in triplicate. (c) Efflux of all-trans RA was measured in NHEK pre-incubated with 20 ng per mL IL-6 and 1 g per mL sIL-6R for 24 h. Experiments were performed in hexaplicate after incubation with 1.4 10^-8 mol per liter all-trans [20-methyl-³H] RA for 60 min, and median values were used for data analysis.

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A novel transport assay for the detection of MRP-mediated efflux activity in NHEK was established using all-trans [20-methyl-³H] retinoic acid (RA) as a substrate. As seen with calcein-AM, treatment of cells with IL-6 and sIL-6R (Figure 6c) increased the active efflux transport of all-trans [20-methyl-³H] RA in NHEK by 25% in comparison with control transport measured in the absence of the retinoid.

Discussion

Dysregulated expression of transport proteins like the MRP has since many years been associated with increased resistance of various cancer cells to chemotherapeutic drugs (^{König et al, 1999;Kruh and Belinsky, 2003}). In recent years, it has become clear that MRP are not only involved in exporting xenobiotica, but also seem to be involved in the export of lipid mediators like leukotrienes or prostaglandins (^{König et al, 1999;Reid et al, 2003}). Therefore, they seem to be target genes of potential interest when studying the inflammatory response. Indeed, first hints that cytokines involved in coordinating the body's inflammatory reaction as well as bacterial products might be involved in the regulation of MRP came from studies in the liver (^{Lee and Piquette-Miller, 2001;Hartmann et al, 2002}), but nothing was known about the regulation of these transporters in human skin cells. The presence of MRP1 and MRP3–7 in human skin has been verified in earlier studies (^{Baron et al, 2001}). Here, we could demonstrate that in primary NHEK as well as dermal fibroblasts the inflammatory cytokines IL-6/sIL-6R and OSM are able to upregulate the expression of different MRP family members.

Lesional psoriatic skin (^{Sehgal, 1990;Bonifati et al, 1998}) or keratinocytes isolated from patients with lichen planus (^{Fayyazi et al, 1999}) are known to display elevated levels of IL-6-type cytokines. The highest level of IL-6 expression was observed in the basal and suprabasal keratinocytes of the involved skin. The role of these IL-6-type cytokines in the pathogenesis of psoriatic lesions is completely unknown. However, an in vitro study has indicated that OSM can directly induce GM-CSF and IL-6 production by endothelial cells and the endothelium seems to be involve d early in the psoriatic process (^{Bonifati et al, 1998}).

In our experiments, specimens from normal human skin revealed a low basal expression and activity of MRP efflux proteins in cultured keratinocytes (Figure 2b, c and Figure 5a). This expression was enhanced in lesional skin of patients with lichen planus (Figure 5b) and psoriasis (Figure 5c), indicating both increased transcription and protein synthesis of the MRP transporters. This increase in MRP visible at the cell membrane was then analyzed in vitro using freshly isolated NHEK and dermal fibroblasts. Stimulation of NHEK with IL-6, in combination with its agonistically acting soluble receptor (sIL-6R), led to a strong upregulation of MRP3, and to a lesser extent of MRP1 (Figure 2a, b, and d). Interestingly, in an inflammatory situation infiltrating leukocytes can indeed secrete a soluble form of IL-6R or induce shedding of membrane-bound IL-6R (^{Jones et al, 2001}). Assessment of other cytokines such as IL-1, TNF, and TGF expressed in inflammatory skin tissue revealed no effect on the expression of these proteins in NHEK (Figure 2c).

By analyzing the keratinocyte–fibroblast interaction in a two-chamber cell cultivation system^{Boxman et al. (1993)}, we were able to show that fibroblasts are also an important source of IL-6 in normal human skin. This expression can be significantly upregulated by IL-1, which is released from keratinocytes after skin injury. Our data indicate that stimulation of keratinocyte–fibroblast cocultures with IL-1 leads to an upregulation of IL-6 production in fibroblasts, which could subsequently induce upregulation of MRP expression in NHEK in a paracrine manner (Figure 3). Stimulation of a keratinocyte monoculture with IL-1 had no effect on the MRP expression of these cells (Figure 3). As seen in the experiments before, direct induction with IL-6/sIL-6R allowed increased expression of MRP in NHEK (Figure 3). These findings support the importance of a keratinocyte–fibroblast communication for the observed regulatory mechanism.

