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The Journal of Immunology, 2006, 176: 3780-3787.
Copyright © 2006 by The American Association of Immunologists

Regulation of IL-1 Family Cytokines IL-1{alpha}, IL-1 Receptor Antagonist, and IL-18 by 1,25-Dihydroxyvitamin D3 in Primary Keratinocytes1

Juan Kong*, Sergei A. Grando{dagger} and Yan Chun Li2,*

* Department of Medicine, University of Chicago, Chicago, IL 60637; and {dagger} Department of Dermatology, University of California Davis Medical Center, Sacramento, CA 95817


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-1 family cytokines are key mediators of inflammatory response. Excessive production of these cytokines by keratinocytes has been implicated in inflammatory and hyperproliferative skin diseases. Given the immunosuppressive role of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its clinical application in treatment of psoriasis, we investigated the effect of 1,25(OH)2D3 on the expression of IL-1{alpha}, intracellular IL-1 receptor antagonist (icIL-1Ra), and IL-18 in mouse primary keratinocytes. Treatment of keratinocytes with 1,25(OH)2D3 increased the expression of IL-1{alpha} and icIL-1Ra and decreased the expression of IL-18 in dose- and time-dependent manners. The magnitude of icIL-1Ra induction was much greater than that of IL-1{alpha} so that the ratio of icIL-1Ra to IL-1{alpha} was markedly increased, leading to repression of IL-1 activity. The regulation of these three cytokines by 1,25(OH)2D3 was mediated by vitamin D receptor (VDR), as 1,25(OH)2D3 had no effect in VDR–/– keratinocytes, whereas the effect was restored in cells derived from VDR–/– mice expressing human VDR. 1,25(OH)2D3 appeared to use different mechanisms to regulate the biosynthesis of IL-1{alpha} and icIL-1Ra: it increased IL-1{alpha} mRNA stability whereas it enhanced icIL-1Ra gene transcription. The basal IL-18 expression and activity were much higher in VDR–/– keratinocytes and skin, underscoring the importance of the repressive role of vitamin D in IL-18 production. Similar regulation of these cytokines was also seen in primary human keratinocytes. Collectively, these results suggest that vitamin D modulates cutaneous inflammatory reactions, at least in part, by increasing the IL-1Ra to IL-1{alpha} ratio and suppressing IL-18 synthesis in keratinocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-1 family cytokines, including IL-1{alpha} and IL-1beta, IL-1 receptor antagonist (IL-1Ra)3 and IL-18, are regulatory and inflammatory cytokines that play key roles in inflammatory response (1). These cytokines are constitutively produced in many cell types including keratinocytes (2, 3, 4). Excessive production of IL-1{alpha} or IL-18 in keratinocytes has been implicated in psoriasis and other inflammatory and hyperproliferative skin diseases (5, 6, 7).

IL-1{alpha} is synthesized as a 33-kDa procytokine that is cleaved into a 17-kDa bioactive molecule (8, 9). IL-1{alpha} exerts its biological effects through binding to specific membrane receptors IL-1RI and IL-1RII. IL-1 signaling depends on IL-1RI and IL-1RI accessory protein (1), whereas IL-1RII is considered as a decoy activity down-modulating receptor (10). Human keratinocytes express both IL-1{alpha} and beta isoforms, whereas mouse keratinocytes express only IL-1{alpha} (11). IL-1 is well known to play key roles in local inflammation.

IL-1Ra is another member of the IL-1 family that binds to the same IL-1Rs without inducing intracellular response, thus inhibiting IL-1 activity (12). It is known that the activity of IL-1 is largely determined by the ratio of IL-1Ra and IL-1 in a particular tissue or cell. Two isoforms of IL-1Ra have been identified that are generated from alternative splicing of two distinctive first exons of the IL-1Ra gene (13, 14, 15). The secreted isoform (sIL-1Ra) is predominantly expressed in the peripheral blood cells, lung, spleen, and liver, whereas the intracellular isoform (icIL-1Ra), which remains in the cytoplasm after synthesis, is produced in large quantities in keratinocytes and other epithelial cells (14).

IL-18 is a member of the IL-1 cytokine family that was initially isolated as an IFN-{gamma}-inducing factor (16). IL-18 potently stimulates IFN-{gamma} production by T lymphocytes and NK cells (16, 17, 18), and thus plays a crucial role in Th1 cytokine responses and cytotoxicity. IL-18 is synthesized as an inactive precursor (pro-IL-18) of 24-kDa polypeptide, and like IL-1beta, is cleaved by the IL-1beta-converting enzyme (also known as caspase-1) into a biologically active 18-kDa monomer (19). IL-18 acts through the membrane receptors IL-18R heterodimer complex, which is composed of an IL-18-binding {alpha}-chain and a nonbinding, signal-transducing beta-chain (20, 21, 22). IL-18 is expressed in many cell types including keratinocytes (4). Previous studies have shown that the basal keratinocytes constitutively express the pro-IL-18 protein, but whether the pro-IL-18 is processed and released as the mature and active form is still controversial (23, 24, 25). Interestingly, it has been reported that the expression of IL-18 is markedly increased in mature keratinocytes (26) and in keratinocytes from psoriatic lesions (6, 23). Enhanced IL-18 expression is also reported in skin tumors (7).

