Activation of the IL-10 gene promoter following photodynamic therapy of murine keratinocytes - PubMed (original) (raw)

Activation of the IL-10 gene promoter following photodynamic therapy of murine keratinocytes

S O Gollnick et al. Photochem Photobiol. 2001 Feb.

Abstract

Photodynamic therapy (PDT), an anticancer treatment modality, has recently been shown to be an effective treatment for several autoimmune disease models including antigen-induced arthritis. PDT was found to induce the expression of IL-10 messenger RNA (mRNA) and protein in the skin, and this expression has similar kinetics to the appearance of PDT-induced suppression of skin-mediated immune responses such as the contract hypersensitivity (CHS) response. Some aspects of the UVB-induced suppression of the immune response have been linked to the induction of IL-10. IL-10 has been shown to inhibit the development and activation of Th1 cells, which are critical for many cell-mediated immune responses, including CHS. We have examined the effect of PDT and UVB irradiation on the activity of the IL-10 gene promoter and on IL-10 mRNA stability using the murine keratinocyte line, PAM 212. In vitro PDT induces IL-10 mRNA and protein expression from PAM 212 cells, which can be correlated with an increase in AP-1 DNA binding activity and activation of the IL-10 gene promoter by PDT. Deletion of an AP-1 response element from the IL-10 gene promoter was shown to abrogate the PDT-induced promoter activity indicating that the AP-1 response element is critical to IL-10 induction by PDT. In addition, PDT results in an increase in IL-10 mRNA stability, which may also contribute to the increased IL-10 expression in PAM 212 cells following PDT. In vitro UVB irradiation also results in activation of the IL-10 promoter. However, in contrast to PDT, UVB-induced activation of the IL-10 promoter is not AP-1 dependent and did not increase IL-10 mRNA stability.

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Figures

Figure 1

Figure 1

Induction of IL-10 mRNA expression in PAM 212 cells by PDT. (A) PAM 212 cells were treated in vitro, with 2.5 μg/mL Photofrin® in complete medium for 24 h, followed by 3 h drug efflux in complete medium and illumination at 630 nm with 0.4 J/cm2 (~LD20). Total RNA was isolated from cells harvested immediately or at 1, 3, 6 and 24 h post-PDT, and 2 μg from each sample were subjected to RT-PCR analysis as described in “Materials and Methods.” Drug only controls were exposed to Photofrin® and were harvested after 6 h. Results are presented as in relative IL-10 mRNA levels ± SEM following normalization to the actin mRNA levels and represent the results of three independent experiments. (B) PAM 212 cells were treated in vitro, with 2.5 μg/mL Photofrin® in complete medium for 24 h, followed by 3 h drug efflux in complete medium and illumination at 630 nm with 0.2–0.8 J/cm2. Total RNA was isolated from cells harvested 4 h post-PDT and 2 μg from each sample were subjected to RT-PCR analysis as described in “Materials and Methods.” Drug only and light only controls were exposed to Photofrin® or 0.8 J/cm2 light only, respectively. Results are presented as in relative IL-10 mRNA levels ± SEM as described in “Materials and Methods” and represent the results of three independent experiments. The LD levels corresponding to each light dose are shown below the J/cm2 dose levels.

Figure 2

Figure 2

Induction of IL-10 secretion from PAM 212 cells by PDT. PAM 212 cells were treated in vitro, with 2.5 μg/mL Photofrin® in complete medium for 24 h, followed by 3 h drug efflux in complete medium and illumination at 630 nm with 0.4 J/cm2 (~LD20). Immediately following PDT, complete medium was replaced by serum-free medium and the cells were incubated at 37°C in 5%CO2. Supernatants were harvested at 6, 24, 48 and 72 h post-PDT, cells were removed by centrifugation and the supernatants were concentrated 10-fold and the amount of IL-10 present was determined by ELISA as described in “Materials and Methods.” Drug only controls were exposed to Photofrin® and harvested at the same time as the treated cells. Results are reported as the mean ± SEM ng of IL-10 per microgram of total protein.

Figure 3

Figure 3

PDT induces IL-10 gene promoter activity. PAM 212 cells were transiently transfected with pIL10pro(−1626)CAT followed by either PDT or UVB treatment as described in “Materials and Methods.” Cells were harvested 48 h after treatment and CAT assays were performed with 100 μg of cell lysates. A representative experiment is shown. The numbers above each lane reflect the results [normalized percent conversion ± (SEM)] of a minimum of three independent experiments. Controls are represented as transfected untreated cells (UT) and transfected cells subjected to either light alone (light) or Photofrin® alone (drug). *Significant compared to controls.

Figure 4

Figure 4

Identification of a 593 bp region of the IL-10 promoter required for PDT induction. (A) PAM 212 cells were transiently transfected with 5′-deletion constructs of the IL-10 promoter (pIL10pro(−1033)CAT, pIL10pro(−885)CAT, pIL10pro(−863)CAT, pIL10pro(−125)CAT), the full length promoter pIL10pro(−1626)CAT construct or the pCAT3basic vector, followed by PDT or UVB irradiation. CAT assays were performed as above. Results are presented as normalized mean percent conversion of chloramphenicol to acetylated chloramphenicol ± SEM.

Figure 5

Figure 5

PDT induces AP-1 DNA binding activity in PAM 212 cells. (A) Nuclear extracts were isolated from PAM 212 cells at 0, 0.5, 3, 6, 24 h post-PDT and subjected to EMSA with radiolabeled AP-1, oligonucleotide as described in “Materials and Methods.” Nuclear extracts isolated from untreated cells (UT) and cells treated with Photofrin® (D) alone were used as controls. Free probe is also shown (F). Results from a representative experiment are shown. Extracts isolated from three independent experiments showed similar results. An arrow indicates the AP-1 complex. (B) Nuclear extracts were isolated 3 h post-PDT treatment were subjected to EMSA with radiolabeled AP-1 oligonucleotide in the presence or absence of 100 or 500-fold excess cold oligonucleotides to test for protein:DNA complex specificity. (C) Nuclear extracts were isolated 3 h post-PDT treatment and subjected to EMSA with radiolabeled AP-1 oligonucleotide in the presence or absence of 4 μg of anti-c-fos, anti-c-jun/AP-1 or control rabbit IgG, C, to identify proteins within the protein: DNA complex. The AP-1 and supershifted (ss) complexes are indicated with arrows.

Figure 6

Figure 6

Deletion of the AP-1 response element from the IL-10 promoter abrogates the PDT responsiveness. PAM 212 cells were: (A) transiently transfected with pIL10(−1626)CAT, pIL10(ΔAP1)CAT or pCAT3basic followed by PDT; or (B) UVB irradiation as described above. CAT assays were done as described in “Materials and Methods.” A representative experiment is shown; the numbers above each lane reflect the results [normalized percent conversion (SEM)] of a minimum of three independent experiments.

Figure 7

Figure 7

PDT prolongs IL-10 mRNA half-life. Actinomycin D (10 μg/mL) was added to PAM 212 cells 1 or 4 h following UVB irradiation or PDT treatment, respectively. Total RNA was isolated from cells harvested immediately (0) or at 1, 2 or 4 h post-actinomycin D addition and 2 μg from each sample were subjected to RT-PCR analysis as described in “Materials and Methods.” Results are presented as in relative IL-10 mRNA levels ± SEM following normalization to the actin mRNA levels. Control values refer to untreated controls for the UVB sample and Photofrin® treatment only for the PDT sample. Controls and treatment samples were harvested at 1 h for the UVB samples and 4 h for the PDT samples.

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