The characterization of signaling events initiated by IL-6/sIL-6R and OSM in dermal fibroblasts demonstrated the activation of the Jak/STAT-, the MAPK- as well as the PI3-kinase pathway (Figure 4a). Over the past decade, the analysis of many genes induced by IL-6-type cytokines has highlighted the involvement of STAT factors in transcription initiation. For MDR genes, however, recent studies have proposed a regulatory role for the MAP-kinases and their downstream targets (^{Sukhai and Piquette-Miller, 2000}). Using the commercially available MEK-1 inhibitor U0126, we were able to abrogate Erk1/2 as well as p38 activation in response to IL-6 or OSM in dermal fibroblasts (not shown). Interestingly, analysis of the MRP mRNA levels demonstrated that this compound did not prevent the MRP upregulation in dermal fibroblasts (Figure 4b). This clearly demonstrates that Erk1/2 and p38 are not involved in the stimulatory effect of IL-6/sIL-6R or OSM. When analyzing the activation of PKB/Akt, it became evident that OSM is a much stronger inducer of PKB/Akt serine phosphorylation than IL-6 in dermal fibroblasts (Figure 4a). This was particularly interesting with respect to the more pronounced stimulation of MRP gene transcription in response to OSM compared with the one induced by IL-6/sIL-6R in dermal fibroblasts (Figure 1). Indeed, pre-treatment of the cells with the PI3-kinase inhibitor LY294002 abolished the stimulatory effect of OSM on the transcription of MRP4 (Figure 4c). This result is in line with recent findings, which described the involvement of the PI3-kinase pathway in the regulation of P-glycoprotein (P-gp) expression in response to the hepatocarcinogen 2-acetylaminofluorene (^{Kuo et al, 2002}). Additionally, it was shown that inhibition of PI3-kinase or PKB/Akt activity by expression of their dominant-negative forms sensitizes breast cancer cells to the induction of apoptosis by chemotherapeutic agents (^{Knuefermann et al, 2003}). Unfortunately, we could not analyze the effect of LY294002 on MRP1 or MRP3 gene regulation since the incubation of fibroblasts with this substance increased the mRNA levels of these two transporters even in the absence of any further stimulus (not shown). Therefore, we cannot exclude the possibility that other transport proteins might be regulated via the Jak/STAT pathway, particularly since STAT1 and STAT3 show a prolonged tyrosine phosphorylation in response to IL-6/sIL-6R and OSM (Figure 4a).

Efflux activity of NHEK was determined either fluorimetrically using the calcein-AM efflux assay or radioactively using all-trans [20-methyl-³H] RA. Therefore, untreated cells were compared with cells pre-incubated with IL-6/sIL-6R for 3 d (Figure 6a, b). The pre-incubated cells revealed a lower retention of the fluorescent calcein reflecting a higher functional activity of MRP transport proteins since calcein-AM is actively pumped out of the cell before it can be hydrolyzed to hydrophilic calcein by cellular esterases.

^{Kizaki et al. (1996)} reported that RA-resistant HL-60 cells express the efflux transport protein P-gp and these cells differentiated to mature granulocytes in response to incubation with all-trans RA and verapamil, an efflux transport inhibitor. Using a novel uptake-transport assay, we were able to show for the first time that not only P-gp – which is not expressed in NHEK – (^{Baron et al, 2001}) but also MRP proteins mediate the active efflux transport of all-trans RA [20-methyl-³H] in NHEK (Figure 6c). This efflux can be specifically inhibited by indomethacin, a known inhibitor of MRP proteins (data not shown). Pre-stimulation of NHEK with IL-6/sIL-6R resulted in a higher activity of MRP proteins and a lower concentration of all-trans RA in the cells (Figure 6c). Since the effect of IL-6/sIL-6R on the efflux transport of all-trans RA is only modest, further studies need to be performed analyzing the effect of IL-6-type cytokines on the MRP-dependent efflux transport of other RA isomers and their 4-oxo-metabolites, which might be better substrates for the MRP.

