It is well established that solar UV radiation (UVR) suppresses cutaneous cell-mediated immunity in humans. trans-Urocanic acid (trans-UCA) is a major UVR-absorbing skin molecule that undergoes a photoisomerization to its cis-isomer following UVR exposure. Animal studies have demonstrated that cis-UCA plays a role in UVR-induced immune suppression, but the molecular mechanisms of action of cis-UCA are not fully understood. In this study, we examined changes in gene expression and synthesis of cytokines and PGE2 following UCA treatment of primary human keratinocytes. A limited microarray analysis of keratinocytes from two donors indicated that ∼400 genes were induced by solar-simulated radiation (SSR), 16 of which were also up-regulated by cis-UCA. In contrast, trans-UCA had little or no effect on gene expression. The genes up-regulated by both cis-UCA and SSR were associated with apoptosis, cell growth arrest, cytokines, and oxidative stress. Further studies using primary keratinocytes from four new donors showed that PG-endoperoxide synthase-2 was dramatically induced by cis-UCA, resulting in an enhanced secretion of PGE2 into the cell culture supernatant. cis-UCA also increased cytokine protein production such as that of TNF-α, IL-6, and IL-8 in a dose-dependent manner. SSR had the same effect as cis-UCA, but trans-UCA had no effect. In addition, activation of NF-κB and lipid peroxidation were induced by cis-UCA and SSR, but not trans-UCA, suggesting possible upstream events of the gene expression changes. The data suggest that the induction of immune suppression by cis-UCA may involve the initiation of gene transcription of immunomodulatory mediators in primary human keratinocytes.

Skin cancer is the most common type of cancer in fair-skinned populations and its incidence has increased steadily over the past few decades (1). Many epidemiological studies have indicated that solar UV radiation (UVR)3 (∼295–400 nm) exposure is the primary risk factor for skin cancer (2). As well as being carcinogenic, UVR also suppresses cutaneous cell-mediated immune responses. Animal models have shown that UVR-induced immune suppression plays an important role allowing UVR-transformed cells to escape tumor surveillance mechanisms and develop into skin cancer (3). Organ transplant patients undergoing immunosuppressive therapy have an elevated risk of both nonmelanoma skin cancer and malignant melanoma, especially if they have a history of high sun exposure (4), suggesting that a similar mechanism occurs in humans. Furthermore, UVR exposure suppresses immune responses to infectious agents in rodent models and therefore has implications for susceptibility to infectious diseases and vaccine efficacy in humans (5). Previously we have shown that low doses of solar-simulated radiation (SSR), typically experienced after 1 h of exposure to midday summer sunlight at mid-latitudes, can suppress contact hypersensitivity (CHS) responses in healthy humans (6). Hence, it is important to elucidate the mechanisms of UVR-induced immune suppression to better understand the biological impact of human exposure to UVR.

UVR-induced biological responses are initiated by absorption by specific molecules (chromophores) located in the skin. trans-Urocanic acid (trans-UCA; (2E)-3-(1H-imidazol-4-yl)prop-2-enoic acid), a deamination product of histidine, is a major chromophore present at a high concentration in the epidermal stratum corneum. Upon exposure to UVR, trans-UCA undergoes a photoisomerization to its cis-isomer until equilibrium is reached with the two isomers being in approximately equal quantities. In humans, this occurs after ∼1 minimal erythema dose (MED) of UVR, which is the lowest dose required to induce a just visibly perceptible erythema (7).

De Fabo and Noonan, using a mouse model of CHS, originally reported that trans-UCA shows a similar UVR-absorption spectrum as the UV wavelength dependence for immune suppression. This observation was the basis for proposing trans-UCA as a photoreceptor for UVR-induced immune suppression (8). Subsequent studies in mice have demonstrated that topical or systemic application of cis-UCA mimics many aspects of UVR-induced immune suppression, including suppression of CHS and delayed-type hypersensitivity responses (9, 10, 11, 12), tumor Ag presentation (13, 14), depletion of Langerhans cells (15), and inhibition of allograft rejection and graft-vs-host disease (16, 17). Neutralization studies using a mAb against cis-UCA also restored many of the effects of UVR on the immune responses (18, 19).

In addition to alterations in Ag presentation and processing, the immunosuppressive effects of UVR are mediated by immunomodulatory molecules (20, 21). These include both proinflammatory and anti-inflammatory compounds such as PGE2, TNF-α, and IL-10. There is some evidence that cis-UCA also affects the production of those mediators in PBMC and CD4+ T cells (22, 23, 24). In the epidermal keratinocyte, a possible site of cis-UCA activity, a synergistic effect of cis-UCA and histamine on PGE2 production was observed (25), although it has not been demonstrated that cis-UCA alone increases PGE2 or cytokine secretion. Because UVR initiates signal transduction and activates transcription factors that result in changes of gene expression of cytokines and their receptors (26), the immunosuppressive effects of cis-UCA may be induced by its ability to initiate gene transcription. Despite considerable research into the effects of UVR on the activation of transcription factors and gene expression (27, 28, 29, 30, 31, 32), a comprehensive analysis of the effects of cis-UCA on those events has not been conducted.

In this study, we examined changes in the gene expression and synthesis of immunomodulatory mediators following UCA treatment of primary human keratinocytes using DNA microarrays, RT-PCR, and ELISA. We report that cis-UCA, but not trans-UCA, induces gene transcription that leads to enhanced production of PGE2 and cytokines in primary human keratinocytes.

