Human keratinocytes are major producers of IL-18: predominant expression of the unprocessed form.

ARTICLE

ABBREVIATIONS

DTH: delayed-type hypersensitivity
EC: epidermal cells
ECS: epidermal cell suspension
HBSS: Hanks' buffered saline solution
HLA: human leukocyte antigen
HRP: horseradish peroxidase
HPRT: hypoxantine phosphatidyl ribosyltransferase
ICE: interleukin-1 converting enzyme
IFN-gamma: interferon-gamma
IGIF: interferon-alpha-inducing factor
IL-1beta: interleukin-1beta
IL-18betaP: IL-18 binding protein
LPS: lipopolysaccharide
MEL CR: mixed epidermal cell leukocyte reaction
pAb: psyclonal antibod
PBMC: peripheral blood mononuclear cells
PMA: phorbol 12-myristate 13-acetate
RT: room temperature

INTRODUCTION

IL-18 (interferon-gamma-inducing factor, IGIF) was initially discovered in studies of IFN-gamma production in a Propionibacterium acnes-induced model of toxic shock [1]. The amino acid sequence of IL-18 is distinct from other cytokine sequences, but structural analysis and fold recognition studies suggest that IL-18 is a member of the IL-1 family [2]. This is supported by the observation that IL-18, like IL-1beta, is processed from an inactive precursor molecule into its bioactive form by caspase-1 (interleukin-1-converting enzyme, ICE) [3-5]. Furthermore it has been shown that IL-18 is bound by members of the IL-1R family, namely IL-1Rrp1 and AcPL (6, 7 and Debets et al. manuscript in preparation) and that IL-18 activates classical IL-1 signaling components, such as myD88, IRAK-1, TRAF-6 and NF-kappaB [8-11]. Despite the structural similarity, human IL-18 shows only 15-18% sequence homology with the IL-1 family of cytokines. To date, the most well documented biological effects of IL-18 are induction and enhancement of IFN-gamma production by Th1 cells (in combination with IL-12) [10, 12, 13], enhancement of Th1 proliferation, and stimulation of cytolytic activity of NK-cells [14-16]. Taken together, these data show that IL-18 plays an important role in inflammation.

In skin, interaction between keratinocytes and leukocytes is of vital importance for maintaining homeostasis, especially during defense [17, 18]. The balance between cytokines such as TNF-alpha, IFN-gamma, IL-12, IL-4, IL-10 and members of the IL-1 family plays a pivotal role in maintaining the immune milieu of the skin [19-21]. Expression and activity of these cytokines, expressed by different cell types (including keratinocytes) is disturbed in some skin disorders. Psoriatic lesions, for instance, are characterized by a disturbed balance between agonists and antagonists of the IL-1 system [22] and by a disturbed responsiveness to (increased levels) of IFN-gamma [23, 24]. The main cytokines responsible for the induction and expression of IFN-gamma are IL-12 and IL-18. Upregulation of IL-12 expression has been reported in psoriasis [25, 26]. In contrast, little is known about the regulation of IL-18 expression and processing in human skin and keratinocytes. Given the obvious involvement of the IL-1 system in inflammatory skin diseases and the properties of IL-18, a role for IL-18 in the skin can be expected. Murine keratinocytes constitutively produce IL-18 mRNA and its expression can be upregulated in vivo by contact allergens, but not by irritants. Keratinocytes turn out to be a major source of active protein [27, 28]. One study in human skin on the accessory cell function of epidermal cells (EC) in the mixed epidermal cell leukocyte reaction (MECLR), showed that endogenously produced IL-18 was of minor importance in this system [29]. Because regulation of IL-18 expression might be essential in inflammatory reactions in the skin, i.e. by maintaining the delayed type hypersensitivity (DTH)-like environment and Th1 polarization, we aimed to analyze whether human keratinocytes are able to produce IL-18 and how its production is regulated. In this report we show that IL-18 mRNA and protein are constitutively expressed by human epidermal cells in vitro as well as in vivo, and by the human keratinocyte cell line HaCaT. Intracellular IL-18 in epidermal cells and HaCaT cells is mainly in the unprocessed 24 kD pro-form. We observed considerably higher levels of pro-IL-18 in normal epidermal cells and HaCaT cells than in peripheral blood leukocytes and the bronchial epithelial cell line BEAS2B. Finally, strong stimulation of HaCaT cells, BEAS2B cells and PBMC by PMA, LPS or IL-1beta did not affect intracellular or released levels of total IL-18 protein. These results show that human keratinocytes are major producers of IL-18, which is predominantly expressed in the unprocessed form.