In summary, our studies demonstrate that the expression and functional activity of efflux pumps in NHEK as well as fibroblasts can be significantly enhanced by IL-6-type cytokines. The analysis of signals initiated by IL-6/sIL-6R or OSM in fibroblasts showed a strong activation of the Jak/STAT-, the PI3-kinase pathway as well as of Erk1/2 and p38. Using an inhibitor specific for the MAP kinase kinase MEK-1, we could exclude the Erk1/2 or p38 pathway as an essential component for initiating enhanced transcription of the MRP transporters. Inhibition of the PI3-kinase, however, suggested a role of this pathway in the regulation of MRP4 gene expression in response to OSM. Future studies using alternative biochemical and molecular approaches will also determine whether MRP1 and MRP3 are regulated in a similar manner or whether the activation of STAT1 or STAT3 might be sufficient for their increased gene transcription. Since MRP have recently been shown to function as prostaglandin efflux transporters and IL-6-type cytokines are known to induce the expression of COX-2 in certain cell types (^{Bernard et al, 1999;Repovic et al, 2003}), it is tempting to speculate that increased prostaglandin efflux might be one additional means by which IL-6 or OSM can contribute to cutaneous inflammation.

Materials and Methods

Chemicals and cytokines

All reagents were of highest grade and purity commercially available. Recombinant IL-6 and sIL-6R were prepared as described (^{Arcone et al, 1991;Weiergräber et al, 1995}). Recombinant human OSM was purchased from Cell Concepts (Umkirch, Germany), IL-1, TGF, and TNF from Roche (Mannheim, Germany). The MEK inhibitor U0126 as well as the PI3-kinase inhibitor LY294002 were obtained from Calbiochem (Merck AG, Darmstadt, Germany). The polyclonal STAT1-pY701 (9171); STAT3-pY705 (9131), Akt-pS473 (9271), and Erk1/2 (9102) antisera as well as the monoclonal Erk1/2-active (9106) antibody were purchased from Cell Signaling Technology (New England Biolabs, Frankfurt, Germany), and the polyclonal p38-active (V1211) antiserum was from Promega (Madison, Wisconsin). The monoclonal STAT3 (610189) antibody was obtained from Pharmingen (San Diego, California), and the polyclonal antisera against STAT1 p84/p91 (E-23), Akt1/2 (N-19), and p38 (C-20) were from Santa Cruz Biotechnology (Santa Cruz, California).

Keratinocytes

NHEK were obtained from foreskin specimen by dispase (Roche) separation of the epidermal sheet from the dermis and subsequent trypsin/ethylenediaminetetraacetic acid (EDTA) (PAA, Linz, Austria) digestion of the epidermis. Samples from patients with psoriasis and lichen planus were obtained in the Department of Dermatology, RWTH Aachen. All studies were approved by the ethical committee of the University Hospital, RWTH Aachen. Participants gave their written informed consent, and the study was conducted according to Declaration of Helsinki Principles. NHEK were cultured in a low calcium (0.09 mM), serum-free keratinocyte medium with bovine pituitary gland extract, recombinant human epidermal growth factor, insulin, gentamycin sulfate, and amphotericin B as described by the manufacturer (KGM, Clonetics, San Diego, California). Cells were subcultivated using the manufacturer's detach-kit with Hank's balanced salt solution and trypsin/EDTA. The medium was replaced regularly three times a week. For this study, proliferating keratinocytes in the first and second passage were used.

