Results

Expression of IL-18R, IL-12R1, and IL-12R2 on human primary keratinocytes

IL-18R was found to be expressed on human primary keratinocytes. In all experiments, there was a low basal expression of IL-18R on keratinocytes. IL-18R was moderately upregulated by IFN (5–10 ng per mL) alone (Figure 1). When receptor expression of IFN-stimulated keratinocytes was compared with expression of matched unstimulated cells, upregulation of IL-18R was significant as determined by a paired the t test (p=0.03). Expression was increased more than 3-fold by IFN in combination with tumor necrosis factor (TNF) and more than 2-fold by IFN in combination with poly I:C (Figure 1). In contrast to the results described for cells of the lymphocyte lineage, we could not find any effect of IL-12 or IL-23 (in a dose range from 1 to 50 ng per mL) on the expression of IL-18R (not shown). This may reflect different responsiveness and signalling toward IL-12 in keratinocytes as compared with T cells or NK cell. IL-1 or GM-CSF also did not show significant effects on the expression level of IL-18R.

Figure 1.

Expression of interleukin (IL)-18R on human primary keratinocytes. IL-18R expression was determined on normal human primary keratinocytes by flow cytometry 24 h after stimulation. A histogram of one representative experiment is shown in (A). (B) Receptor expression determined in individual experiments was normalized to the corresponding medium value to allow summary of different experiments. Original (unprocessed) geometric mean data were submitted to the t test and compared with the expression level of non-stimulated (ns) cells. For all stimulation shown, at least six independent experiments were performed. Interferon (IFN) (10 ng per mL), IL-18 (50 ng per mL), IL-12 (50 ng per mL), tumor necrosis factor (TNF) (200 U per mL), poly I:C (200 ng per mL). (C, D) (n=3), the effect of IL-4 on IL-18R expression is shown. Keratinocytes were incubated with IL-4 (50 ng per mL in C) 16 h before addition of IFN+second signal. MFI, mean fluorescense intensity; pI:C, poly I:C.

Full figure and legend (49K)

IL-12R1 was found to be expressed on keratinocytes and slightly but reproducibly upregulated by IFN (mean 6.9, standard error of the mean (SEM) 1.36; non-stimulated: mean 5.3, SEM 0.839; p=0.03 as determined by the paired t test; n=10), IFN (200 U per mL; mean 11.7, SEM 1.5; non-stimulated: mean 9.5, SEM 2.0; p=0.035 as determined by a paired the t test; n=7) or IFN+TNF (mean 6.4, SEM 1.4, non-stimulated: mean 5.6, SEM 1.17; p=0.02 as determined by a paired the t test; n=9), but not by IL-18, IL-12, TNF, or poly I:C (data not shown). Upregulation of IL-12R1 by these stimuli was much lower (i.e. less than 2-fold) than upregulation of IL-18R. In a total of ten independent experiments, IL-12R2 was not detectable on keratinocytes with or without addition of stimuli (IL-1, IL-18, IL-12, TNF, IFN, poly I:C, IFN, IFN, combination of IFN with cytokines, or addition of cyclosporin A;^{van Rietschoten et al, 2001}).

IL-4 downregulates the expression of IL-18R on human keratinocytes

Many inflammatory skin diseases are associated with a local "mixed" cytokine pattern both of Th2 and Th1 cytokines. In a subsequent series, we thus tested the influence of the major "Th2" cytokine IL-4 on IL-18R expression. Given alone, IL-4 did not exert any significant effect on receptor expression. But IL-4 given prior (16 h) to incubation with the pairs of cytokine that had the strongest effects on IL-18R expression on keratinocytes led to a receptor downregulation (Figure 1c). The effect was concentration dependent, with 50–100 ng per mL of IL-4 having the most pronounced effects (Figure 1d). When IL-4 was given simultaneously with the stimulation, downregulation of the receptor was not evident.