METHODS

Epidermal cells

Dermatome specimens were obtained after informed consent from normal skin of patients undergoing breast or abdominal plastic surgery in the University Hospital Rotterdam. The epidermis was detached from the dermis by trypsinisation. Epidermal cell suspensions (ECS) were prepared from epidermal sheets by incubating them at 37° C for 45 min in trypsinisation buffer (0.025% trypsin and 0.1% EDTA in PBS), to which 0.25% DNAse was added for the last 15 min of the incubation (Boehringer Mannheim, Mannheim, Germany). The cell suspension was filtered through a 30 mum mesh gauze and suspended in PBS containing trypsin inhibitors (Boehringer Mannheim), and adjusted to a concentration of 4 x 10⁶ EC per ml.

HLA-DR⁺ cells were depleted from total ECS, using para-magnetic "Dynabeads" coated with anti-HLA-DR mAb (Dynal, Oslo, Norway). The mixture of EC and dynabeads was centrifuged gently for 10 min (200 x g at room temperature (RT)) and incubated for 30 min on ice. The HLA-DR⁺ cells were removed from the total ECS using the Dynal magnetic particle concentrator (MPC^®-1). ECS depleted of Langerhans and other HLA-DR⁺ cells (ECS^­) were resuspended in 1 ml of Hanks' balanced salt solution (HBSS) (Gibco BRL, Paisley, Scotland), supplemented with a broad mixture of protease inhibitors (Complete™, Boehringer Mannheim). Intracellular proteins were extracted by 4 freeze-thaw cycles and stored at ­ 80° C.

Leukocytes, PBMC and monocytes

Heparanized blood was drawn by venipuncture of healthy volunteers after informed consent. Leukocytes were isolated using a standard procedure. In brief, 2 ml of whole blood was diluted in 50 ml lysis buffer (NH₄Cl, pH 7.4), incubated on ice for 20 min and centrifuged for 5 min (450 x g at RT) followed by washing with PBS. Leukocytes were counted using a hemacyto-counter (Coulter ZM, Beckman Coulter, Fullerton, CA) and the cells were resuspended in HBSS, supplemented with a broad mixture of protease inhibitors (Boehringer Mannheim) and adjusted to a concentration of 75 x 10⁶ cells/ml. PBMC were isolated using standard Ficoll gradient centrifugation. Normal human monocytes were kindly provided by Prof. Drexhage (Dept. of Immunology, Erasmus University Rotterdam, The Netherlands) and were isolated by layering PBMC on a percoll solution followed by centrifugation for 40 min (300 x g at RT). Cells at the interface were isolated and counted. Monocyte isolation resulted in an average purity of 88% (range: 83-93%). The intracellular proteins from the cells were isolated by 4 freeze-thaw cycles.

Cell lines and stimulation experiments

HaCaT cells [30] were cultured in RPMI 1640 (GibcoBRL) supplemented with 5% fetal calf serum (FCS) (BioWhittaker, Walkersville, MD, USA) at 37° C and 5% CO₂. The cells were passaged every 5 days. Prior to an experiment, HaCaT cells were detached using trypsinisation buffer, rinsed in PBS, taken up in IMDM (GibcoBRL) containing 1% human serum (HS) (Sigma, St. Louis, MO, USA) and plated at 5 x 10⁵ cells per well in 24-well plates (Nunc, Roskilde, Denmark). The cells were allowed to adhere to the plates for 18 hours, after which they were rinsed with PBS. IMDM containing 1% HS with or without stimuli was then added to the cells which were subsequently cultured for different periods of time (0-72 hours). HaCaT cells were stimulated with 10 mug/ml lipopolysaccharide (LPS) (Brunschwig, Amsterdam, The Netherlands), 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) or 250 U/ml IL-1beta (Glaxo, Research Triangle Park, NC, USA). Supernatants were collected after centrifugation for 1 min (17,000 x g at 4° C) and stored at ­ 80° C until further analysis. Cells were detached as described above, washed in PBS and resuspended in 400 mul HBSS, supplemented with a broad mixture of protease inhibitors (Boehringer Mannheim). To obtain intracellular proteins, the cells were subjected to four cycles of freeze thawing, and the extracts were stored at ­ 80° C until analysis. The bronchial epithelial cell line BEAS2B was cultured as described previously [31] and PBMC were isolated as described above. Both cell types served as a control and were cultured and stimulated identicaly to the HaCaT cells.

