Bert-Jan Vermeer

Determination of the photoprotective efficacy of a topical sunscreen against UVB-induced DNA damage in human epidermis

The ability of a chemical sunscreen with a sun protection factor of ten to protect human skin in situ against UVB-induced DNA damage (cyclobutyl thymine dimers) was evaluated. Biopsies were taken from the left buttock of ten human volunteers prior to UVB (280-315 nm) exposure. Subsequently, a sunscreen (n = 6) or vehicle (n = 4) was applied to a delineated area on the right buttock. After a period of 30 min, the entire buttock area was irradiated in a UVB cabin with one minimal erythema dose. Immediately after irradiation, biopsy specimens were obtained from the UVB-exposed sunscreen- or vehicle-treated right buttock and from the non-treated UVB-exposed left buttock. Dimers were assayed in skin sections by immunofluorescence microscopy with a monoclonal antibody against the cyclobutyl thymine dimer. The dimer-specific fluorescence from the epidermal cell nuclei, identified by counterstaining with propidium iodide, was quantified through computer-mediated image processing and analysis in skin sections of one sunscreen-treated and one vehicle-treated volunteer. After a single dose of UVB, significant dimer-specific nuclear fluorescence was observed and measured in the non-treated biopsy specimens. No nuclear fluorescence was observed and very little could be measured in the non-UVB-exposed skin and in the sunscreen-treated UVB-exposed skin respectively, indicating that the sunscreen offered good protection against the induction of cyclobutyl thymine dimers by UVB. This visual scoring is in general semiquantitative, but quantification through computer-mediated image processing was performed in one case for sunscreen-treated skin and in one case for vehicle-treated skin. Both assessments resulted in similar conclusions.(ABSTRACT TRUNCATED AT 250 WORDS)

The UV action spectra for the clone-forming ability of cultured human melanocytes and keratinocytes.

Abstract Melanocytes (skin type 2) and keratinocytes were irradiated with UV light of 254, 297, 302, 312 and 365 nm and the survival was measured. Clone‐forming ability was chosen as the parameter for cell survival. Melanocytes were found to be less sensitive to UV light than keratinocytes (a difference of a factor 1.22‐1.92 for the UV‐C and UV‐R wavelengths (254, 297, 301 and 312 nm) and a factor 6.71 for the UV‐A wavelength (365 nm). Because melanin does not appear to protect against the induction of pyrimidine dimers the difference between melanocytes and keratinocytes in the UV‐C and UV‐B region could not be explained by the presence of melanin in the melanocytes. The relatively small difference can be explained by the longer cell cycle of melanocytes, which provides more time for the melanocytes to repair UV damage. In the UV‐A region the difference between melanocytes and keratinocytes was much larger, suggesting that besides the longer cell cycle some additional factors must be involved in protection against UV‐A light.

THE ACTION SPECTRA FOR UV‐INDUCED SUPPRESSION OF MLR and MECLR SHOW THAT IMMUNOSUPPRESSION IS MEDIATED BY DNA DAMAGE

‐Ultraviolet‐B (UVB,280–320 nm) radiation can promote the induction of skin cancer by two mechanisms: damage of epidermal DNA and suppression of the immune system, allowing the developing tumor to escape immune surveillance. The mixed lymphocyte reaction (MLR) and the mixed epidermal cell lymphocyte reaction (MECLR) are commonly used methods to study the immunosuppressive effects of UVB radiation. To obtain a better understanding of the mechanism by which UVB radiation decreases the alloactivating capacity of in vitro‐irradiated cells, action spectra for the MLR and MECLR were determined. Suspensions of peripheral blood mononuclear cells or epidermal cells were irradiated with monochromatic light of 254, 297, 302 or 312 nm and used as stimulator cells in the MLR or MECLR. Using dose‐response curves for each wavelength, the action spectra were calculated. Both MLR and MECLR action spectra had a maximum at 254 nm and a relative sensitivity at 312 nm that was a thousand times lower than at 254 nm. Strikingly, the action spectra corresponded very closely to the action spectra that were found by Matsunaga et al. (Photochem. Photobiol. 54,403–410, 1991) for the induction of thymine dimers and (6‐4)photoproducts in irradiated calf thymus DNA solutions, strongly suggesting that the UV‐induced abrogation of the MLR and MECLR responses is mediated by UV‐induced DNA damage. Furthermore, the action spectra for the MLR and MECLR were similar, suggesting that they share a common mechanism for UV‐induced suppression.

In Situ Action Spectra Suggest that DNA Damage is Involved in Ultraviolet Radiation-induced Immunosuppression in Humans

Abstract— The mixed epidermal cell lymphocyte reaction (MECLR) is a commonly used method to study the immunomodulatory effects of UV radiation. The in vitro action spectrum for the MECLR showed that the UV‐induced suppression of the MECLR responses is associated with UV‐induced DNA damage. To investigate whether in vivo DNA damage also leads to the abrogation of the MECLR, in situ action spectra were made for the MECLR and the induction of thymine dimers (T<>T). Human skin, obtained from plastic surgery, was exposed to monochromatic light of 254, 297, 302 and 312 nm. After irradiation, epidermal cells were isolated and used as stimulator cells in the MECLR or processed for flow cytometric detection of T<>T. On the basis of dose‐response curves for each wavelength, the action spectra for suppression of the MECLR and the induction of T<>T were calculated. These spectra showed close similarities, suggesting that, also in situ, UV‐induced DNA damage is involved in the UV‐induced suppression of the MECLR. Both action spectra showed a small decline from 254 nm to 302 nm, followed by a steep decline to 312 nm. These data show that, in situ, UVC can efficiently induce DNA damage and modulate cutaneous immune responses.