Primary cultures of human normal epidermal keratinocytes were prepared from biopsies taken from the unexposed buttock skin of six Caucasian volunteers. The profile of the donors are shown in Table I. The procedure was approved by St Thomas’ Hospital Ethics Committee (London, U.K.) and the donors gave written informed consent. Briefly, the biopsies were treated with 2 mg/ml dispase (Invitrogen) in PBS at 6°C overnight. The epidermal sheets were separated from the dermis and dissociated in 0.5% trypsin in PBS at 37°C. The keratinocyte suspension was centrifuged and the pellet was resuspended in serum-free keratinocyte medium supplemented with 50 μg/ml pituitary extract, 2.5 ng/ml epidermal growth factor, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The cells were maintained at 37°C in 5% CO2 and passaged at 60% confluency. The medium was changed every 2 days. For all experiments, keratinocytes in the third passage were grown to 80% confluency (1.5–2 × 106 cells per 75-cm2 flask). They were fed 24 h before treatment and 24 h after treatment were washed with PBS once and then harvested by trypsinization. For RNA isolation, cells were lysed directly in the flask by the addition of RNA-Bee reagent. The number of viable cells was counted using a trypan blue dye exclusion assay.

Table I.

The profile of the donors

DonorAgeSexSkin TypeaMEDb (J/cm2)Analysis
23 Microarray, RT-PCR 
24 III 12 Microarray, RT-PCR 
30 ELISA 
62 I/II ELISA, RT-PCR 
26 III/IV ELISA 
30 III/IV ELISA 
DonorAgeSexSkin TypeaMEDb (J/cm2)Analysis
23 Microarray, RT-PCR 
24 III 12 Microarray, RT-PCR 
30 ELISA 
62 I/II ELISA, RT-PCR 
26 III/IV ELISA 
30 III/IV ELISA 
a

Modified Fitzpatrick skin type system: I, white skin always burns, never tans; II, white skin always burns, tans minimally; III, white skin sometimes burns, always tans; IV, white skin rarely burns, tans well.

b

Individual SSR MED on previously unexposed buttock skin. The experimental dose of 12 J/cm2 used in vitro represents a range of 1–3 MEDs on these donors.

trans-UCA (99% purity) was obtained from Sigma-Aldrich. cis-UCA was prepared from trans-UCA as previously described and was >99% pure as determined by HPLC (33). UCA isomers were dissolved in PBS and added to keratinocyte cultures at a final concentration of 10, 50, or 100 μg/ml. PBS served as a baseline control. The baseline levels of both isomers, trans and cis, are negligible because UCA formation and the histidase activity that converts histidine to trans-UCA are almost completely restricted to the stratum corneum (34). It has also been reported that no UCA or histidase activity was found in medium or cell extracts in primary human keratinocyte culture (35).

SSR was generated by a 1-kilowatt xenon arc solar simulator (Oriel Corporation), and we have previously published details of its emission spectrum (filter 2) (36). Irradiance was routinely determined with a wide-band thermopile radiometer (Medical Physics) calibrated against a DM150 double monochromator Bentham spectroradiometer (Bentham Instruments). Keratinocytes were washed with PBS twice and then irradiated in PBS at a dose of 12 joules (J)/cm2. The cells were maintained on a water-cooled plate during the irradiation. The PBS was then replaced with the original conditioned medium. A dose of 12 J/cm2 can be readily obtained on a horizontal surface (i.e., sunbathing) within 40 min of solar exposure based on summer noon solar spectral irradiance measurements (290–400 nm) made at 38°N (Albuquerque, NM) and 38°S (Melbourne, Australia) (37). A similar dose on the face would require <2 h of exposure (assuming one-third irradiance of horizontal plane).

Total RNA was extracted using RNA-Bee (Tel-Test) according to the manufacturer’s instructions. For hybridization to the HG-U133A microarrays (Affymetrix), biotinylated cRNA was prepared following Affymetrix expression analysis technical instructions with minor modifications. Briefly, 5–10 μg of total RNA from each sample was reverse-transcribed using T7-oligo(dT) primer (Affymetrix). After cDNA synthesis, in vitro transcription amplification and biotin labeling were performed using an in vitro transcription labeling kit (Affymetrix) at 37°C for 16 h. The biotin-labeled cRNA was purified using cRNA spin columns (Affymetrix) and then fragmented. Ten micrograms of the fragmented cRNA was hybridized to the array followed by washing and staining with streptavidin-phycoerythrin on an Affymetrix fluidics station 450. Fluorescent images were scanned using the GeneChip Scanner 3000 (Affymetrix).

Microarray data from the scanner were analyzed using Affymetrix GeneChip operating software (GCOS). The average (trimmed mean) signal of each array was linearly scaled to a target signal value of 100. We used GCOS because this software uses a combination of qualitative and quantitative measures of transcript performance that greatly reduces the false positive rate compared with a single parameter (38). GCOS uses three statistical algorithms, detection, change, and signal log ratio algorithms, which were developed using standard statistical techniques, i.e., the Wilcoxon’s signed-rank test and one-step Tukey’s biweight estimate.