Immunohistochemistry

Biopsies were obtained from 5 patients undergoing breast or abdominal plastic surgery. The biopsies were snap frozen in liquid nitrogen and cryosections were cut using a cryostat (Jung Frigocut 2800 E, Leica, Rijswijk, The Netherlands). Sections were fixed in acetone for 10 min and blocked for 10 min with PBS containing 0.05% Tween 20 (Merck, Whitehouse Station, NJ, USA) and 1% HS at RT. The fixed tissue was subsequently incubated for 18 hours at 4° C with a mouse anti-human IL-18 specific primary mAb (MAB318, R&D Systems, Minneapolis, MN, USA), followed by an incubation for 30 min with a phosphatase-linked secondary rabbit anti mouse polyclonal antibody (pAb) (DAKO, Carpinteria, CA, USA). Isotype controls were stained with an irrelevant antibody of the same isotype as the IL-18 specific antibody (anti-KLH IgG_2A mAb, MAB003, R&D Systems). 3-amino-9-ethylcarbazole (Sigma) was used as the chromogen.

Western blotting and immunodetection of IL-18

Proteins were separated using 15% SDS-PAGE gels according to Laemmli [32]. The proteins were blotted onto Hybond-C membranes (Amersham, Little Chalfont, UK) using an electroblot system (BioRad, Hercules, CA, USA). The membranes were blocked with Tris buffered saline (TBS) containing 5% low fat milk and 0.05% Tween 20 for 1 hour at room temperature (RT). Blots were stained with a primary antibody against IL-18 (MAB318, R&D systems), followed by a secondary biotin-labelled anti-mouse polyclonal antibody (Amersham) and streptavidin poly-horseradish peroxidase (HRP) (CLB, Amsterdam, The Netherlands). Isotype controls were stained with an irrelevant antibody of the same isotype as the IL-18 specific antibody (anti-KLH IgG_2A mAb, MAB003, R&D Systems). IL-18 specific staining was detected using a chemoluminescence substrate (Pierce, Rockford, IL, USA).

Cytokine specific ELISA

Maxisorb ELISA plates (Nunc, Roskilde, Denmark) were coated for 18 hours at 4° C with 100 mul of either 2 mug/ml anti-human-IL-18 mAb (MAB318, R&D systems) or 0.5 mug/ml anti-human-IL-6 mAb (Biosource, Camarillo, CA, USA) followed by blocking with 1% BSA (Sigma). One hundred mul of IL-18 standard or sample and 50 mul of either 0.2 mug/ml biotin-linked anti-human-IL-18 pAb (BAF318, R&D systems) or 0.2 mug/ml biotin-linked anti-human-IL-6 pAb (Bioscource) detection antibody was simultaneously added to each well. The standards were diluted in sample buffer (HBSS or IMDM + 1% HS). Samples, standards and detection antibodies were incubated for 2 hours at RT. Cytokines were detected by using Streptavidin linked HRP (CLB) and TMB peroxidase substrate (Kirkegaard & Perry, Gaithersburg, MD, USA). The OD was measured at 450 nm.

RNA isolation and RT-PCR

RNA was isolated from 1 x 10⁶ cells using the guanidine thiocyanate extraction procedure [33]. RNA was reversed transcribed into cDNA and PCR reactions were performed as previously described [22, 31]. In each sample hypoxantine phosphatidyl ribosyltransferase (HPRT) cDNA was measured as a control.