Fibroblasts

Normal human fibroblasts were obtained from foreskin specimen. After excision, the specimens were washed three times in sterile phosphate-buffered saline (PBS; PAA) containing antibiotics (penicillin/streptomycin; PAA) and antimycotics (amphotericin B; Roche, Mannheim, Germany), and digested in dispase solution (2 U per mL, GibcoBRL, Karlsruhe, Germany) for 20 h at 4°C and subsequently for 2 h at 37°C. Then, the epidermis was removed and, for adherence to the polystyrene surface, the dermis was placed in a dry cell culture multi-well plate (FALCON-Becton Dickinson, Franklin Lakes, New Jersey) for 20 min. Subsequently, Dulbecco's minimum essential medium with high glucose and L-glutamine (DMEM, GibcoBRL), 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) was added. The plates were transferred to a CO₂ incubator (MCO-17AI, SANYO, Osaka, Japan) at 37°C in a humidified atmosphere with 5% CO₂. Monitoring fibroblast outgrowth by light microscopy (LEICA DMIL; Leitz, Wetzlar, Germany), the dermis was removed after 5–7 d. After reaching the state of early confluence, cells were washed with PBS and incubated in 1 mL trypsin/EDTA (PAA) for 3 min at 37°C. The enzymatic digestion was stopped by addition of 3 mL FCS. Then, cells were centrifuged, the supernatant was discarded, and the cells were suspended in FCS or autologous serum and subcultivated in cell culture flasks (NUNC, Roskilde, Denmark).

Keratinocyte–fibroblast coculture

For coculture experiments, proliferating NHEK and dermal fibroblasts were cultured in a TW (Transwell-COL; COSTAR, Bodenheim, Germany) using keratinocyte medium as described above. Fibroblasts were plated on a porous membrane and NHEK cells on the bottom of the well. Both cell types were first allowed to attach for 24 h and afterwards cytokines were added to the medium and cells were incubated for 24 h.

RNA isolation

mRNA was extracted using 5 10⁶ keratinocytes from different donors with the Oligotex Direct mRNA-purification kit (Qiagen, Hilden, Germany) using the mRNA-enrichment protocol (^{Kuribayashi et al, 1988}). Total RNA was isolated using peqGOLD RNAPure (peqlab, Erlangen, Germany) as described by the manufacturer. RNA concentration of each sample was measured using the Nanodrop system (NanoDrop Technologies, Rockland, Delaware) and equal amounts of mRNA were used for RT.

Total RNA from human dermal fibroblasts or keratinocytes was isolated using the high pure RNA isolation kit (Roche) according to the manufacturer's instructions, including the DNase step.

RT-PCR

RT and PCR were performed with the GeneAmp RNA PCR kit (Perkin-Elmer, Weiterstadt, Germany) according to the manufacturer's instructions. All RT-PCR experiments were performed in duplicate for each donor. Detection of specific mRNA for MRP1–7 was achieved by using primers designed to amplify at least one intron in the gene to exclude contamination of cDNA with genomic DNA (^{Baron et al, 2001}). -actin was used as an internal standard as described before (^{Baron et al, 1998}). Amplification was carried out with 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 53°C, and 1 min extension at 72°C. Amplification was terminated with an extension step of 10 min duration at 72°C after the last cycle. PCR products were separated on 1.8% agarose gels (1 TBE) and stained with ethidium bromide.

Quantitative real-time PCR

Purified RNA was reverse transcribed with the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Weiterstadt, Germany) with random hexamers as primers. TaqMan experiments were carried out on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) using Assay-on-Demand gene expression products for MRP1 (Hs00219905), MRP3 (Hs00358656), and MRP4 (Hs00195260) according to the manufacturer's recommendations. The primer/probe set for human MRP5 was designed using Primer Express 2.0.0 software (Applied Biosystems) based on published sequences (accession number U83661). An Assay-on-Demand product for human -actin (Hs99999903) or 18S rRNA (Hs99999901) was used as an internal reference to normalize the target transcripts. MRP, -actin, and 18S rRNA sequences were amplified independently in separate reaction wells in triplicate. The real-time PCR efficiencies were determined for each primer/probe set from standard curves generated from serial dilutions of a cDNA sample of unstimulated dermal fibroblasts. The relative differences between the MRP expression of unstimulated and stimulated cells were calculated using the mathematical model developed by^{Pfaffl (2001)} including the PCR efficiencies.