In a comparison expression analysis, genes deemed absent or marginal by the detection algorithm in both control and treated samples were eliminated because they are uninformative. We then selected genes according to the following criteria: 1) the expression difference between treated and PBS control is 2-fold or more as calculated from the signal log ratio (SLR) values (SLR ≥ 1 or ≤ −1); and 2) change p values marked with “increasing” or “decreasing” in treated samples compared with control samples. The change p value ranges in scale from 0.0 to 1.0 and provides a measure of likelihood of change and direction. Each change p value is computed by the Wilcoxon’s signed-rank test, which uses the differences between perfect match and mismatch probe intensities as well as the differences between perfect match probe intensities and background. Values close to 0.0 indicate an increase in the sample array compared with the control, whereas close to 1.0 indicate a decrease. We set the change p value threshold for increase to p < 0.0045 and that for decrease to p > 0.9955.

We performed two sets of single microarray analysis using keratinocytes derived from two different donors and selected only genes that met the above criteria in both donors to minimize false positive data.

cDNA was generated from the total RNA of each sample with TaqMan reverse transcription reagents (Applied Biosystems) following the manufacturer’s protocol. Expression level of mRNA was analyzed by real-time quantitative RT-PCR on an ABI Prism 7700 sequence detection system (Applied Biosystems) with the following TaqMan probes (gene name and reference sequence no.): ADAMTS1, Hs00199608_m1; GADD45B, Hs00169587_m1; HSPA1B, Hs00271244_s1; IGFBP3, Hs00181211_m1; IL-1α, Hs00174092_m1; IL-1β, Hs00174097_m1; IL-6, Hs00174131_m1; IL-8, Hs00174103_m1; IL-10, Hs00174086_m1; PMAIP1, Hs00560402_m1; PTGS2 (PG-endoperoxide synthase 2), Hs00153133_m1; SERPINB2, Hs00234032_m1; TNF, Hs00174128_m1; and TNFAIP3, Hs00234713_m1 as provided by the manufacturer. GAPDH served as an endogenous control gene.

For measurement of apoptotic cell death, cell death detection ELISAplus (Roche Diagnostics) was used according to the manufacturer’s instructions. The enrichment of mononucleosomes and oligonucleosomes released into the cytoplasm was calculated as an enrichment factor using the following formula: absorbance of treated sample/absorbance of PBS control.

Supernatants were collected at 24 h by centrifugation. TNF-α, IL-6, and IL-8 concentrations were measured using duo set ELISA kits (R&D Systems). PGE2 concentration was determined by Parameter PGE2 assay (R&D Systems). For the measurement of 8-isoprostane, an 8-isoprostane enzyme immunoassay (EIA) kit (Cayman Chemical) was used.

Nuclear extract was prepared with nuclear extract kit (Active Motif), and NF-κB activities were measured using an Active Motif TransAm NF-κB p65 ELISA kit according to the manufacturer’s instructions.

For all analyses, apart from the microarray data, Student’s unpaired t test was performed using SPSS 11.5 software. Values of p < 0.05 were considered significant.

For microarray analysis, keratinocytes derived from two donors were treated with 10 μg/ml trans- or cis-UCA or with SSR at 12 J/cm2, which is equivalent to 2∼3 MED for fair skin. The total UCA content within the 70-μm-thick human epidermis ranges from ∼2–62 nmol/cm2, which is equivalent to 0.3–8.9 mM (40–1230 μg/ml) (39). Because a maximum of approximately half of the trans-UCA is converted to cis-UCA following 1MED of SSR, 10 μg/ml cis-UCA can be achieved in vivo after treatment with SSR at 12 J/cm2. In all treatments, abnormal morphology or significant cell death was not visibly observed. Table II shows the number of genes with at least a 2-fold difference 24 h after treatment. cis-UCA regulated more genes than trans-UCA but not as many as SSR. The response to cis-UCA was different between the two donors; more genes were regulated in donor 2 than in donor 1. To eliminate false positive results, only genes common to both donors were considered regulated. cis-UCA up-regulated 20 genes and down-regulated 12 genes in both donors. SSR regulated >1,000 genes in both donors of which 397 were induced and 322 were suppressed in donors 1 and 2. Among the 20 genes up-regulated by cis-UCA, 16 were also up-regulated by SSR in both donors, whereas only two genes were down-regulated by both cis-UCA and SSR. In contrast, few genes were regulated by trans-UCA, but because none of these genes overlapped in donors 1 and 2, trans-UCA was considered to have little or no effect on gene regulation.

Table II.

The number of genes regulated by urocanic acid or solar simulated UVR in primary human keratinocytesa

DonorUp-RegulatedDown-Regulated
trans-UCA (10 μg/ml)cis-UCA (10 μg/ml)SSR 12 (J/cm2)cis-UCA and SSRbtrans-UCA (10 μg/ml)Cis-UCA (10 μg/ml)SSR (12 J/cm2)Cis-UCA and SSRb
43 547 26 31 709 
16 263 492 114 191 574 38 
1 and 2c 20 397 16 12 322 
DonorUp-RegulatedDown-Regulated
trans-UCA (10 μg/ml)cis-UCA (10 μg/ml)SSR 12 (J/cm2)cis-UCA and SSRbtrans-UCA (10 μg/ml)Cis-UCA (10 μg/ml)SSR (12 J/cm2)Cis-UCA and SSRb
43 547 26 31 709 
16 263 492 114 191 574 38 
1 and 2c 20 397 16 12 322 
a

Genes with the expression level differences >2-fold and change p < 0.0045 (up-regulated) or more than 0.9955 (down-regulated) relative to PBS control were counted.

b

The number of genes common to treatment with either cis-UCA or SSR.

c

The number of genes common to both donors.