IL-18R expression is upregulated in skin lesions of psoriasis and AD

All surface markers used in the study were verified to stain for trypsin-insensitive epitopes (as assessed for the trypsin concentration and incubation period used). We isolated keratinocytes from skin biopsies taken from lesional psoriasis—which is now considered to be "Th1" associated—and spontaneous lesions of AD—which is both Th2 and Th1 associated or from normal skin. Upon isolation, keratinocytes were analyzed within 2 h for expression of IL-18R. The same instrument settings were used for each experiment. A more than 5-fold higher expression of the receptor could be observed on keratinocytes from lesional psoriasis and a 2-fold higher expression was observed on keratinocytes from AD skin as compared with normal skin (Figure 2).

Figure 2.

Expression of interleukin (IL)-18R in chronic inflammatory skin diseases. IL-18R expression was determined on freshly isolated keratinocytes derived from lesional atopic dermatitis (AD) (n=3) or psoriasis skin (n=5) or from normal skin (n=4). Geometric mean of matched isotype was subtracted from geometric mean obtained with the specific IL-18R antibody in each experiment. Exemplary histograms of IL-18R expression on ex vivo keratinocytes from a healthy individual and from lesional AD and psoriasis are depicted.

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IL-18 signals via NF-B in human keratinocytes

Keratinocytes showed activation of the NF-B-signalling pathway upon IL-18 stimulation as detected by nuclear translocation. This was demonstrated by using a FACS analysis method as described before (^{Foulds, 1997}). IL-18 alone was able to induce nuclear translocation. The IL-18 effect was blocked by the presence of neutralizing anti-IL-18 antibody during the stimulation procedure (Figure 3).

Figure 3.

Interleukin (IL)-18 signals via nuclear factor B (NF-B) in human primary keratinocytes. NF-Bp65 was stained in intact nuclei isolated from normal human keratinocytes. Cells were stimulated with IL-18 (50 ng per mL) for 30 min. Where indicated, stimulation was performed in the presence of blocking antibody (10 g per mL). The left histogram depicts one representative experiment performed with a blocking anti-IL-18R antibody. ns, non-stimulated.

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IL-18 induces CXCL10 production, but not IL-6, IL-8 production, or expression of costimulatory or adhesion molecules in human primary keratinocytes

Keratinocytes are potent producers of the chemokine CXCL10 that attracts CXCR3-expressing Th1 cells (^{Bonecchi et al, 1998;Boorsma et al, 1998}). IFN is a known strong inducer of CXCL10 production by keratinocytes, which was confirmed in this study. As shown here, IL-18-induced CXCL10 about 3-fold in the picogram per milliliter range after overnight incubation. Moreover, IL-18 could further increase CXCL10 production by more than 50% in IFN-treated keratinocytes (Figure 4a). CXCL10 induction was also observable in freshly isolated keratinocytes from lesional psoriasis skin incubated overnight with IL-18. The induction level of unstimulated to stimulated keratinocytes was significantly higher in psoriasis as compared with normal keratinocytes (Figure 4b).

Figure 4.

Interleukin (IL)-18 induction of CXCL10 production in human keratinocytes. CXCL10 production of cultured normal keratinocytes in passage one to four cultured in 24-well plates in 500 L of culture medium were left unstimulated (ns), stimulated with IL-18 (50 ng per mL) alone, or in combination with interferon (IFN) (10 ng per mL) (A; n=14 (left), n=11 (right)). Supernatants were harvested after 24 h and analyzed by ELISA. Freshly isolated keratinocytes from lesional psoriasis skin or healthy skin were stimulated overnight with IL-18 (B). The fold induction of CXCL10 secretion in IL-18-stimulated compared with non-stimulated keratinocytes is given (n=7 in each group).

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In contrast to data found for human DC (^{Gutzmer et al, 2003}) and human fibroblast/endothelial cells (^{Morel et al, 2001}), we could not find an increased CD54 expression induced by IL-18 on human primary keratinocytes. Costimulatory molecules were not affected either (CD40, CD80, CD86). Negative results were also obtained for CD44 and TLR2 (data not shown). Using the cytometric bead array (CBA), we could not observe consistently increased IL-8, IL-6, IL-10, IL-12p40, or IL-1 in the supernatant of human primary keratinocytes stimulated with IL-18. FasL expression or apoptosis induction—as described for other cell types such as NK cells—were not observed in human primary keratinocytes under the influence of IL-18.