Vitamin D is well known to have anti-inflammatory and anti-proliferative activities. These activities are, at least in part, mediated through regulating and interacting with cytokines and growth factors (27). For example, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and vitamin D analogs modulate inflammatory responses by suppressing the production of IL-2 and IFN-{gamma} in T cells at the transcriptional level (28, 29, 30). Vitamin D also inhibits IL-12 production by both macrophages and dendritic cells (31), a key cytokine critical for Th1 cell development. In human keratinocytes, vitamin D and analogs have been shown to suppress the TNF-{alpha}-induced increase in IL-1{alpha} and IL-8 production (32). Given the potent proinflammatory activity of IL-1 and IL-18 and their possible involvement in inflammatory skin disease, the immunosuppressive activity of 1,25(OH)2D3 (33) and its therapeutic effect on psoriasis (34), in the present study we investigated the effect of 1,25(OH)2D3 on the expression of IL-1 family cytokines IL-1{alpha}, icIL-1Ra, and IL-18 in primary keratinocytes. We found that 1,25(OH)2D3 markedly increases the icIL-1Ra to IL-1{alpha} ratio whereas it directly suppresses IL-18 production, suggesting that 1,25(OH)2D3 exerts its anti-inflammatory action in the skin, at least in part, by suppressing IL-1 and IL-18 activities in keratinocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
cDNA probes, cytokines, and Abs

IL-1{alpha} cDNA probe was released from a human IL-1{alpha} cDNA plasmid purchased from the American Type Culture Collection. Cloning of mouse IL-18 cDNA probe has been described previously (26). The icIL-1Ra cDNA probe was obtained by RT-PCR according to the mouse IL-1Ra cDNA sequence deposited in the GenBank database. The PCR primer sequences were as follows: 5'-TCACCCATGGCTTCAGAGGCAGCC-3' (forward), and 5'-GGCCTTTCTCAGAGCGGATGAAGG-3' (reverse). PCR products were cloned into the pSK(+) vector (Stratagene) and the identity of icIL-1Ra was confirmed by DNA sequencing. Recombinant IL-1{alpha} and IL-18, and Abs against mouse IL-1{alpha}, IL-1Ra, and IL-18 were obtained from R&D Systems.

Cell culture

Isolation and culture of mouse primary keratinocytes have been described previously (35). Briefly, skins were harvested from 2- to 3-day-old wild-type, VDR–/– (36) and human (h) VDR/VDR–/– mice (37), and treated overnight with 0.25% trypsin (Invitrogen Life Technologies) at 4°C. The epidermis was then collected, and epidermal cells were released and cultured in low calcium media containing 0.045 mM Ca2+ supplemented with 4% chelex-treated FBS and 7.5 ng/ml epidermal growth factor at 34°C and 7% CO2 in collagen-coated plates. The use of animals was approved by the Institutional Animal Care and Use Committee at the University of Chicago. Primary human keratinocytes were isolated from normal neonatal foreskins as described previously (38) and were cultured in keratinocyte-serum-free medium supplemented with 5 ng/ml recombinant epidermal growth factor and 50 µg/ml bovine pituitary extract (Invitrogen Life Technologies). Treatment of keratinocytes was detailed in each experiment.

Northern blot and RNA protection assays

Northern blot analysis was performed as described previously (39). Briefly, total RNA was isolated from skin or cultured keratinocytes using the TRIzol reagent (Invitrogen Life Technologies) and separated on a 1.2% agarose gel containing 0.6 M formaldehyde, transferred onto a nylon membrane (MSI) and cross-linked in a UV cross-linker (Bio-Rad). The membranes were hybridized with 32P-labeled cDNA probes according to the method of Church and Gilbert (40). The relative amount of mRNA was quantified using a PhosphorImager (Molecular Dynamics) and variations in RNA loading were normalized with GAPDH or 36B4 cDNA probe. IL-18 mRNA levels in the skin were determined by RNase protection assays using a commercial RPA kit from Ambion according to manufacturer’s instructions.