The sequences of the primers were as follows: IL-18 (forward): 5'-GTC TTC GTT TTG AAC AGT GAA-3'; IL-18 (reverse): 5'-TAC TTT GGC AAG CTT GAA TCT-3'; HPRT (forward): 5'-GTG ATG ATG AAC CAG GTT ATG ACC TT-3'; HPRT (reverse): 5'-CTT GCG ACC TTG ACC ATC TTT GGA-3'. The predicted sizes of the PCR products were 470 bp for IL-18, and 454 bp for HPRT. The products were separated on a 1% agarose gel containing ethidium bromide, visualised by UV light and the gels were photographed.

RESULTS

IL-18 is constitutively produced by keratinocytes in normal skin

Biopsies from normal skin were taken to investigate expression of IL-18 in normal epidermis in vivo using immunohistochemistry. IL-18 expression was observed in both the dermis and epidermis (Figure 1A). In the dermis, strong positive staining in the cytoplasm of cells with dendritic morphology was seen (Figure 1B). The epidermis showed diffuse staining, but also local staining of some strongly positive cells with dendritic morphology, probably Langerhans cells (Figure 1B). Keratinocytes were more diffusely stained. Incubation of normal skin sections with an isotype control antibody did not show any reaction (Figure 1C). These results indicate that keratinocytes express IL-18 in vivo.

IL-18 mRNA and protein are constitutively expressed by epidermal cells and HaCaT cells in vitro

IL-18 mRNA expression by normal epidermal cells (EC), HaCaT cells, PBMC and cells from the bronchus epithelial cell line BEAS2B, was analyzed using RT-PCR. IL-18 mRNA was expressed by all cell types, and no marked visual differences in mRNA levels were detected (Figure 2). Detection of IL-18 protein in cellular lysates of HaCaT cells (by use of ELISA) revealed that HaCaT cells constitutively expressed intracellular IL-18 protein, i.e. extracts of 1 x 10⁵ freshly harvested HaCaT cells contained 816 ± 140 pg IL-18. As a comparison, extracts of 1 x 10⁵ EC contained about 100 pg `IL-18, and EC suspensions depleted of HLA-DR⁺ cells contained similar levels of IL-18 (see below). The intracellular IL-18 concentration in HaCaT cells did not change significantly during culture in medium (Figure 3A). To assess whether human keratinocytes also secrete IL-18 protein, supernatants of HaCaT cells cultures were analyzed. After 3 hours of culture (without stimulation), an average of 115 ± 5 pg/ml IL-18 was measured. After subsequent culture for 24 hours, the IL-18 concentration had dropped to 86 ± 4 pg/ml and decreased further to 39 ± 1 pg/ml after 48 hours (Figure 3B). This decreased detection of IL-18 secretion could be due to epitope blocking because of the binding of IL-18 to IL-18 binding protein (IL-18BP) or the IL-18 receptor. These data show that normal keratinocytes and HaCaT cells constitutively express IL-18 mRNA and protein.

Intracellular-produced IL-18 in epithelial cells is mainly in the unprocessed form

To determine if the IL-18 produced by EC, wether or not depleted of HLA-DR⁺ cells, HaCaT and BEAS2B cells, was in the processed or the unprocessed form, we analyzed extracts and supernatants of these cells using Western blotting and an mAb specific for both the processed and unprocessed forms of IL-18 (MAB318). All extracts contained the 24 kD unprocessed IL-18 and no processed IL-18 could be detected (Figure 4). As illustrated in figure 4, HaCaT cells contained the most pronounced amount of pro-IL-18, followed by EC and BEAS2B cells. ELISA (data not shown) confirmed these data. The levels of secreted IL-18 were below the detection limits of the Western blotting assay used. These data show that normal keratinocytes, HaCaT and BEAS2B cells constitutively express unprocessed IL-18.

Normal human epidermal cells produce more (pro)-IL-18 than leukocytes, PBMC, monocytes and bronchial epithelial cells