The 20 genes up-regulated by cis-UCA in both donors are listed in Table III. Several different cellular processes were up-regulated by cis-UCA. trans-UCA did not induce any of these genes. cis-UCA up-regulated both proapoptotic (GADD45B, IGFBP3, and PMAIP1) and anti-apoptotic genes (CRYAB, SERPINB2, and TNFAIP3). These genes were also highly induced by SSR. Besides their proapoptotic function, GADD45B and IGFBP3 are associated with cell growth arrest. cis-UCA and SSR also induced ADAMTS1, which is a negative regulator of cell proliferation. The inhibitory effect of cis-UCA and SSR on cell growth was confirmed by cell count. After 24 h of incubation, cell proliferation was inhibited ∼20 and 30% by cis-UCA and SSR, respectively. We also measured mononucleosome and oligonucleosome release into the cytoplasm as a marker for apoptosis (results not shown). cis-UCA and SSR resulted in a small increase in nucleosome release. The enrichment factors of cis-UCA and SSR were 1.59 ± 0.69 and 1.93 ± 0.83, respectively. trans-UCA had no effect on cell count or nucleosome release.

Table III.

Genes up-regulated by cis-urocanic acid in primary human keratinocytesa

GenBankSymbolGene NameFold Change from PBS Control
trans-UCA (10 μg/ml)Cis-UCA (10 μg/ml)SSR (12 J/cm2)
Donor 1Donor 2Donor 1Donor 2Donor 1Donor 2
Apoptosis         
 AF007162.1 CRYAB Crystallin, α B 1.1 1.2 3.2 7.5 24.3 24.3 
 NM_002575.1 SERPINB2 Serpin peptidase inhibitor, clade B (ovalbumin), member 2 1.0 1.0 2.1 2.1 3.0 2.6 
 NM_006290.1 TNFAIP3 Tumor necrosis factor, α -induced protein 3 1.1 1.1 2.3 2.6 7.0 4.6 
 AI857639 PMAIP1 Phorbol-12-myristate-13-acetate-induced protein 1 1.3 1.0 2.3 2.6 27.9 18.4 
         
Apoptosis and cell growth  arrest         
 NM_015675.1 GADD45B Growth arrest and DNA-damage-inducible, β 1.1 0.8 2.0 2.1 97.0 55.7 
 M31159.1 IGFBP3 Insulin-like growth factor binding protein 3 0.8 1.0 4.3 8.0 11.3 9.8 
         
Cell proliferation         
 AK023795.1 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 1.7 1.1 4.0 2.5 42.2 7.5 
         
Oxidative stress related         
 NM_001353.2 AKR1C1 Aldo-keto reductase family 1, member C1 0.7 0.9 2.6 4.3 3.2 4.3 
 U05598.1 AKR1C2 Aldo-keto reductase family 1, member C2 0.8 0.9 2.3 3.5 2.6 3.0 
 NM_002064.1 GLRX Glutaredoxin (thioltransferase)* 1.1 1.0 2.1 2.1 1.6 1.1 
 NM_005345.3 HSPA1A Heat shock 70-kDa protein 1A 0.9 1.0 2.5 5.3 10.6 10.6 
 NM_005346.2 HSPA1B Heat shock 70-kDa protein 1B 0.9 0.9 2.0 4.0 8.6 10.6 
 BC001606.1 NCF2 Neutrophil cytosolic factor 2 (65-kDa, chronic granulomatous disease, autosomal 2)* 1.1 1.2 3.2 4.6 4.0 1.2 
 NM_000963.1 PTGS2 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) 1.0 1.1 5.7 19.7 78.8 39.4 
         
Cell adhesion and cytoskeleton  associated         
 NM_021101.1 CLDN1 Claudin 1* 1.0 1.1 2.8 4.3 3.5 1.9 
 NM_014547.1 TMOD3 Tropomodulin 3 (ubiquitous)* 1.1 1.3 2.0 2.1 0.9 1.4 
         
Others         
 NM_000574.1 CD55 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 1.1 0.8 2.0 2.3 6.1 3.2 
 U90065.1 KCNK1 Potassium channel, subfamily K, member 1 1.0 1.5 3.7 3.2 6.1 5.3 
 NM_005729.1 PPIF Peptidylprolyl isomerase F (cyclophilin F) 1.1 0.9 2.0 4.0 11.3 9.2 
 NM_003447.1 ZNF165 Zinc finger protein 165 2.8 2.8 7.5 4.9 8.6 22.6 
GenBankSymbolGene NameFold Change from PBS Control
trans-UCA (10 μg/ml)Cis-UCA (10 μg/ml)SSR (12 J/cm2)
Donor 1Donor 2Donor 1Donor 2Donor 1Donor 2
Apoptosis         
 AF007162.1 CRYAB Crystallin, α B 1.1 1.2 3.2 7.5 24.3 24.3 
 NM_002575.1 SERPINB2 Serpin peptidase inhibitor, clade B (ovalbumin), member 2 1.0 1.0 2.1 2.1 3.0 2.6 
 NM_006290.1 TNFAIP3 Tumor necrosis factor, α -induced protein 3 1.1 1.1 2.3 2.6 7.0 4.6 
 AI857639 PMAIP1 Phorbol-12-myristate-13-acetate-induced protein 1 1.3 1.0 2.3 2.6 27.9 18.4 
         