Real-time RT-PCR

Expression of IL-1{alpha}, IL-1Ra, and IL-18 in primary human keratinocytes was quantified by real-time RT-PCR. Briefly, first strand cDNAs were synthesized from 2 µg of total RNAs using a ProtoScript First Strand cDNA Synthesis kit (New England Biolab) according to manufacturer’s instruction. The cDNAs were then used as the template (1.5 µl/reaction) for real-time PCR amplification. Real-time PCR was conducted using the 7300 Real-Time PCR System (Applied Biosystems) and a SYBR Green PCR Reagents kit (Applied Biosystems). The PCR primers used were the following: IL-18: 5'-ATGGCTGCTGAACCAGTAGAAG-3' (forward), and 5'-CAGCCATACCTCTAGGCTGGC-3' (reverse) (41); IL-1{alpha}: 5'-ATCAGTACCTCACGGCTGCT-3' (forward), and 5'-TGGGTATCTCAGGCATCTCC-3' (reverse); and icIL-1Ra: 5'-GCGAGAACAGAAAGCAGGAC-3' (forward), and 5'-CCTTCGTCAGGCATATTGGT-3' (reverse). These primers were designed according to the corresponding cDNA sequences deposited in the GenBank database. GAPDH was used as the internal control for each reaction. GAPDH primers are 5'-CAACTTTGGCATTGTGGAAGG-3' (forward), and 5'-ACACATTGGGGGTAGGAACAC-3' (reverse). All primers were tested for their specificity by conventional PCR before being used for the real-time PCR quantitative studies. The cycle threshold (Ct) value for each gene was obtained from the real-time PCRs, and the starting amount of each target mRNA was calculated based on a calibration curve and the Ct value. The amount of mRNA for each gene was normalized to GAPDH mRNA and presented as relative values.

Western blot

Keratinocytes were lysed with the Laemmli sample buffer (42), and protein concentrations were determined as described (39). The cell lysates were separated by SDS-PAGE, and proteins were then transferred onto an Immobolin membrane. The membrane was incubated with an Ab against mouse IL-1{alpha}, IL-1Ra, or IL-18. After another incubation with peroxidase-conjugated second Ab, the Ag was visualized using a chemiluminescent kit (K&P Laboratories).

Measurement of IL-1 activity

IL-1 biological activity was determined by measuring its stimulation of murine thymocyte proliferation as described previously (2). Briefly, mouse thymocytes were plated at 1.5 x 106 cells/well in a 96-well plate and cultured in RPMI 1640 supplemented with 10% FBS and 1 µg/ml PHA. Recombinant IL-1{alpha} (at 0, 10, 20, 80, 160, and 320 pg/ml, for standard curve) or keratinocyte lysates (5 or 10 µg/well) treated with ethanol or 1,25(OH)2D3 for 24 h were added into the thymocyte culture media in triplicates. In another experiment, the cell lysates were preincubated with Ab against mouse IL-1{alpha} or normal serum for 1 h on ice before the assay. After 72 h, thymocyte proliferation was quantified by MTT (Sigma-Aldrich) assay at 630 nm as described (2).

Measurement of IL-18 activity

IL-18 activity was determined by measuring IFN-{gamma} release from mouse splenocytes based on the method described previously (4, 23) with modifications (26). Briefly, keratinocytes grown in 10-cm dishes were treated with ethanol or 2 x 10–8 M 1,25(OH)2D3 for 24 h, and the media were collected and stored at –70° C until use. For IL-18 activity assay, mouse splenocytes were seeded into 96-well plates at 5 x 105 cells/well in RPMI 1640 medium supplemented with 10% FBS and 0.5 µg/ml Con A. After 24 h, the medium was replaced with a 50:50 mixture of RPMI 1640 and the keratinocyte-conditioned medium. After continued culture for 24 h, 150 µl of the culture supernatant was collected and assayed for IFN-{gamma} production by ELISA using a commercial kit (R&D Systems). The IL-18 activity was expressed as picograms of IFN-{gamma} per milliliter of media.

RNA stability assays

VDR+/+ keratinocytes were treated with ethanol or 2 x 10–8 M 1,25(OH)2D3 for 24 h. The media were then replaced with the same fresh media but containing actinomycin D (0.5 µg/ml) to stop new RNA synthesis. Total cellular RNAs were extracted at 0, 2, 4, and 8 h after actinomycin D treatment (for IL-1{alpha} assay), or at 0, 6, 12, and 18 h after actinomycin D treatment (for icIL-1Ra assay). IL-1{alpha} and icIL-1Ra mRNA levels were determined by Northern blot analyses, and the relative amount of mRNA was quantified using a PhosphorImager.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We used primary mouse keratinocytes to investigate the effect of vitamin D on the expression of IL-1 family cytokines. As shown in Fig. 1, in keratinocytes treated with increasing doses of 1,25(OH)2D3 for 24 h, both IL-1{alpha} and icIL-1Ra mRNA levels were increased in a dose-dependent manner, with the maximal stimulation seen at 10–8 M (Fig. 1A). As expected, the protein levels of pro-IL-1{alpha} and icIL-1Ra were also dose-dependently increased in cell lysates (Fig. 1B). The induction of IL-1{alpha} and icIL-1Ra by 1,25(OH)2D3 was also increased with time, apparently peaking by 12 h of treatment (Fig. 2, A and B). Interestingly, the magnitude of induction over the basal level was much higher for icIL-1Ra than for IL-1{alpha}, so that the ratio of icIL-1Ra to IL-1{alpha} was markedly increased after 1,25(OH)2D3 treatment (Figs. 1C and 2B). Because IL-1Ra antagonizes IL-1 activity and IL-1 activity is determined by the IL-1Ra to IL-1 ratio, this observation suggests a repression of IL-1{alpha} activity by 1,25(OH)2D3. To confirm this, we measured IL-1 activity in cell lysates because keratinocyte-derived IL-1{alpha} and icIL-1Ra are both intracellular. Indeed, IL-1 activity in keratinocyte lysates determined by thymocyte proliferation assays confirmed an ~50% reduction of IL-1 activity after 24 h of 1,25(OH)2D3 treatment (Fig. 2C). The activity in the cell lysates was blocked by Ab against mouse IL-1{alpha}, confirming the specificity of this assay (Fig. 2D).