IL-18 induces MHC class I and II expression on human keratinocytes

IL-18-induced MHC class I and II expression on human keratinocytes, known as non-professional antigen-presenting cells. Overnight incubation of keratinocytes with 5 ng per mL IL-18 resulted in upregulation of cell surface expression of MHC class I (Figure 5). In contrast, IL-18 did not consistently induce an upregulation of MHC I in human blood monocytes in six different healthy donors tested (data not shown). Higher concentration of IL-18 failed to further increase MHC I expression on keratinocytes.

Figure 5.

Interleukin (IL)-18 induces major histocompatibility complex (MHC) class I upregulation in human keratinocytes. Human keratinocytes were stimulated with IL-18 (5 ng per mL) for 24 h. MHC I expression was determined by flow cytometry. The histogram depicts an exemplary experiment performed with the blocking IL-18R antibody (10 g per mL).

Full figure and legend (26K)

IL-18 alone had no marked effect on MHC class II. In the presence of low doses of IFN (5–10 ng per mL), however, IL-18 was able to increase cell surface expression of MHC class II by more than 50% on human keratinocytes (Figure 6a and b).

Figure 6.

Interleukin (IL)-18 induces functionally active major histocompatibility complex (MHC) class II expression in human keratinocytes. Keratinocytes were incubated overnight with interferon (IFN) (5 ng per mL) and IL-18 (50 ng per mL). MHC class II was upregulated by IL-18 as determined by flow cytometry (A, B). (B) Eleven independent experiments are summarized. MHC II was significantly upregulated by IFN and IFN+IL-18 as determined by the t test. The difference between IFN- and IFN+IL-18-stimulated keratinocytes in MHC II expression was significant as determined by paired the t test (p=0.03). (C) Purified CD4+ T cells from healthy donors were incubated with cultured keratinocytes from healthy individuals that had been exposed to IFN or IFN+IL-18. These stimuli were carefully washed out before start of the coculture. Coculture was performed in the presence of the superantigen staphylococcal enterotoxin B (SEB) (100 pg per mL) for 2 h in keratinocyte growth medium. CD4+ T cells (in six experiments autologous, and in four heterologous highly purified CD4+ T cells were used) were then transferred to IAB medium and cultured for another 40 h. Supernatants were harvested and IFN was determined by ELISA. Values found for CD4+ T cells cultured with IFN pre-treated keratinocytes in the presence of SEB were set to 100%. Difference in IFN of CD4+ T cells cocultured with IFN-treated as compared with IFN+IL-18-treated keratinocytes was significant as determined by a paired the t test with the raw data (p=0.02). Where indicated, cell-to-cell contact was averted using a 0.2 m transwell.

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IL-18-induced MHC class II expression in human keratinocytes was of functional significance in a coculture system with CD4+ T cells

The IL-18-induced MHC II expression was of functional significance as indirectly determined by stimulation of purified CD4+ T cells (10 independent experiments were performed; in six experiments, autologous CD4+ T cells were used and in four experiments heterologous highly purified CD4+ T cells) in the presence of the staphylococcal enterotoxin B (SEB), which functions as a superantigen: T cells were stimulated with SEB in the presence of cultured keratinocytes that had been pretreated with IFN alone or with IFN+IL-18 (stimuli were carefully washed out before start of the coculture). IFN+IL-18 pre-treatment of keratinocytes led to higher production of IFN by T cells after coincubation in the presence of SEB (Figure 6c). In addition, T cells produced significantly higher amounts of IFN when cell-to-cell contacts to keratinocytes were allowed compared with T cells that were separated from keratinocytes in a transwell system (Figure 6c).

IL-12 does not act synergistically with IL-18 on human keratinocytes

We carefully tested for synergistic effects of IL-12 or IL-23 and IL-18. For the parameters examined (CXCL10 production, MHC class I and II expression), IL-12 did not act in synergy with IL-18 on human keratinocytes. For none of the IL-18 effects found we could observe a cooperation of the two cytokines as it has been described for numerous other effects. This may be because of different IL-12-signalling pathways in keratinocytes as compared with blood-derived cells.