Next, we asked whether cells other than EC also produced (pro) IL-18, and how this production relates to the amounts produced by epidermal cells. Cellular extracts from 3 x 10⁵ leukocytes, PBMC, monocytes, total EC and HLA-DR⁺ cell-depleted EC were analyzed by Western blot for their IL-18 content. Amounts of (pro) IL-18, detected in extracts of total leukocytes, PBMC or monocytes, were relatively low as compared to EC, depleted of HLA-DR⁺ cells or not, these extracts contained high amounts of unprocessed IL-18 (Figure 5). ELISA results show that the extracts of 1 x 10⁵ total EC and HLA-DR⁺ cell-depleted EC contained 99.89 + 23.48 pg (range: 83.25-116.49 pg) and 81.66 + 12.43 pg (range: 68.71-95.85 pg) of IL-18 respectively. The extracts of 1 x 10⁵ leukocytes, PBMC and monocytes, however, contained 0.81 ± 0.61 pg (range: 0.99-1.39 pg), 3.36 ± 5.13 pg (range: 0.39-9.29 pg) and 1.22 ± 0.40 (range: 0.88-1.79 pg) of IL-18, respectively. During culture in normal medium, intracellular IL-18 expression in PBMC (Table 1) and BEAS2B cells (data not shown) was about 150 fold and 10 fold lower on average respectively, when compared to IL-18 expression in HaCaT cells. IL-18 secretion by BEAS2B cells and PBMC was 6 fold lower on average than the production by HaCaT cells.

These data show that normal keratinocytes and HaCaT cells produce a considerably larger amount of total IL-18 protein than peripheral blood leukocytes and BEAS2B cells.

Stimulation of keratinocytes with LPS, PMA and IL-1beta

To investigate whether IL-18 protein expression by keratinocytes could be altered by robust stimuli like LPS, PMA or IL-1beta, HaCaT cells were cultured for different periods of time in the presence of these compounds. BEAS2B cells, representing bronchial epithelial cells, and PBMC were cultured under similar conditions to the HaCaT cells, for comparison. Stimulation of HaCaT cells with LPS, PMA or IL-1beta (Figure 3) and PBMC (Table 1) or BEAS2B (data not shown) with LPS or PMA did not alter intracellular expression of total IL-18 protein, nor did it alter secreted levels of IL-18 protein. To confirm that the cells were able to respond to the given stimuli, IL-6 levels were measured in the supernatants. As expected, significant levels of IL-6 were detected after LPS or PMA stimulation, ranging from 9 to 10 times the secretion of non-stimulated cells (data not shown), confirming the activation of the cells.

DISCUSSION

This is the first study which provides in vitro and in vivo evidence that human keratinocytes are a potent source of pro-IL-18 when compared to other epithelial cells, leukocytes, PBMC or monocytes. Our studies on the regulation of mRNA and protein expression, as well as the processing of pro-IL-18 into biologically active IL-18 revealed that keratinocytes preferentially store large amounts of pro-IL-18 intracellularly, and potent stimuli such as LPS, PMA and IL-1beta have no effect on total IL-18 protein expression. We could not show a clear processing of IL-18 after stimulation, in the cell extracts or culture supernatants by the methods used here.

Stoll et al. [27] showed that in murine skin, keratinocytes are the major source of IL-18. The data presented here show that human keratinocytes express and release IL-18 as well. Because bioactive IL-18 may promote IFN-gamma synthesis in normal skin, we investigated whether IL-18 was expressed in the unprocessed (24 kD) or processed (18 kD) form. Our results show that IL-18 expressed by HaCaT and normal keratinocytes was essentially in the non-active, unprocessed 24 kD form. This is in accordance with the expression of IL-1beta, which in normal skin is also synthesized in the unprocessed 32 kD form [34]. Expression of bioactive IL-1beta in keratinocytes is dependent on caspase-1 activity [35]. Whether keratinocytes express caspase-1 has been a matter of controversy, until Zepter et al. [35] showed that IL-1beta in human keratinocytes is processed by caspase-1 which in turn is upregulated upon stimulation by urushiol and irritant chemicals. Pro-IL-18 may be converted into its active form in the same way. If so, keratinocytes are a reservoir of non-active IL-18, which can be processed by caspase-1 directly after stimulation. However, pro-IL-18 may also be processed by other proteases as is the case for IL-1beta in the epidermis [36, 37]. An important difference between IL-18 and IL-1beta in keratinocytes is that intracellular concentrations of pro-IL-18 are much higher than those of pro-IL-beta. Pro-IL-18 levels correspond better with the intracellular concentrations of IL-1ra and IL-1alpha in skin, which are also high when compared to IL-1beta [38]. Therefore, like IL-1beta, IL-18 may have other functions in addition to its role in the inflammatory response [39].