Apoptosis and cell growth  arrest         
 NM_015675.1 GADD45B Growth arrest and DNA-damage-inducible, β 1.1 0.8 2.0 2.1 97.0 55.7 
 M31159.1 IGFBP3 Insulin-like growth factor binding protein 3 0.8 1.0 4.3 8.0 11.3 9.8 
         
Cell proliferation         
 AK023795.1 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 1.7 1.1 4.0 2.5 42.2 7.5 
         
Oxidative stress related         
 NM_001353.2 AKR1C1 Aldo-keto reductase family 1, member C1 0.7 0.9 2.6 4.3 3.2 4.3 
 U05598.1 AKR1C2 Aldo-keto reductase family 1, member C2 0.8 0.9 2.3 3.5 2.6 3.0 
 NM_002064.1 GLRX Glutaredoxin (thioltransferase)* 1.1 1.0 2.1 2.1 1.6 1.1 
 NM_005345.3 HSPA1A Heat shock 70-kDa protein 1A 0.9 1.0 2.5 5.3 10.6 10.6 
 NM_005346.2 HSPA1B Heat shock 70-kDa protein 1B 0.9 0.9 2.0 4.0 8.6 10.6 
 BC001606.1 NCF2 Neutrophil cytosolic factor 2 (65-kDa, chronic granulomatous disease, autosomal 2)* 1.1 1.2 3.2 4.6 4.0 1.2 
 NM_000963.1 PTGS2 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) 1.0 1.1 5.7 19.7 78.8 39.4 
         
Cell adhesion and cytoskeleton  associated         
 NM_021101.1 CLDN1 Claudin 1* 1.0 1.1 2.8 4.3 3.5 1.9 
 NM_014547.1 TMOD3 Tropomodulin 3 (ubiquitous)* 1.1 1.3 2.0 2.1 0.9 1.4 
         
Others         
 NM_000574.1 CD55 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 1.1 0.8 2.0 2.3 6.1 3.2 
 U90065.1 KCNK1 Potassium channel, subfamily K, member 1 1.0 1.5 3.7 3.2 6.1 5.3 
 NM_005729.1 PPIF Peptidylprolyl isomerase F (cyclophilin F) 1.1 0.9 2.0 4.0 11.3 9.2 
 NM_003447.1 ZNF165 Zinc finger protein 165 2.8 2.8 7.5 4.9 8.6 22.6 
a

Genes with at least 2-fold changes in expression in donors 1 and 2 24 h after treatment with cis-urocanic acid were listed. Note that all genes apart from those marked with an asterisk (∗) were also up-regulated by SSR.

Many oxidative stress-related genes were induced by cis-UCA. These include AKR1C, HSPA1, NCF2, PTGS2, and GLRX, of which PTGS2 (also called COX-2) was the most highly up-regulated. NCF2 and GLRX were not induced by SSR based on 2-fold change. cis-UCA also induced adhesion and cytoskeleton-associated genes (CLDN1 and TMOD3).

To confirm the array results, the expression levels of eight representative genes up-regulated by cis-UCA and SSR were quantified independently using TaqMan real-time RT-PCR. As shown in Table IV, all of these genes gave similar results using the two methods.

Table IV.

Confirmation of the array results with RT-PCRa

Donortrans-UCA (10 μg/ml)cis-UCA (10 μg/ml)SSR (12 J/cm2)
Donor 1Donor 2Donor 1Donor 2Donor 1Donor 2
SERPINB2 1.0 1.2 3.7 5.0 6.9 4.9 
TNFAIP3 1.0 1.3 3.3 6.1 18.7 6.9 
ADAMTS1 1.1 1.3 4.0 3.4 65.6 8.3 
HSPA1B 0.9 0.9 3.2 10.2 136 106 
PTGS2 0.9 1.2 4.4 12.7 49.9 18.2 
GADD45B 0.9 1.4 1.9 3.8 172 105 
IGFBP3 1.1 1.1 41.9 34.3 37.6 137 
PMAIP1 0.9 1.4 1.8 5.2 74.4 33.2 
Donortrans-UCA (10 μg/ml)cis-UCA (10 μg/ml)SSR (12 J/cm2)
Donor 1Donor 2Donor 1Donor 2Donor 1Donor 2
SERPINB2 1.0 1.2 3.7 5.0 6.9 4.9 
TNFAIP3 1.0 1.3 3.3 6.1 18.7 6.9 
ADAMTS1 1.1 1.3 4.0 3.4 65.6 8.3 
HSPA1B 0.9 0.9 3.2 10.2 136 106 
PTGS2 0.9 1.2 4.4 12.7 49.9 18.2 
GADD45B 0.9 1.4 1.9 3.8 172 105 
IGFBP3 1.1 1.1 41.9 34.3 37.6 137 
PMAIP1 0.9 1.4 1.8 5.2 74.4 33.2 
a

Relative mRNA expression levels to PBS control.

Microarray and RT-PCR analyses showed that cis-UCA strongly up-regulated PTGS2. Because PTGS2 is involved in the regulation of prostaglandin synthesis, we measured PGE2 secretions into the cell culture supernatant at 24 h. As expected, a dose-dependent increase in the release of PGE2 was seen after treatment with cis-UCA. cis-UCA at 50 and 100 μg/ml were more effective than SSR, but trans-UCA at 100 μg/ml had no effect (Fig. 1,A). Total RNA was extracted from the cells 24 h after treatment, and the mRNA expression level of PTGS2 was measured to confirm its up-regulation. cis-UCA and SSR up-regulated PTGS2 expression, but trans-UCA had no effect. The expression level of PTGS2 after treatment with cis-UCA at 50 and 100 μg/ml was greater than that at 10 μg/ml (Fig. 1 B).