Discussion

Keratinocytes represent the major constituent of the skin and participate actively in the skin immune system by producing various soluble mediators. Keratinocyte-derived chemokines are important for the recruitment and activation of immune cells, and the formation of the T cell-rich infiltrate that characterizes chronic inflammatory skin diseases such as allergic contact dermatitis, psoriasis, or AD. In these skin diseases, T cells actually invade the epidermal compartment, thus allowing T cell keratinocyte interaction. Keratinocytes are viewed as non-professional, tolerizing APC in normal skin. Activated keratinocytes, however, produce inflammatory cytokines and express MHC class II molecules and CD54. They also express costimulatory molecules such as CD80 (only upon stimulation) and CD40. Thus, there is increasing evidence that keratinocytes directly participate in cutaneous immunologic diseases (^{Fan et al, 2003}).

IL-18 has been shown to have a broad range of effector functions beyond lymphocyte activation that implicate IL-18 as an important regulator of both innate and acquired immunity (^{Akira, 2000;McInnes et al, 2000}). IL-18-mediated processes have been implicated in a number of chronic inflammatory disorders. Our study further highlights the role that IL-18 may play in chronic inflammatory skin diseases by its capacity to activate keratinocytes.

Expression of IL-18R and IL-18R on human keratinocytes has been shown on the mRNA level (^{Mee et al, 2000}), but upregulation by any stimulus has not been investigated so far. In this work, we show in support of the mRNA data constitutive expression of IL-18R at the protein level, which can be markedly upregulated by IFN in combination with either TNF or double-stranded RNA (poly I:C).

The IL-18R complex can be upregulated on naïve T cells, Th1, NK cells, and B cells by IL-12 (^{Yoshimoto et al, 1998;Sareneva et al, 2000}). By contrast, IL-12 did not upregulate the expression of IL-18R on keratinocytes on the protein level as shown in our study, nor on the mRNA level (^{Mee et al, 2000}). Although a response of keratinocytes toward IL-12 has been described (^{Schwarz et al, 2002}), it has not been shown so far that they express the IL-12 receptor. Here, we could show that human keratinocytes express the IL-12R1, but not the high-affinity receptor IL-12R2. Different receptor expression and signalling toward IL-12 in keratinocytes as compared with lymphocytes could be responsible for the lack to interfere with the "IL-18 system".

On the other hand, IL-4 was effective in downregulating the receptor as it has been described for lymphocytes (^{Sareneva et al, 2000;Smeltz et al, 2001,2002}). It is known that keratinocytes are good responders to IL-4, whereas the spectrum of the biological effects of this cytokine to keratinocytes is quite broad (^{Albanesi et al, 2000}) and includes pro-inflammatory as well as anti-inflammatory effects.

The IL-18R complex-induced-signalling pathways are shared with other IL-1R family members. These involve MyD88, IRAK, TRAF6, and NF-B nuclear translocation (^{Adachi et al, 1998;Kojima et al, 1999}). Dominant-negative transfectants of IB have been shown to inhibit IL-18-dependent IB degradation and NF-B activation in KG-1 cells (^{Kojima et al, 1999}). In addition to IRAK/TRAF6 signalling, evidence suggests a role for mitogen-activated protein kinase (MAPK) in IL-18 signalling. Thus, activation of the MAPK p38 and ERK by IL-18 was detected in a human NK cell line (^{Kalina et al, 2000}). IL-18 signalling via NF-B, but not MAPK, has been shown for human monocytes (^{Dai et al, 2004}); activation of both NF-B and MAPK pathways has been shown in synovial fibroblasts (^{Morel et al, 2002}). Our data extend the notion that IL-18 signals via NF-B pathway also in human keratinocytes. In the study by^{Lee et al (2004)}, human epithelial cells (A549) were stably transfected with the IL-18R chain and responded to IL-18 with increased production of IL-1, IL-6, and IL-8. Signalling via the MAPK p38 but not NF-B pathway was observed in the study. Differences from our observations might be explained by different culture systems. We used human primary keratinocytes and have not been working with cell lines. Furthermore, our cells were not transfected and were cultured in serum-free medium.