Figure 1
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  		FIGURE 1. Induction of IL-1{alpha} and icIL-1Ra by 1,25(OH)2D3 in primary mouse keratinocytes. A, Dose-dependent induction of IL-1{alpha} and icIL-1Ra mRNAs by 1,25(OH)2D3. B, Dose-dependent induction of pro-IL-1{alpha} and icIL-1Ra proteins by 1,25(OH)2D3. Keratinocytes were treated for 24 h with increasing concentrations of 1,25(OH)2D3 as indicated, and total cellular RNAs or cell lysates were extracted and analyzed by Northern (20 µg of RNA/lane) (A) or Western blotting (30 µg of protein/lane) (B). C, Comparison of IL-1{alpha} and icIL-1Ra mRNA induction by increasing doses of 1,25(OH)2D3. Note the much greater magnitude of induction for icIL-1Ra.

 

Figure 2
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  		FIGURE 2. Effect of 1,25(OH)2D3 on IL-1{alpha} and icIL-1Ra expression and IL-1 activity in mouse keratinocytes. A, Time-dependent induction of IL-1{alpha} and icIL-1Ra expression. Mouse keratinocytes were treated with ethanol (–) or 2 x 10–8 M 1,25(OH)2D3 (+) for 6, 12, or 24 h, and total cellular RNAs were analyzed by Northern blotting. B, Calculated fold of induction of IL-1{alpha} and icIL-1Ra mRNAs by 1,25(OH)2D3 treatment at different time points. C, Intracellular IL-1 activity. Mouse keratinocytes were treated with ethanol (E) or 2 x 10–8 M 1,25(OH)2D3 (VD) for 24 h, and cell lysates were used to determine IL-1 activity using the thymocyte proliferation assay as described in Materials and Methods. IL-1 activity was expressed as picograms of IL-1 per microgram of lysate proteins. D, Blockade of IL-1 activity by Ab against mouse IL-1{alpha}. Cell lysates as treated in (C) were preincubated with normal serum (Control) or anti-IL-1{alpha} Ab (Ab) for 1 h before IL-1 activity was determined.

 
To investigate whether vitamin D receptor (VDR) is required to mediate the effect of 1,25(OH)2D3 on IL-1{alpha} and icIL-1Ra expression, keratinocytes were derived from wild-type and VDR–/– mice as well as VDR–/– mice that express the hVDR transgene in the skin (37) and treated with 1,25(OH)2D3. Because the VDR–/–-hVDR mice are in a C57BL/6/CD-1 mixed background, we also included cells derived from wild-type and VDR–/– mice in this mixed background. As shown in Fig. 3, the basal IL-1{alpha} mRNA level is relatively higher in C57BL/6 cells than in C57BL/6/CD-1 cells, suggesting that genetic background influences IL-1{alpha} expression. 1,25(OH)2D3 regulated IL-1{alpha} and icIL-1Ra expression in the same manner in these two genetic backgrounds. In contrast to wild-type cells, 1,25(OH)2D3 treatment had no effect on IL-1{alpha} or icIL-1Ra mRNA expression in VDR–/– keratinocytes in both backgrounds; however, 1,25(OH)2D3 stimulation of IL-1{alpha} and icIL-1Ra expression was restored in VDR–/– cells carrying the hVDR transgene (Fig. 3). These results demonstrate that vitamin D regulation of both IL-1{alpha} and icIL-1Ra expression is mediated by VDR.