Cell lysates and western blotting

Normal human dermal fibroblasts were stimulated for the indicated periods of time with 20 ng per mL IL-6, in combination with 1 g per mL soluble IL-6R, or 20 ng per mL OSM. Immediately after stimulation, cells were lysed in Triton lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 5 mg per mL aprotinin, and 5 mg per mL leupeptin), scraped off the dish, and left on ice for 30 min. Lysates were centrifuged with 16,000 g for 10 min at 4°C and equal amounts of whole-cell extracts were boiled for 5 min in Laemmli buffer at 95°C. Proteins were separated by SDS-PAGE in 10% gels, followed by electroblotting onto a polyvinylidene difluoride membrane (PALL, Dreieich, Germany). Western blot analysis was conducted using the indicated antibodies and the enhanced chemiluminescence kit (Amersham Biosciences, Freiburg, Germany) according to the manufacturer's instructions. Before reprobing, blots were stripped in 2% SDS, 100 mM -mercaptoethanol in 62.5 mM Tris-HCl (pH 6.7) for 20 min at 75°C.

Efflux assay (calcein-AM)

calcein-AM (Molecular Probes/MoBITec, Göttingen, Germany) was used to study the efflux activity of the MRP (^{Baron et al, 2002}). Proliferating NHEK or dermal fibroblasts were seeded in six-well plates for 3 d with and without addition of IL-6/sIL-6R. Briefly, cells were trypsinized and washed once with Hepes-buffered RMPI 1640 (HPMI) buffer (120 mM NaCl, 5 mM KCl, 0.4 mM MgCl₂, 10 mM HEPES-Na (pH 7.4), 10 mM NaHCO₃, 10 mM glucose, and 5 mM Na₂HPO₄). They were then resuspended in the HPMI buffer and incubated with 0.25 mM calcein-AM at 37°C for 15–60 min (concentration of solvent in medium, 0.5% DMSO). Incubation was terminated by centrifugation, followed by washing of the cell pellet with HPMI buffer. The final pellet was resuspended in 1 mL of lysis solution (0.25 M sucrose, 10 mM Hepes (pH 7.6), and 1 mM EDTA). Fluorescence was measured at 493/515 nm. A high level of fluorescence indicates a high intracellular retention of calcein, reflecting a low activity of efflux transport proteins such as MRP.

Transport assay (all-trans RA)

Proliferating NHEK were cultured in 24-well plates for 2 d and 24 h before starting the uptake experiment, the cell culture medium was replaced by serum-free medium. Both media contained IL-6/sIL-6R. Untreated NKEK served as control. Cells were washed once with pre-warmed PBS and then incubated with 500 L of serum-free medium. After incubation for 30 min at 37°C, another 500 L medium containing 1.4 10^-8 mol per liter of the tritium-labeled substrate all-trans [20-methyl-³H] RA (Perkin-Elmer) was added. After incubation for 60 min at 37°C, the radioactive medium was removed and cells were washed three times with PBS. Cells were lysed by adding Optiphase "Supermix" (Wallac, Turku, Finland). The cell-associated radioactivity was determined by transferring 500 L aliquots of the lysate into a flexible 24-well plate and counting radioactivity using a cross-talk preventing tape and a Wallac 1450 MicroBeta TriLux liquid scintillation counter (Wallac).

Immunohistochemistry

Paraffin-embedded skin specimen from normal human skin as well as skin specimen from patients with lichen planus or psoriasis were cut into 8 m sections, mounted on superfrost slides (Menzel, Braunschweig, Germany), deparaffinized, and rehydrated. To unmask antigens, the specimens were pretreated with Target Retrieval pH 6.1 (DAKO, Glostrup, Denmark) as indicated in the data sheet, rinsed in distilled water, and placed in Tris-buffered saline (TBS). Subsequently, specimens were incubated for 30 min with primary antibody, either the monoclonal antibody specific for MRP1 (MRPr1, IQProducts, Groningen, the Netherlands) or the isotypic control, and were then rinsed in TBS for 10 min (^{Baron et al, 2001}). For visualization of the staining, the DAKO EnVision system was used as suggested by the manufacturer. Finally, specimens were counterstained with hematoxylin and were mounted with cover slips. Examination and photodocumentation were performed using an inverse photomicroscope (LEICA DMIL, Leitz).

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Acknowledgments

These studies were supported by a grant from the Deutsche Forschungsgemeinschaft (BA 1803/4-1; SFB 542, TP C11) and the Fonds der Chemischen Industrie (Frankfurt am Main, Germany).