Keratinocytes seem to have a unique IL-18R regulation and response pattern toward IL-18 compared with other cell types. Here we report on secretion of CXCL10 upon IL-18 stimulation by human keratinocytes, which has not been described before. As mentioned above, clear differences exist in receptor regulation compared with cells of the lymphocyte lineage/NK cells. Also, the effects described here are not all comparable with those found for IL-18 in non-lymphocytic cells. In contrast to data found on human DC (^{Gutzmer et al, 2003}) and human fibroblast/endothelial cells (^{Morel et al, 2001}), we could not find an increased CD54 expression induced by IL-18 on human primary keratinocytes. Costimulatory molecules were not affected either (CD40, CD80, CD86). IL-18 induces IL-6, IL-8, CD54, and matrix metalloproteinase expression in vascular smooth muscle cells, endothelial cells, and macrophages (^{Gerdes et al, 2002}). Using the CBA, we could not observe a consistent IL-18-induced production of IL-8, TNF, IL-1, IL-10, IL-12p40, or IL-6 in human primary keratinocytes. Furthermore, we could not detect increased apoptosis in our cultured keratinocytes stimulated with IL-18, nor could we detect induction of FasL by IL-18 as it has been described for NK cells.^{Yamanaka et al (2000)} have shown in skin-specific IL-1 converting enzyme-transgenic mice, which constitutively secrete mature IL-18, spontaneous development of skin ulcers with massive keratinocyte apoptosis. According to our data, IL-18 does not seem to play a direct role in skin apoptosis in the human system.

Our data support the notion that IL-18 is involved in the pathogenesis of local Th1 responses in chronic inflammatory skin diseases such as psoriasis and AD (^{Ohta et al, 2001;Novak et al, 2004}). Here we show that IL-18R is upregulated in both diseases and that keratinocytes from lesional psoriasis are more responsive to IL-18 than cells from normal skin. In eczematous skin diseases, the development of skin lesions from acute to chronic is accompanied by a switch from a more Th2 cytokine pattern to a Th1-like cytokine pattern expressed in the cutaneous lesions (^{Leung and Bieber, 2003}). The process of the shift from Th2 (^{Wittmann et al, 2004}) to Th1 dominance in the lesions is largely unknown but it is speculated that IL-12 (from FcRI-activated inflammatory dendritic epidermal cells;^{Novak et al, 2004}, skin invading cells such as macrophages or eosinophils) together with IL-18 leads to the activation of Th1 cells in this process. IL-18 (together with IL-12) may not only act directly on infiltrating T cells to produce IFN, but keratinocytes may be directly involved in perpetuating a Th1-like reaction pattern. At sites of acute inflammation, the IL-18R is upregulated on keratinocytes, rendering them highly responsive toward IL-18. By production of CXCL10, CXCR3 expressing IFN-producing T cells are attracted to the site of inflammation. Here they encounter MHC class II expressing keratinocytes, which in combination with superantigens—which have been shown to be present in AD epidermis and psoriasis (^{Leung et al, 1995})—induce an T cell stimulation resulting in high IFN production.

As acute flareup lesions of AD have been investigated in this study, the presence of the Th2 cytokines IL-4/IL-13 might be responsible for the lower expression level of IL-18R on AD keratinocytes as compared with keratinocytes freshly isolated from psoriasis lesions. Presumably, a higher expression level might be found in the more Th1-like chronic phase of the disease.

The data presented here also support an involvement of epithelial cells themselves in the protective effect of IL-18 in the situation of epidermal viral infection by upregulation of MHC class I. It is known that IL-18 plays a part in the clearance of viruses. IL-18 is protective in a murine model of Herpes simplex virus (HSV) infection (^{Fujioka et al, 1999;Harandi et al, 2001;Malmgaard and Paludan, 2003}). Downmodulation of IL-18-induced immune responses by human papilloma virus (HPV) oncoproteins may contribute to viral pathogenesis or carcinogenesis. This may arise via HPV binding to the IL-18R, thus preventing IL-18 induction of IFN (^{Cho et al, 2001}). So, in epidermal viral infections such as HSV, HPV, and varicella-zoster virus, in addition to inducing IFN, and activating CD8+ T cells, IL-18 produced by keratinocytes may act in an autocrine fashion.