Figure 3
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  		FIGURE 3. Requirement of VDR to mediate the effect of 1,25(OH)2D3 on IL-1{alpha} and icIL-1Ra expression. Keratinocytes were isolated from wild-type (VDR+/+), VDR-null–/– mice, and VDR-null mice expressing the hVDR transgene in the skin (hVDR/VDR–/–) in C57BL/6 and C57BL/6/CD-1 mixed background as indicated. The cells were treated with ethanol (–) or with 2 x 10–8 M 1,25(OH)2D3 (+) for 24 h. The mRNA levels of IL-1{alpha} and icIL-1Ra were determined by Northern blot analyses. Note the basal IL-1{alpha} level in the C57BL/6 background is higher than in the mixed background, suggesting genetic background influences IL-1{alpha} expression.

 
To understand the mechanism whereby 1,25(OH)2D3 stimulates IL-1{alpha} and icIL-1Ra synthesis, we investigated the effect of 1,25(OH)2D3 on IL-1{alpha} and IL-1Ra mRNA stability. To this end, keratinocytes were first cultured in the presence or absence of 1,25(OH)2D3 for 24 h and then treated with actinomycin D to block new RNA synthesis. The old, synthesized mRNA levels of IL-1{alpha} and icIL-1Ra in these cells were monitored at different time points (from 0 to 18 h for icIL-1Ra, and from 0 to 8 h for IL-1{alpha}) by Northern blot analyses following actinomycin D treatment. When new RNA synthesis was blocked, the amount of existing icIL-1Ra mRNA decreased with time regardless of the presence and absence of 1,25(OH)2D3, even though icIL-1Ra mRNA had been markedly induced by 1,25(OH)2D3 (Fig. 4A); in fact, the decay rate of icIL-1Ra mRNA was the same with or without 1,25(OH)2D3 treatment (Fig. 4B), suggesting that 1,25(OH)2D3 has no effect on icIL-1Ra mRNA stability. In contrast, 1,25(OH)2D3 treatment prevented the degradation of IL-1{alpha} mRNA, whereas IL-1{alpha} mRNA was quickly degraded within 4–8 h in the absence of 1,25(OH)2D3 (Fig. 4, C and D), indicating that 1,25(OH)2D3 increases IL-1{alpha} mRNA stability. Therefore, 1,25(OH)2D3 has very different effects on the stability of icIL-1Ra and IL-1{alpha} mRNAs in keratinocytes.


Figure 4
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  		FIGURE 4. Effect of 1,25(OH)2D3 on IL-1{alpha} and icIL-1Ra mRNA stability. A, 1,25(OH)2D3 has no effect on icIL-1Ra mRNA decay. Keratinocytes were cultured in the presence of ethanol or 2 x 10–8 M 1,25(OH)2D3 for 24 h, and then continuously cultured in the same media containing 0.5 µg/ml actinomycin D. Total cellular RNAs were isolated at 0, 6, 12, and 18 h after actinomycin D treatment, and icIL-1Ra mRNA levels were determined by Northern blot analyses. B, Phosphorimaging quantitative data showing the decay rate of icIL-1Ra mRNA in the presence of ethanol or 1,25(OH)2D3. The amount of icIL-1Ra mRNA at each time point is presented as percentage of the amount at time 0. C, 1,25(OH)2D3 increases IL-1{alpha} mRNA stability. After keratinocytes were cultured in the presence of ethanol (–) or 2 x 10–8 M 1,25(OH)2D3 (+) for 24 h, 0.5 µg/ml actinomycin D was added into the media. Total cellular RNAs were extracted at 0, 2, 4, and 8 h following actinomycin D treatment and IL-1{alpha} mRNA levels were determined by Northern blot. D, Quantitative data showing the decay rate of IL-1{alpha} mRNA in the presence of ethanol or 1,25(OH)2D3. The amount of IL-1{alpha} mRNA at each time point is presented as percentage of the amount at time 0. E, New protein synthesis is required for 1,25(OH)2D3 stimulation of icIL-1Ra. Keratinocytes were treated with 2 x 10–8 M of 1,25(OH)2D3 for 24 h in the presence of increasing doses of cycloheximide (CHX), and icIL-1Ra mRNA levels were determined by Northern blot.

 
To further investigate the mechanism of vitamin D regulation of icIL-1Ra expression, keratinocytes were treated with 1,25(OH)2D3 in the presence of cycloheximide, an inhibitor of new protein synthesis. As shown in Fig. 4E, cycloheximide completely abolished the induction of icIL-1Ra by 1,25(OH)2D3, suggesting that new protein synthesis is required for 1,25(OH)2D3 stimulation of icIL-1Ra in keratinocytes (Fig. 4E). Our preliminary study of the IL-1Ra gene promoter suggested that 1,25(OH)2D3 regulates icIL-1Ra expression at the transcriptional level (data not shown).

It has been reported that phorbol ester stimulates IL-1Ra expression in human dermal fibroblasts (43). When keratinocytes were treated with PMA or 1,25(OH)2D3 alone, stimulation of icIL-1Ra was seen as expected; the combined treatment with PMA and 1,25(OH)2D3 stimulated icIL-1Ra expression even more (Fig. 5A). Further studies are required to confirm whether PMA and 1,25(OH)2D3 stimulate icIL-1Ra through different pathways, and whether their effects were additive or synergistic.