Differential Suppression of the Human Mixed Epidermal Cell Lymphocyte Reaction (MECLR) and Mixed Lymphocyte Reaction (MLR) by Cis‐Urocanic Acid

Abstract— Cis‐urocanic acid (UCA), formed in the stratum corneum by UV irradiation of trans‐UCA has been proposed as a mediator of UV‐induced immunosuppression in the skin. In this study, we examined the in vitro effect of cis‐UCA (6‐100 μg/mL) on the human mixed lymphocyte reaction (MLR) and the mixed epidermal cell lymphocyte reaction (MECLR). Addition of cis‐UCA (purified or in a mixture with trans‐UCA) did not affect the MLR but was able to induce a 20% suppression of the MECLR responses. Because this effect of cis‐UCA on the MECLR was not as strong as could be expected from previous in vivo results, we designed a set of experiments in order to enhance the in vitro immunosuppressive capacity of cis‐UCA. Firstly, we preincubated epidermal cells with UCA (50 u.g/mL) for 3 or 6 days before culture in the MECLR because in vivo repeated UV exposure can lead to a photostationary state, where cis‐UCA may be present for several weeks. This pretreatment with cis‐UCA resulted in a maximal decrease of the MECLR responses of 27%, whereas trans‐UCA had no effect. Secondly, we investigated whether UVB irradiation of epidermal cells could make cells more sensitive to cis‐UCA. However, addition of trans‐ or cis‐UCA did not potentiate the reduced alloac‐tivating capacity of UVB‐irradiated cells. Finally, we examined the possibility of a synergistic effect of cis‐UCA with histamine. Addition of histamine suppressed the MLR and MECLR responses, but neither cis‐ nor trans‐UCA were able to modulate this decrease. We conclude that cis‐UCA can partly downregulate the human MECLR but not the MLR. The mechanism involved in this differential downregulation is not known. In this respect it is striking that cis‐UCA does not potentiate the UVB‐ or histamine‐induced suppression of the MECLR.

UVB-induced suppression of the mixed epidermal cell lymphocyte reaction is critically dependent on irradiance.

The mixed epidermal cell lymphocyte reaction (MECLR) is a commonly used method to study the effects of ultraviolet B (UVB) radiation on the skin immune system. In UVB experiments dosimetry is very important. The influence of irradiance on the MECLR was studied in vitro using Philips FS40 lamps with variable UV intensities. Irradiation of isolated epidermal cells with high irradiance impaired the alloactivating capacity more than irradiation with low irradiance. In vivo, the influence of long‐term UVB exposure on the MECLR was studied by treating normal healthy volunteers with suberythemagenic doses of UVB thrice weekly during 4 weeks. The first set of experiments, using low irradiance Sylvania UV‐21 F75/85 W lamps, resulted in a decrease of MECLR responses of 83.1%. In the second set of experiments performed a year later, employing an identical protocol except for the use of high irradiance Waldmann UV‐21 F85/100 W lamps, an increase of MECLR responses of 99.7% was observed. Volunteers of both sets of experiments received equal doses of UVB. In conclusion, this study shows that in vitro UVB‐induced suppression of the MECLR is critically dependent on irradiance and therefore might explain contradictory results described in the literature. The in vivo data suggest that, comparable to the in vitro experiments, irradiance may influence the effects of UVB irradiation in vivo. Further experiments should prove whether this is indeed the case.

Decreased expression of decay-accelerating factor on endothelial cells of immune complex-mediated vasculitic skin lesions.

Endothelial cells may be damaged directly by the membrane attack complex of complement in immune complex vasculitis of the skin. However, for endothelial cell membrane injury to occur, normal regulatory mechanisms must fail. One of the main complement regulatory proteins of endothelial cells is decay-accelerating factor, a surface protein which interferes with either the classical or alternative pathway C3 and C5 convertases. We have investigated the expression of decay-accelerating factor in 4 patients with histologically proven cutaneous immune complex vasculitis, using an immuno-electronmicroscopic technique. We demonstrated that endothelial cells of upper dermal vessels in vasculitic lesions were almost completely devoid of decay-accelerating factor. By contrast, the expression of this protein on endothelial cells in uninvolved skin of the patients was the same as in skin of healthy volunteers. As yet, the mechanism responsible for depletion of decay-accelerating factor is not clear. Absence of decay-accelerating factor may follow enzymatic release from the phosphatidylinositol anchor, proteolytic stripping from the cell membrane or a down-regulation of decay-accelerating factor synthesis. Regardless of mechanism, endothelial cell injury or death could serve a phlogistic function to facilitate complement-mediated destruction of endothelial cells for removal and repair.

Presence and interpretation of vascular immune deposits in human skin: The value of direct immunofluorescence

Direct immunofluorescence investigation of the skin is an easy and valuable technique to establish the diagnosis immune complex vasculitis. Vascular immune deposits can be found in 60-80% of all cases. Absence of vascular immune deposits, however, does not exclude vasculitis per se, since the dynamics of the vasculitic process limit their presence in time. Knowledge of these dynamics is indispensable for both the clinician and the interpreter. Several practical options are discussed that may increase sensitivity. The specificity of vascular immune deposits has become a complex matter. Different immunoglobulin classes have different specificity, indicating that specificity also depends on the relative incidence of individual immunoglobulin classes. Some of these relative incidences seem to have changed over the years. Furthermore, several non-vasculitic diseases and conditions have now been described, that may show fluorescent pictures similar to vasculitis and thus decrease specificity.