But important questions remain. To completely understand the "IL-18" system in the skin and the outcome of IL-18 "activity" on the skin immune response, it is necessary to further investigate the complex interaction of the players in this system: caspases 1 and 3 (^{Akita et al, 1997}) activity, proteinase 3 activity, IL-18R binding IL-1H (^{Pan et al, 2001}), and secretion of IL-18 BP (^{Muhl et al, 2000}). Finally, the position of IL-18 in the functional hierarchy of pro-inflammatory cytokines in chronic inflammation is not fully resolved.

Materials and Methods

Cytokines and reagents

All cytokines were used as purified recombinant human preparations. Human recombinant IL-13 (50 ng per mL), TNF (200 U per mL), and CXCL10 (10 or 100 ng per mL) were purchased from Tebu/Peprotech (Frankfurt a. M., Germany); IFN (5–10 ng per mL), IFN1a (300 U per mL), and IL-1 (200 U per mL) from ImmunoTools (Friesoythe, Germany); IL-4 (20–50 ng per mL), IL-12 (1 or 10 ng per mL), IL-23 (10–50 ng per mL), IL-18 (5 or 50 ng per mL), and mouse anti-human IL-18R mAb (clone 132,016 for neutralizing of IL-18 effects: 10 g per mL) from R&D Systems (Wiesbaden, Germany); and GM-CSF (500 U per mL, Essex Pharma, Munich, Germany), poly I:C (200 ng per mL, Sigma-Aldrich, Deisenhofen, Germany), IFN2a (200 U per mL, Roche Applied Science, Mannheim, Germany), and SEB (100 pg per mL) were purchased from Toxin Technologies/Alexis (Gruenberg, Germany).

Cell isolation and culture

Peripheral blood mononuclear cells were separated by Ficoll–Hypaque density gradient centrifugation. CD4+ T cells were isolated using a negative selection kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Purity of the resulting CD4+ T cells was 95% as verified by flow cytometry analysis of CD4+ T cells (mouse anti-human CD4 monoclonal antibody; Beckman-Coulter, Krefeld, Germany). After separation, cells were resuspended in Iscove medium (Biochrom, Berlin, Germany) supplemented with 4% human AB serum.

Primary cultures of normal human keratinocytes were prepared from foreskin of children undergoing surgery. Skin specimens were cut into small pieces and incubated overnight in dispase II (2.4 U per mL, Roche Applied Science) at 4°C. Epidermis was detached from the dermis with fine forceps and incubated in Hank's solution with 0.25% trypsin (Sigma-Aldrich) for 20 min at 37°C. Trypsin activity was stopped by addition of FCS (Life Technolologies, Karlsruhe, Germany). The cell suspension was passed through a 40 m sterile gauze. The obtained keratinocytes were washed twice and single-cell suspension of keratinocytes was cultured in serum-free keratinocytes growth medium (Keratinocyte Growth Medium 2 Kit; PromoCell GmbH, Heidelberg, Germany). All cell cultures were incubated in a humidified atmosphere containing 5% CO₂ at 37°C and were used at the passage two to five. Hydrocortisone-free medium was used for all experiments. The purity of keratinocytes was verified by the expression of the epithelial marker cytokeratin (mouse anti-human cytokeratin antibody, clone: MNF-116, DakoCytomation, Hamburg, Germany) and the fibroblast-specific marker ASO2 (CD90, Dianova GmbH, Hamburg, Germany). All cells (more than 95%) were found to be uniformly positive for cytokeratin but not for CD90.