That human keratinocytes can produce bioactive IL-18 after stimulation with LPS or PMA has recently been demonstrated by Naik et al. [40]. They also showed that IL-18 protein concentration is not altered after stimulation with PMA and LPS, which is confirmed by our studies. In addition we show that keratinocytes express significantly higher levels of IL-18 than bronchial epithelial cells, normal human leukocytes, PBMC and monocytes.

Constitutive expression of pro-IL-18 has also been reported to occur in other cell types. Pizarro et al. [41] showed that, like keratinocytes, gut epithelial cells only express the unprocessed form of IL-18. They also found that in Crohn's disease, in which the Th cell balance is skewed towards the Th1 pole, pro IL-18 is processed into the 18 kD form. A predominant Th1 environment is also apparent in psoriasis, a human inflammatory skin disease. Analagously to Crohn's disease, one would expect processing of IL-18 in psoriatic lesional skin. However, Western blot data did not reveal the 18 kD mature form of IL-18 in extracts of psoriatic lesional skin (unpublished data). Constitutive pro IL-18 expression was also detected in human chondrocytes [42] and stimulation of these chondrocytes with IL-1beta generated the processed form of IL-18. Finally, Puren et al. [43] showed constitutive pro-IL-18 expression in human PBMC. In our experiments, the IL-18 concentration in the PBMC extracts and culture supernatants could be detected by ELISA but was below the detection limit of the Western blot method used. Therefore, it was not possible to determine whether the IL-18 present was in the processed or unprocessed form.

The considerably lower expression of (pro) IL-18 in monocytes compared to keratinocytes, might be explained by the severe and undesirable systemic side effects that may occur if high amounts of pro-IL-18 were to be processed and released upon stimulation by monocytes. In skin, IL-18 could have a more local effect, in analagously to the IL-1 cytokine family. Excess production of IL-18 could then be overcome by the IL-18 BP, which has recently been described by Novick et al. [44]. IL-18BP is also expressed by human keratinocytes, at least on the RNA level (R. Groves, personal communication).

During skin inflammation, the local, intracellulaly stored pro-IL-18 may be rapidly processed, followed by the release of bioactive IL-18 by the keratinocytes. The released IL-18 may then skew T cells towards a Th1 phenotype, characterized by IFN-gamma secretion. Therefore, IL-18, together with IL-12, may be a key cytokine for maintaining the Th1 environment in the skin after proinflammatory stimuli.

CONCLUSION

In conclusion, our data provide additional evidence that IL-18 might play a key role in facilitating the maintenance of a local Th1-like environment in skin during inflammation.

Acknowledgements. The authors wish to thank Dr. J.D. Laman and Dr. H.J.F. Savelkoul for discussing the paper and Mr. Van Os for preparing the figures.

REFERENCES

REFERENCES

1. Nakamura K, Okamura H, Wada M, Nagata K, Tamura T. 1989. Endotoxin-induced serum factor that stimulates gamma interferon production. Infect. Immun. 57: 590.

2. Bazan J F, Timans J C, Kastelein R A. 1996. A newly defined interleukin-1? (letter) Nature 379: 591.

3. Akita K, Ohtsuki T, Nukada Y, Tanimoto T, Namba M, Okura T, Takakura-Yamamoto R, Torigoe K, Gu Y, Su M S S, Fujii M, Satoh-Itoh M, Yamamoto K, Kohno K, Ikeda M, Kurimoto M. 1997. Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells. J. Biol. Chem. 272: 26595.

4. Ghayur T, Banerjee S, Hugunin M, Butler D, Herzog L, Carter A, Quintal L, Sekut L, Talanian R, Paskind M, Wong W, Kamen R, Tracey D, Allen H. 1997. Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature 386: 619.

5. Fantuzzi G, Dinarello C A. 1999. Interleukin-18 and interleukin-1 beta: two cytokine substrates for ICE (caspase-1). (Review) J. Clin. Immunol. 19: 1.

6. Born T L, Thomassen E, Bird T A, Sims J E. 1998. Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signaling. J. Biol. Chem. 273: 29445.