Figure 5
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  		FIGURE 5. Effect of PMA, vitamin D, and calcium on icIL-1Ra expression. Keratinocytes were cultured in the presence of 60 nM PMA, 2 x 10–8 M 1,25(OH)2D3 (VD) or both (P+D) for 24 h (A), or cultured in normal media (C, 0.045 mM Ca2+) or high calcium media (Ca2+, 0.15 mM) for 24 h (B), and icIL-1Ra mRNA levels were determined by Northern blot analyses.

 
Because 1,25(OH)2D3 is well known to induce keratinocyte differentiation in vitro (44, 45), it is possible that the induction of icIL-1Ra by 1,25(OH)2D3 is differentiation-related. To test this possibility, we determined icIL-1Ra expression in cells cultured in 0.15 mM Ca2+, a much more potent inducer of keratinocyte differentiation (46). As shown in Fig. 5B, in contrast to 1,25(OH)2D3, high calcium in the media actually slightly reduced icIL-1Ra expression (Fig. 5B), indicating that 1,25(OH)2D3 induces icIL-1Ra expression by a mechanism independent of cell differentiation.

IL-18 is another proinflammatory cytokine that is highly expressed in keratinocytes and has been implicated in inflammatory skin disease (6). We therefore also examined the effect of vitamin D on IL-18 expression in mouse keratinocytes. As shown in Fig. 6, treatment of keratinocytes with 1,25(OH)2D3 suppressed IL-18 mRNA expression in dose- and time-dependent manners; treatment with 10–8 M of 1,25(OH)2D3 for 24 h reduced IL-18 mRNA levels by 90% (Fig. 6).


Figure 6
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  		FIGURE 6. Suppression of IL-18 expression by 1,25(OH)2D3. A, Dose-dependent inhibition of IL-18 mRNA levels in keratinocytes. Keratinocytes were treated with increasing doses of 1,25(OH)2D3 as indicated for 24 h. B, Keratinocytes were treated with 2 x 10–8 M 1,25(OH)2D3 for 12 and 24 h. IL-18 mRNA expression was determined by Northern blot.

 
Interestingly, consistent with the repressive role of VDR in IL-18 regulation, the presence and absence of VDR had a profound effect on the basal expression of IL-18 in keratinocytes. In VDR–/– keratinocytes derived from VDR–/– mice, the basal levels of IL-18 mRNA (Fig. 7A) and protein (both pro-IL-18 and IL-18, Fig. 7B) were markedly elevated compared with wild-type cells, whereas hVDR was able to restore the low basal levels of IL-18 mRNA and protein in hVDR-expressing VDR–/– (hVDR/VDR–/–) cells (Fig. 7, A and B). Furthermore, the basal IL-18 mRNA level in the skin of VDR–/– mice was also markedly up-regulated compared with wild-type mice (Fig. 7D). It was reported that keratinocytes mainly produce unprocessed, biologically inactive pro-IL-18 (25); however, VDR–/– keratinocytes appeared to be able to generate a small amount of processed IL-18 (Fig. 7B). Consistently, IL-18 activity released into the media by VDR–/– keratinocytes was significantly higher compared with wild-type keratinocytes (Fig. 7C). Interestingly, comparison of the epidermis of VDR+/+ and VDR–/– mouse skins showed marked thickening of the basal layer in VDR–/– mice. In contrast to a single layer of basal keratinocytes normally seen in VDR+/+ mice, VDR–/– mice exhibited multiple layers of keratinocytes in the basal layer of the epidermis (Fig. 7E). These results were consistent with the inhibitory role of VDR in IL-18 expression and basal keratinocyte proliferation. However, this stimulatory effect of VDR ablation on IL-18 expression appeared to be tissue- or cell-specific, as the basal IL-18 mRNA level was not significantly changed in other tissues such as liver and spleen from VDR–/– mice (data not shown).


Figure 7
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  		FIGURE 7. Elevated basal IL-18 expression in keratinocytes lacking the VDR. A, IL-18 mRNA levels in keratinocytes derived from VDR+/+, VDR–/– and hVDR/VDR–/– mice. The hVDR/VDR–/– mice are VDR–/– mice expressing the hVDR transgene in the skin. The membrane was hybridized with hVDR, IL-18, and GAPDH cDNA probes. B, Western blot showing pro-IL-18 and IL-18 protein levels in VDR+/+, VDR–/–, and hVDR/VDR–/– keratinocytes. C, IL-18 activity in the media of VDR+/+ and VDR–/– keratinocytes, determined by measuring the release of IFN-{gamma} from mouse splenocytes. D, IL-18 mRNA levels in the skin of 3-mo-old wild-type +/+ and VDR–/– mice, determined by RNase protection assay. Each lane represents one individual animal. E, Histology of the epidermis of 3-mo-old VDR+/+ and VDR–/– mouse skin. Note the multiple layers of keratinocytes in the basal layer in VDR–/– mice.