Assessment of IL-18R in fresh human skin biopsies

Punch skin biopsies of 4 mm were obtained from patients with active psoriasis, AD, and healthy individuals after informed written consent. All psoriasis patients (type II psoriasis) suffered from plaque psoriasis (no psoriasis arthritis); AD patients were affected by the extrinsic form and presented with acute flareup lesions. None of the patients received any systemic immunosuppression or anti-histamine treatment. The study was approved by the Ethics committee of the Hannover Medical School, Hannover and was conducted according to the Declaration of Helsinki Principles. For obtaining fresh keratinocytes, skin biopsies were immediately cut into small pieces and incubated in 0.25% trypsin (Sigma-Aldrich) for 20 min at 37°C. The digestion was stopped by addition of FCS (Life Technologies). The single-cell suspension was prepared after passing the cells through a 40 m sterile gauze. Assessment of IL-18R on keratinocytes was performed by flow cytometry. Keratinocytes were identified by cytokeratin-positive staining. More than 85% of the cells were positive for cytokeratin. Eight to 15% CD3+ cells could be detected, but no CD1a+ cells.

Flow cytometric analysis of intracellular and membrane molecules

Expression of surface antigens was assessed as described previously (^{Wittmann et al, 2002}). The following PE- or FITC-labelled monoclonal anti-human antibodies were used: IL-18R (clone 70,625 R&D Systems), IL-12R1 (clone 2.4E6), IL-12R2 (clone 2B6/122), and MHC class II (clone G46-6 (L243), BD Biosciences, Heidelburg, Germany), MHC class I (clone W6/32, Biosource, Solingen, Germany), CD54 (clone 84H10), and CD40 (clone MAB89, Beckman-Coulter). All surface markers used in the study were verified to stain for trypsin-insensitive epitopes (as assessed for the trypsin concentration and incubation period used). Some keratinocyte cultures did not respond to IFN (less than 1.5-fold increase of MHC I or II expression as determined by shift in geometric mean); these experiments were excluded from analysis. Cytokeratin (DakoCytomation) was detected by intracellular staining using the Cytofix/Cytoperm kit (BD Biosciences). Stained cells were measured by flow cytometry (FACSCalibur) and analyzed using CELLQuestPro software (BD Biosciences). Geometric mean values were analyzed to determine differences in receptor expression.

NF-B staining

We have used a flow cytometric technique to study translocation of NF-B from the cytoplasm to the nuclei of the cell as published previously (^{Foulds, 1997}). In brief, after stimulation of the cells (as indicated), intact nuclei were isolated using the CycleTest PLUS DNA Reagent Kit (BD Biosciences) according to the manufacturer's instruction. 2.5 g per mL of mouse monoclonal anti-human NF-B (p65) antibody (clone 112A1021, Imgenex, San Diego, California) or isotype-matched controls were then added for 20 min at room temperature followed by further 20 min incubation with 1 l goat anti-mouse FITC antibody (Beckman-Coulter). Two hundred microliters of cold propidium iodide (PI) solution was added to the nuclei and the preparation was incubated for further 10 min. Acquisition of stained nuclei was carried out on a FACS Calibur (BD Biosciences) equipped with an electronic doublet discrimination module. The singlet population was analyzed for FITC staining.

Quantification of cytokines and chemokines

IFN in the cell-free supernatants of cultured human CD4+ T cells was measured after 48 h. IFN was detected by ELISA (Ready-Set-Go human IFN ELISA, Biocarta, Hamburg, Germany). The concentration of human CXCL10 was determined in supernatants of human keratinocytes according to the manufacturer's instruction (IP-10, OptEIA, BD Biosciences). For analysis of CXCL10 production by IL-18 alone, experiments with a very high spontaneous CXCL10 production (>150 pg per mL CXCL10 production by non-stimulated cultured keratinocytes) were excluded. The CBA human inflammation kit allowing simultaneous detection of IL-12p40, IL-1, IL-6, IL-8, and TNF, and IL-10 assay was performed according to the manufacturer&aposs instruction (BD Biosciences).

Statistical analysis

Comparative data were analyzed using the t test (data are depicted in columns) or paired t test (paired data are depicted). The software used to perform the statistical analysis was SigmaStat for Windows version 2.03. Mean valuesSEM are depicted. In the figures, ^*p-value<0.05, ^**p<0.02, and ^***p<0.01.

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Acknowledgments

Special thanks to Dr med. Kolb for continuous support of our keratinocyte project. We also thank Tanja Stünkel for excellent technical assistance. This study was supported by DFG grant SFB 566, A6.