7. Torigoe K, Ushio S, Okura T, Kobayashi S, Taniai M, Kunikata T, Murakami T, Sanou O, Kojima H, Fujii M, Ohta T, Ikeda M, Ikegami H, Kurimoto M. 1997. Purification and characterization of the human interleukin-18 receptor. J. Biol. Chem. 272: 25737.

8. Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami H, Nakanishi K, Akira K. 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9: 143.

9. Kojima H, Takeuchi M, Ohta T, Nishida Y, Arai N, Ikeda M, Ikegami H, Kurimoto M. 1998. Interleukin-18 Activates the IRAK-TRAF6 pathway in mouse EL-4 cells. Biochem. Biophys. Res. Commun. 244: 183.

10. Robinson D, Shibuya K, Mui A, Zonin F, Murphy E, Sana T, Hartley S B, Menon S, Kastelein R, Bazan F, O'Garra A. 1997. IGIF does not drive Th1 development but synergizes with IL-12 for interferon-gamma production and activates IRAK and NFkappaB. Immunity 7: 571.

11. Thomassen E, Bird T A, Renshaw B R, Kennedy M K, Sims J E. 1998. Binding of interleukin-18 to the interleukin-1 receptor homologous receptor IL-1Rrp1 leads to activation of signaling pathways similar to those used by interleukin-1. J. Interferon Cytokine Res. 18: 1077.

12. Yoshimoto T, Takeda K, Tanaka T, Ohkusu K, Kashiwamura S, Okamura H, Akira S, Nakanishi K. 1998. IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-gamma production. J. Immunol. 161: 3400.

13. Ahn H J, Maruo S, Tomura M, Mu J, Hamaoka T, Nakanishi K, Clark S, Kurimoto M, Okamura H, Fujiwara H. 1997. A mechanism underlying synergy between IL-12 and IFN-gamma-inducing factor in enhanced production of IFN-gamma. J. Immunol. 159: 2125.

14. Dao T, Mehal W Z, Crispe I N. 1998. IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells. J. Immunol. 161: 2217.

15. Kohno K, Kataoka J, Ohtsuki T, Suemoto Y, Okamoto I, Usui M, Ikeda M, Kurimoto M. 1997. IFN-gamma-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J. Immunol. 158: 1541.

16. Kanakaraj P, Ngo K, Wu Y, Angulo A, Ghazal P, Harris C A, Siekierka J J, Peterson P A, Fung-Leung W P. 1999. Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice. J. Exp. Med. 189: 1129.

17. Williams I R, Kupper T S. 1996. Immunity at the surface: homeostatic mechanisms of the skin immune system. (Review) Life Sci. 58: 1485.

18. Barker J N, Mitra R S, Griffiths C E, Dixit V M, Nickoloff B J. 1991. Keratinocytes as initiators of inflammation. (Review) Lancet 337: 211.

19. Prinz J C, Gross B, Vollmer S, Trommler P, Strobel I, Meurer M, Plewig G. 1994. T cell clones from psoriasis skin lesions can promote keratinocyte proliferation in vitro via secreted products. Eur. J. Immunol. 24: 593.

20. Horrocks C, Holder J E, Berth-Jones J, Camp R D. 1997. Antigen-independent expansion of T cells from psoriatic skin lesions: phenotypic characterization and antigen reactivity. Br. J. Dermatol. 137: 331.

21. Schlaak J F, Buslau M, Jochum W, Hermann E, Girndt M, Gallati H, Meyer zum Buschenfelde K H, Fleischer B. 1994. T cells involved in psoriasis vulgaris belong to the Th1 subset. J. Invest. Dermatol. 102: 145.

22. Debets R, Hegmans J P, Croughs P, Troost R J, Prins J B, Benner R, Prens E P. 1997. The IL-1 system in psoriatic skin: IL-1 antagonist sphere of influence in lesional psoriatic epidermis. J. Immunol. 158: 2955.

23. Schmid P, Itin P, Cox D, McMaster G K, Horisberger M A. 1994. The type I interferon system is locally activated in psoriatic lesions. J. Interferon Res. 14: 229.

24. Uyemura K, Yamamura M, Fivenson D F, Modlin R, Nickoloff B J. 1993. The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response. J. Invest. Dermatol. 101: 701.