FIGURE 1.

cis-Urocanic acid enhances PGE2 synthesis. Keratinocytes were treated with PBS, trans-UCA (100 μg/ml), cis-UCA (10, 50, and 100 μg/ml), or solar-simulated UVR (12 J/cm2) as indicated. A, PGE2 secretion into the supernatant 24 h following treatment was analyzed by ELISA. B, RNA was isolated 24 h after treatment and PTGS2 expression was quantified using real-time RT-PCR (TaqMan). Results are expressed as mean ± SD (n = 3) of one representative experiment of four. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs PBS control.

FIGURE 1.

cis-Urocanic acid enhances PGE2 synthesis. Keratinocytes were treated with PBS, trans-UCA (100 μg/ml), cis-UCA (10, 50, and 100 μg/ml), or solar-simulated UVR (12 J/cm2) as indicated. A, PGE2 secretion into the supernatant 24 h following treatment was analyzed by ELISA. B, RNA was isolated 24 h after treatment and PTGS2 expression was quantified using real-time RT-PCR (TaqMan). Results are expressed as mean ± SD (n = 3) of one representative experiment of four. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs PBS control.

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Although the induction of TNF-α mRNA by cis-UCA was not seen on the microarray, the up-regulation of TNF-α-induced protein implies that TNF-α synthesis occurred after cis-UCA treatment. In addition, several chemokine- and cytokine-related genes such as IL-8 and IL-1 and IL-6 receptors were induced by cis-UCA and SSR in at least one donor (data not shown). Taken together, cis-UCA is likely to enhance cytokine production in keratinocytes. To further assess the effect of cis-UCA on cytokine synthesis, mRNA expression levels of major cytokines induced by SSR were quantified individually using TaqMan real-time RT-PCR, which is more sensitive. As shown in Table V, cis-UCA increased mRNA expression levels of IL-6 and IL-8 in both donors and TNF-α in donor 2. SSR also increased their levels in both donors. The expression level of IL-1α or IL-1β was not altered by cis-UCA. SSR increased IL-1α expression in donor 1 but not IL-1β expression in either donor. It has been reported that IL-1α and IL-1β mRNA expressions peak within 6 h post-UVR and return to the steady-state level 24 h after treatment (40, 41). Thus, the IL-1 expression level may have dropped to baseline level 24 h after SSR treatment. IL-10 mRNA expression was also analyzed but not detected (data not shown).

Table V.

mRNA expression levels of cytokines by RT-PCRa

Donortrans-UCA (10 μg/ml)cis-UCA (10 μg/ml)SSR (12 J/cm2)
Donor 1Donor 2Donor 1Donor 2Donor 1Donor 2
TNF-α 1.2 1.0 1.3 2.3 12.6 5.0 
IL-1α 0.9 0.8 1.5 1.0 2.3 0.6 
IL-1β 1.0 1.0 1.1 1.3 1.2 0.6 
IL-6 1.2 1.5 2.2 9.1 211 68.6 
IL-8 0.9 1.4 4.3 27.5 739 301 
Donortrans-UCA (10 μg/ml)cis-UCA (10 μg/ml)SSR (12 J/cm2)
Donor 1Donor 2Donor 1Donor 2Donor 1Donor 2
TNF-α 1.2 1.0 1.3 2.3 12.6 5.0 
IL-1α 0.9 0.8 1.5 1.0 2.3 0.6 
IL-1β 1.0 1.0 1.1 1.3 1.2 0.6 
IL-6 1.2 1.5 2.2 9.1 211 68.6 
IL-8 0.9 1.4 4.3 27.5 739 301 
a

Relative to PBS control.

Having confirmed that cis-UCA enhances keratinocyte cytokine synthesis at the mRNA level, we proceeded to assess secretion of TNF-α, IL-6, and IL-8 into the cell culture supernatant by ELISA. The results are given in Fig. 2. cis-UCA increased TNF-α, IL-6, and IL-8 significantly compared with PBS, and the increases in IL-6 and IL-8 were dose dependent. SSR had the same effect as cis-UCA, but trans-UCA had no effect.

FIGURE 2.

cis-Urocanic acid increases protein secretion of TNF-α, IL-6, and IL-8. Keratinocytes were treated with PBS, trans-UCA (100 μg/ml), cis-UCA (10, 50, and 100 μg/ml), or solar-simulated UVR (12 J/cm2) as indicated. 24 h after treatment, supernatants were collected and analyzed for TNF-α (A), IL-6 (B), and IL-8 (C) by ELISA. Results are expressed as mean ± SD (n = 3) of one representative experiment of four. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs PBS control.

FIGURE 2.

cis-Urocanic acid increases protein secretion of TNF-α, IL-6, and IL-8. Keratinocytes were treated with PBS, trans-UCA (100 μg/ml), cis-UCA (10, 50, and 100 μg/ml), or solar-simulated UVR (12 J/cm2) as indicated. 24 h after treatment, supernatants were collected and analyzed for TNF-α (A), IL-6 (B), and IL-8 (C) by ELISA. Results are expressed as mean ± SD (n = 3) of one representative experiment of four. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs PBS control.