 
The inhibition of IL-18 expression by 1,25(OH)2D3 was VDR-dependent, as no effect on IL-18 mRNA was seen in VDR–/– cells treated with 1,25(OH)2D3, even though the basal level was greatly increased (Fig. 8A). 1,25(OH)2D3 suppression of IL-18 was restored in VDR–/– cells expressing the hVDR transgene, even though these cells were from the mixed C57BL/6/CD-1 background (Fig. 8B).


Figure 8
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  		FIGURE 8. Effects of VDR ablation and calcium on IL-18 expression. A, VDR is required for the suppression of IL-18 x 1,25(OH)2D3. VDR+/+ and VDR–/– keratinocytes were treated with ethanol (–) or 2 x 10–8 M of 1,25(OH)2D3 (+) for 24 h. B, hVDR/VDR–/– keratinocytes were treated with ethanol (–) or 2 x 10–8 M 1,25(OH)2D3 (+) for 24 h. The membrane was hybridized with both hVDR and IL-18 cDNA probes simultaneously. C, VDR+/+ keratinocytes were treated with 2 x 10–8 M 1,25(OH)2D3 (VD), 0.15 mM calcium (Ca) or both (VD+Ca) for 24 h. IL-18 mRNA levels were determined by Northern blot.

 
As we have shown previously (26), a high calcium concentration in the media stimulated IL-18 expression (Fig. 8C); however, combination of 1,25(OH)2D3 and high calcium did not appear to affect the high calcium stimulation. These results suggest that different regulatory pathways are involved in 1,25(OH)2D3 and calcium actions on IL-18, and the regulation of IL-18 by vitamin D is independent of keratinocyte differentiation.

Finally, we asked whether our findings of vitamin D regulation of IL-1{alpha}, icIL-1Ra, and IL-18 can be confirmed in primary human keratinocytes, a better model for psoriasis. As shown in Fig. 9, data obtained from quantitative real-time RT-PCR analyses confirmed that, in keratinocytes derived from human foreskins, treatment with 1,25(OH)2D3 for 24 h led to induction of both IL-1{alpha} and icIL-1Ra, and the induction of icIL-1Ra (~5.8-fold) was much greater than that of IL-1{alpha} (~2.8-fold) (Fig. 9A); in contrast, 1,25(OH)2D3 treatment markedly decreased IL-18 expression (by ~40%) (Fig. 9B). These observations are consistent with the results seen in primary mouse keratinocytes.


Figure 9
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  		FIGURE 9. Regulation of IL-1{alpha}, icIL-1Ra, and IL-18 by vitamin D in primary human keratinocytes. Keratinocytes isolated from neonatal human foreskins were treated with ethanol (E) or 2 x 10–8 M 1,25(OH)2D3 (VD) for 24 h, and the mRNA levels of IL-1{alpha} and icIL-1Ra (A) and IL-18 (B) in these cells were quantified by real-time RT-PCR. The mRNA levels were expressed as relative units.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The skin is an important organ involved in vitamin D metabolism and actions. Not only does the initial reaction in vitamin D biosynthesis take place in the skin (47), but keratinocytes also synthesize 1,25(OH)2D3 (48, 49), the hormonal form of vitamin D. Furthermore, the skin is one of the tissues that express a high level of VDR (50). Thus, 1,25(OH)2D3 may have autocrine or paracrine effects on the skin. In fact, 1,25(OH)2D3 has been shown to inhibit keratinocyte proliferation and induce keratinocyte differentiation (45). As anti-proliferative and immunosuppressive agents, vitamin D and its analogs have been used to treat psoriasis (34), which is believed to be an inflammatory skin disease, and cytokines may well be the targets of vitamin D actions in the skin (51). Thus, understanding the effect of vitamin D on cytokines, particularly proinflammatory cytokines, in keratinocytes is important to understanding the anti-proliferative and anti-inflammatory actions of vitamin D in the skin.

In the present study, we investigated the effect of vitamin D on the synthesis of IL-1{alpha}, IL-1Ra, and IL-18 in primary keratinocytes. IL-1beta was not included in the study because it is not expressed in mouse keratinocytes (11). These are cytokines of the IL-1 family that play crucial roles in local and systemic inflammation. Enhanced production of IL-1{alpha} or IL-18 in keratinocytes has been found in psoriasis (5, 6). Our data show that 1,25(OH)2D3 increases the IL-1Ra/IL-1{alpha} ratio, leading to reduction of IL-1 activity; at the same time, 1,25(OH)2D3 also suppresses the biosynthesis and activity of IL-18. The regulation of these cytokines is mediated by VDR and is independent of keratinocyte differentiation, and similar regulation is seen in mouse cells derived from different genetic backgrounds as well as in human cells. Given the potent stimulatory roles of IL-1 and IL-18 in inflammation and proliferation, it is speculated that vitamin D may modulate skin inflammation, at least in part, by suppressing IL-1 and IL-18 activity. However, one limitation of our study is that the data presented in this report are from unstimulated keratinocytes. Future studies are required to investigate the effects of vitamin D on these cytokines in cells under proinflammatory stimulation, as hyperproliferative skin diseases mostly have a significant underlying proinflammatory stimulus.