25. Taha R A, Leung D Y, Ghaffar O, Boguniewicz M, Hamid Q. 1998. In vivo expression of cytokine receptor mRNA in atopic dermatitis. J. Allergy Clin. Immunol. 102: 245.

26. Yawalkar N, Karlen S, Hunger R, Brand C U, Braathen L R. 1998. Expression of interleukin-12 is increased in psoriatic skin. J. Invest. Dermatol. 111: 1053.

27. Stoll S, Muller G, Kurimoto M, Saloga J, Tanimoto T, Yamauchi H, Okamura H, Knop J, Enk A H. 1997. Production of IL-18 (IFN-gamma-inducing factor) messenger RNA and functional protein by murine keratinocytes. J. Immunol. 159: 298.

28. Xu B, Aoyama K, Yu S, Kitani A, Okamura H, Kurimoto M, Matsuyama T, Matsushita T. 1998. Expression of interleukin-18 in murine contact hypersensitivity. J. Interferon Cytokine Res. 18: 653.

29. Suemoto Y, Ando O, Kurimoto M, Horikawa T, Ichihashi M. 1998. IL-12 promotes the accessory cell function of epidermal Langerhans cells. J. Dermatol. Sci. 18: 98.

30. Boukamp P, Petrussevska R T, Breitkreutz D, Hornung J, Markham A, Fusenig N E. 1988. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106: 761.

31. van der Velden V H, Naber B A, van der Spoel P, Hoogsteden H C, Versnel M A. 1998. Cytokines and glucocorticoids modulate human bronchial epithelial cell peptidases. Cytokine 10: 55.

32. Laemmli U K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680.

33. Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156.

34. Mizutani H, Black R, Kupper T S. 1991. Human keratinocytes produce but do not process pro-interleukin-1 (IL-1) beta. Different strategies of IL-1 production and processing in monocytes and keratinocytes. J. Clin. Invest. 87: 1066.

35. Zepter K, Haffner A, Soohoo L F, De Luca D, Tang H P, Fisher P, Chavinson J, Elmets C A. 1997. Induction of biologically active IL-1 beta-converting enzyme and mature IL-1 beta in human keratinocytes by inflammatory and immunologic stimuli. J. Immunol. 159: 6203.

36. Brattsand M, Egelrud T. 1998. Purification and characterization of interleukin-1 beta from human plantar stratum corneum. Evidence of interleukin 1 beta processing in vivo not involving interleukin-1 beta convertase. Cytokine 10: 506.

37. Lundqvist E N, Companjen A R, Prens E P, Egelrud T. 1998. Biological activity of human epidermal interleukin-1beta: comparison with recombinant human interleukin-1beta. Eur. Cytokine Netw. 9: 41.

38. Phillips W G, Feldmann M, Breathnach S M, Brennan F M. 1995. Modulation of the IL-1 cytokine network in keratinocytes by intracellular IL-1 alpha and IL-1 receptor antagonist. Clin. Exp. Immunol. 101: 177.

39. Maier J A, Voulalas P, Roeder D, Maciag T. 1990. Extension of the life-span of human endothelial cells by an interleukin-1 alpha antisense oligomer. Science 249: 1570.

40. Naik S M, Cannon G, Burbach G J, Singh S R, Swerlick R A, Wilcox J N, Ansel J C, Caughman W. 1999. Human keratinocytes constitutively express IL-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene. J. Invest. Dermatol. 113: 766.

41. Pizarro T T, Michie M H, Bentz M, Woraratanadharm J, Smith M F Jr, Foley E, Moskaluk C A, Bickston S J, Cominelli F. 1999. IL-18, a novel immunoregulatory cytokine, is up-regulated in Crohn's disease: expression and localization in intestinal mucosal cells. J. Immunol. 162: 6829.

42. Olee T, Hashimoto S, Quach J, Lotz M. 1999. IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses. J. Immunol. 162: 1096.

43. Puren A J, Fantuzzi G, Dinarello C A. 1999. Gene expression, synthesis, and secretion of interleukin-18 and interleukin-1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells. Proc. Natl. Acad. Sci. USA 96: 2256.

44. Novick D, Kim S H, Fantuzzi G, Reznikov L L, Dinarello C A, Rubinstein M. 1999. Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 10: 127.