Close modal

As many oxidative stress-related genes were induced by cis-UCA, it was of interest to examine the ability of cis-UCA to induce oxidative stress. We measured 8-isoprostane release for 24 h after UCA or SSR treatment. 8-Isoprostane, a biomarker for photo-oxidative stress, is generated by free radical-catalyzed peroxidation of tissue phospholipids (42). As shown in Fig. 3 A, cis-UCA and SSR significantly increased the release of 8-isoprostane, and cis-UCA showed a dose-dependent trend. Cells treated with cis-UCA at higher concentrations secreted more 8-isoprostane than those treated with SSR. trans-UCA had little effect.

FIGURE 3.

cis-Urocanic acid induces lipid peroxidation and NF-κB activation. Keratinocytes were treated with PBS, trans-UCA (100 μg/ml), cis-UCA (10, 50, and 100 μg/ml), or solar-simulated UVR (12 J/cm2) as indicated. A, 8-Isoprostane release into the supernatant 24 h following treatment was analyzed by ELISA. B, Nuclear extracts were prepared 24 h after treatment and NF-κB activation was measured using TransAM kits (Active Motif). Results are expressed as mean ± SD (n = 3) of one representative experiment of three. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs PBS control.

FIGURE 3.

cis-Urocanic acid induces lipid peroxidation and NF-κB activation. Keratinocytes were treated with PBS, trans-UCA (100 μg/ml), cis-UCA (10, 50, and 100 μg/ml), or solar-simulated UVR (12 J/cm2) as indicated. A, 8-Isoprostane release into the supernatant 24 h following treatment was analyzed by ELISA. B, Nuclear extracts were prepared 24 h after treatment and NF-κB activation was measured using TransAM kits (Active Motif). Results are expressed as mean ± SD (n = 3) of one representative experiment of three. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs PBS control.

Close modal

NF-κB is a key transcription factor that controls the inflammatory and immune response in response to cellular stress and plays an important role in the control of UVR-induced gene expression (43). Increased production of immunomodulatory mediators, oxidative stress, and antiapoptotic and proapoptotic gene expression imply a contribution of cis-UCA to the activation of NF-κB, which was analyzed 24 h after UCA or SSR treatment. As shown in Fig. 3 B, cis-UCA but not trans-UCA induced NF-κB activation significantly at higher doses of 50 and 100 μg/ml. SSR also activated NF-κB.

The keratinocyte is an important candidate for the site of action of cis-UCA. In the present study we used a limited microarray analysis to assist in the identification of any biological effects of cis-UCA on keratinocytes. This revealed that cis-UCA, but not trans-UCA, up-regulated 16 UVR-inducible genes that affect different cellular processes in keratinocytes (Table III). The list of genes transcribed is likely to underestimate the total number of genes regulated by cis-UCA, as we used a single concentration of cis-UCA (10 μg/ml) and assessed one time point after treatment (24 h). In addition, the keratinocytes were obtained from two different skin type donors (Table I). cis-UCA induced gene transcription in both donors, but the responses to cis-UCA were different. There are no data regarding individual variations in sensitivity to cis-UCA, but it has been reported that the concentration of total UCA in Caucasians is not correlated with skin type, pigmentation, MED, age, or sex (44, 45). We have previously shown that susceptibility to SSR-induced immune suppression is skin type dependent within white-skinned Caucasians (6), but any relationship with cis-UCA needs to be determined with a larger number of samples than that used in the present study.

To eliminate “false positive” results, we only selected genes that were up-regulated in both donors (one skin type I and one skin type III) for further analysis (Table III) and then confirmed the genes transcribed by RT-PCR using the same RNA samples (Tables IV and V). The microarray results were extended by estimating protein expression in four further primary keratinocyte cell lines (Figs. 1–3). Among the genes induced by cis-UCA and SSR, PTGS2 was the most dramatically up-regulated (Table III) and resulted in an enhanced secretion of PGE2 (Fig. 1). Shreedhar et al. have proposed that PGE2 released by keratinocytes is an initiator of UVR-induced systemic immune suppression (46). Their findings suggest that PGE2, presumably secreted from UVR-exposed keratinocytes, stimulates serum IL-10 production following IL-4 release by peripheral blood leukocytes that leads to systemic immune suppression. Our data support their theory and suggest that the primary role of cis-UCA in keratinocytes may be the induction of PGE2 release to initiate the cytokine cascade.

We also provide direct evidence that cis-UCA induces cytokine production in all four primary human keratinocyte lines. cis-UCA up-regulated mRNA expression levels of TNF-α, IL-6, and IL-8 (Table V) and significantly increased their secretion into the cell culture supernatant in a dose-dependent manner (Fig. 2). TNF-α has been shown to cause depletion of epidermal Langerhans cells and recruitment of macrophages into the dermis, which are important events in the modulation of alloantigen presentation in human skin (47). The immunosuppressive effects of cis-UCA may be partially mediated via TNF-α, because anti-TNF-α treated or TNF-α receptor knockout mice show a reduced cis-UCA induced suppression of the CHS response (9, 11). In addition, cis-UCA induced several genes associated with apoptosis and cell growth arrest. Induction of apoptosis and DNA repair following UVR exposure are important defense mechanisms for keratinocytes and coordinate a delicate balance to eliminate genetic damage that may cause malignant transformation. Although their molecular pathways have not been fully elucidated, both DNA damage and death receptors on the cell surface are involved. Along with the direct activation of the death receptors by UVR, it has been reported that autocrine release of TNF-α may contribute to UVR-induced apoptosis (48). Thus, increased secretion of TNF-α by cis-UCA may also play a role in the induction of apoptosis. In contrast to PGE2 and TNF-α, cis-UCA was much less effective in IL-6 and IL-8 production than SSR, suggesting that cis-UCA and UVR regulate these cytokines by different mechanisms.