Because IL-1{alpha}, IL-1beta, and IL-1Ra all bind to the same IL-1Rs and IL-1Ra binding does not transduce signals inside the cell, the actual activity of IL-1 is determined by the ratio of IL-1Ra to IL-1 in a particular situation. To this end, it is interesting that even though vitamin D increases both IL-1{alpha} and IL-1Ra synthesis in keratinocytes, it stimulates IL-1Ra to a much greater extent so that the net effect of vitamin D is the suppression of IL-1 activity. Vitamin D suppression of IL-1{alpha} has been reported previously in LPS-induced human monocytes and TNF-{alpha}-induced human keratinocytes (32, 52), whereas vitamin D stimulation of IL-1Ra is a novel finding, to our knowledge, which may represent another important target of vitamin D’s anti-inflammatory action. Because IL-1 and IL-1Ra are coexpressed in many cells, it is important to investigate both cytokines when studying vitamin D’s effect on IL-1.

It is also interesting that vitamin D induces IL-1{alpha} and IL-1Ra by totally different mechanisms. Although 1,25(OH)2D3 has no effects on the decay of icIL-1Ra mRNA, it clearly increases IL-1{alpha} mRNA stability. Our primary data from IL-1Ra gene promoter study suggest that 1,25(OH)2D3 induces icIL-1Ra mRNA by enhancing IL-1Ra gene transcription, which requires new protein synthesis. More studies are needed to fully elucidate the molecular mechanisms involved in the regulation of icIL-1Ra and IL-1{alpha} by vitamin D.

IL-18 is a potent proinflammatory cytokine in that it promotes the release of IFN-{gamma}, TNF-{alpha}, and GM-CSF, synergizes with IL-12 and IL-15, and is involved in many inflammatory and autoimmune diseases (53). Because of these actions, IL-18 is considered as a novel target for immunopharmacological anti-inflammatory intervention (54). Our study suggests that IL-18 is also a previously unrecognized target of vitamin D’s anti-inflammatory actions. In contrast to suppressing IL-1 activity by increasing the amount of its receptor antagonist, 1,25(OH)2D3 directly suppresses IL-18 production. Underscoring the physiological importance of vitamin D suppression in IL-18 expression, keratinocytes lacking the VDR exhibit a dramatic up-regulation of IL-18 mRNA, protein, and activity; moreover, similar IL-18 elevation is also observed in the skin of VDR-null mice. Because of the high basal IL-18 levels, the skin of VDR-null mice might be in an inflammation-susceptible state. Interestingly, regions in the epidermis of adult VDR-null mouse skin exhibit multiple layers of basal keratinocytes. IL-18 has been linked to increased cell proliferation in the skin (7), but it remains to be determined whether the epidermal thickening of the VDR-null skin is a direct consequence of elevated IL-18 production.

The suppression of IL-1 and IL-18 activities by 1,25(OH)2D3 may have broad implications, considering the wide therapeutic potentials of vitamin D and analogs (55) and the possible involvement of these cytokines in a relatively large number of autoimmune and inflammatory diseases. For instance, besides psoriasis, up-regulation of IL-18 has been detected in type I diabetes mellitus, multiple sclerosis, inflammatory bowel disease, and inflammatory arthritis (53, 54), and vitamin D and analogs have been shown to prevent or suppress these diseases (34, 56, 57, 58). This raises the possibility that vitamin D suppression of IL-18 production might be part of the mechanism underlying the therapeutic benefits of vitamin D in many autoimmune and inflammatory diseases. To this end, the present study has opened an avenue for more investigations in the future to test this speculation.


   Acknowledgments
 
We thank Marc Bissonnette for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grant DK59327. Back

2 Address correspondence and reprint requests to Dr. Yan Chun Li, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC4080, Chicago, IL 60637. E-mail address: cyan{at}medicine.bsd.uchicago.edu Back

3 Abbreviations used in this paper: IL-1Ra, IL receptor antagonist; icIL-1Ra, intracellular isoform IL-1Ra; 1,25(OH2)D3, 1,25-dihydroxyvitamin D3; h, human; Ct, cycle threshold; VDR, vitamin D receptor. Back

Received for publication April 13, 2005. Accepted for publication January 6, 2006.


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 Introduction
 Materials and Methods
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