Zak-Prelich et al. previously reported that no induction of TNF-α, IL-10, or TGFβ mRNA by cis-UCA was seen in the murine squamous cell carcinoma cell line PAM-212 (49). However, their results cannot be extrapolated directly to the human system. First, PAM-212 may have a defect in the normal immunoregulatory response to UVR exposure, because up-regulation of TNF-α and TGFβ mRNA by UVR was not seen. In addition, unlike murine keratinocytes, it is debatable whether human keratinocytes produce IL-10 following UVR exposure (50). Some groups have demonstrated the production of IL-10 mRNA and its protein secretion in UV- and non-UV-irradiated keratinocytes (51, 52), whereas others were unable to detect IL-10 mRNA or protein up to 24 h after UV irradiation (53, 54, 55). We were also unable to detect IL-10 mRNA or protein 24 h following UCA or SSR treatment.

Redondo et al. used primary human keratinocytes and showed that cis-UCA up-regulated TNF-α, IL-1α, and IL-1β mRNA after 8 h but had no effect on the cytokine concentration at 24 h in the cell culture supernatant (56). Although significance was not seen, they noted that cis-UCA slightly increased the protein level of IL-8 in two samples. They failed to detect IL-6 mRNA and protein secretion. They used Northern blot analysis, which may not have been sensitive enough to detect a significant difference. We used the more sensitive TaqMan real-time RT-PCR that enabled us to quantify mRNA expression level of the cytokine transcripts.

Our results suggest that cis-UCA-induced gene transcription involves activation of the NF-κB transcription factor (Fig. 3,B). There is substantial evidence that the generation of reactive oxygen species mediates NF-κB activation (43). In his review, Ullrich discussed free radical formation in UVR-induced immune suppression and suggested that NF-κB activation and cytokine transcription are caused by UVR-induced oxidative stress and membrane lipid peroxidation independently of DNA photodamage (57). Interestingly, our microarray analysis revealed that cis-UCA induced several oxidative stress-related genes (Table III), and increased lipid peroxidation was verified by measuring 8-isoprostane release (Fig. 3 A). Steenvoorden and Beijersbergen van Henegouwen showed that topical treatment with the antioxidants α-tocopherol and ascorbic acid inhibited cis-UCA-induced local immune suppression dose-dependently in mice. In their model, lipid soluble α-tocopherol was 500-fold more effective than ascorbic acid, which is a water soluble antioxidant (58). This observation suggests that membrane lipid peroxidation may contribute to the immunosuppressive effects of cis-UCA. Taken together, reactive oxygen species may act as a second messenger in cis-UCA induced immune suppression.

Although NF-κB activation and oxidative stress are suggested as upstream events of cis-UCA induced gene transcription, it is still unknown how cis-UCA participates in signal transduction pathways. A specific keratinocyte receptor for cis-UCA has not yet been identified. cis-UCA has been reported to bind to the 5-hydroxytryptamine receptor, but the biological effects of this binding remain controversial (22, 59). Further study is required to elucidate the identity of a functional receptor for cis-UCA, which may not necessarily be on keratinocytes.

The immunomodulatory properties of UVR are often exploited in the treatment of skin diseases, including psoriasis, polymorphic light eruption, and atopic eczema. The present study showed that cis-UCA can induce immunomodulatory mediators in a similar way as UVR, suggesting that cis-UCA may be of some benefit in the treatment of some immunological skin disorders. A small trial for the treatment of psoriatic stable plaques reported improvement in patients receiving topical cis-UCA, but it was less efficient than tar or dithranol paste, conventional topical ointments. However, cis-UCA was dissolved in 90% acetone, and the authors noted that the delivery system required refinement to ensure adequate penetration into the skin (60). Further investigations regarding effects of topical application of cis-UCA on skin disorders may be worthwhile.

In summary, the results presented here demonstrate that cis-UCA but not trans-UCA induces UVR-inducible genes associated with apoptosis, cell growth arrest, cytokines, and oxidative stress, resulting in enhanced secretion of immunomodulatory mediators in primary human keratinocytes. Activation and translocation of NFκB and lipid peroxidation are possible upstream events of the gene expression changes. Overall, our findings suggest that the induction of immune suppression by cis-UCA may involve the initiation of gene transcription in epidermal keratinocytes.

We thank Dr. Matthew Arno at the Genomics Centre, King’s College London for the Affymetrix microarray assay and statistical advice, and Dr. Roy Palmer for taking the biopsies. We also thank Professor Brian Diffey for assistance with the exposure calculations based on his paper (37).

The authors have no financial conflict of interest.

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 by St. John’s Institute of Dermatology and Clinique Laboratories, Melville, NY.

3

Abbreviations used in this paper: UVR, UV radiation; CHS, contact hypersensitivity; J, joule; MED, minimal erythema dose; PTGS2, prostaglandin-endoperoxide synthase 2; SSR, solar simulated radiation; UCA, urocanic acid.

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