1. ^*Dermatology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
2. ^†Clinical Epidemiology Units, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

Correspondence: Mona Ståhle, MD, PhD, Unit of Dermatology, Department of Medicine, Karolinska Institutet, S-17176 Stockholm, Sweden. Email: mona.stahle@medks.ki.se

Received 21 March 2005; Revised 26 May 2005; Accepted 27 May 2005; Published online 25 October 2005.

To the Editor:

Human antimicrobial protein (hCAP18), is an effector molecule of the innate immune system. Its C-terminal peptide LL-37 has broad antibacterial activity in vitro, and current research indicates that it is important in protecting the integrity of the skin. The in vivo importance of hCAP18/LL-37 in antimicrobial defense is supported by laboratory animal models (^{Nizet et al, 2001}). In addition to its antibiotic effects, hCAP18 is emerging as a multifunctional protein involved in chemotaxis, neo-angiogenesis (^{Koczulla et al, 2003}), and wound healing (^{Heilborn et al, 2003}). hCAP18 is widely expressed in leukocytes and in epithelia, and is upregulated in response to inflammation and injury in the skin (^{Dorschner et al, 2001;Heilborn et al, 2003}).

Molecular mechanisms regulating the expression of hCAP18 in vivo are still poorly understood. We and others have recently shown that vitamin D treatment rapidly and directly upregulates the production of hCAP18 in human keratinocytes in vitro through binding a functional vitamin D responsive element (VDRE), in the hCAP18 gene promoter (^{Wang et al, 2004;Weber et al, 2005}). We have further verified the effect in human skin in vivo following topical application of a vitamin D analog ointment, calcipotriol (^{Weber et al, 2005}). The aim of this study was to explore the physiologic relevance of this pharmacologic effect using UVB as the natural stimulus for vitamin D production in human skin.

Here we demonstrate that irradiation with a single dose of UVB but not of UVA significantly upregulated the expression of hCAP18 and vitamin D receptor (VDR) in the skin of healthy volunteers. These findings indicate that vitamin D may be a natural regulator for hCAP18 expression in the skin.

The study was performed using a protocol for UVB irradiation that was previously shown to induce vitamin D in human skin (^{Lehmann et al, 2003}). Eight healthy Caucasian individuals with no history of skin disease were recruited for the study, which was approved by the Regional Committee of Ethics and was conducted according to the Declaration of Helsinki Principles. Written informed consent was given by all individuals. Only fair-skinned individuals with skin types II and III were selected in order to optimize the vitamin D-inducing effect from UVB. The minimal erythema dose (MED), in response to UVB irradiation was determined for all individuals (Table I). MED is defined as the lowest UVB dose evoking a minimal erythema 24 h after exposure. The UVB source was an Osram Xenon arc lamp with a Schott 295 filter (Carl Zeiss AB, Stockholm, Sweden) emitting 280–315 nm with an intensity of 2.3 mW per cm². The experiment was performed as follows: a single MED dose of UVB was delivered to untanned buttock skin of each individual. At 24–28 h following exposure, a 6 mm² punch biopsy was obtained from the irradiated area that displayed a barely detectable erythema. A control biopsy was obtained from non-irradiated skin, and the tissues were snap-frozen as described (^{Weber et al, 2005}). On the other buttock, a single dose of UVA1 20J per cm² was given as an additional control, and after 24–28 h, a punch biopsy was obtained from the irradiated area as described above. The UVA1 source was a UVASUN 3000 lamp (Mutzhas, Germany) emitting 340–400 nm with an intensity of 61mW per cm². RNA was extracted from the tissues, and hCAP18 mRNA was determined by Real-Time PCR as described (^{Weber et al, 2005}). All measurements were normalized to the expression of 18S RNA, and are presented as arbitrary units (Table I). In addition, the expression of the VDR, was determined using a commercially available kit (Assay on Demand, Applied Biosystems, Foster City, California). The results show that both hCAP18 mRNA and VDR expression were significantly upregulated (signed rank test; p=0.008 and 0.02, respectively) after exposure to UVB (Table I), whereas there were no significant differences between non-irradiated skin and skin exposed to UVA (Table I). Moreover, there was a significant correlation between the responses in hCAP18 and VDR mRNA induced by UVB (Spearman rank correlation p=0.01). There was a substantial variation between individuals in the non-exposed skin for both hCAP18 and VDR expression. But the upregulation was proportional to baseline levels.

Table I - Increased absolute levels of hCAP18 and VDR mRNA expression in skin exposed to the MED dose of UVB compared with non-irradiated skin and 20 J per cm2 of UVA.

Full table

In conclusion, we show that irradiation with UVB according to a protocol that was reported to induce the synthesis of vitamin D in human skin (^{Lehmann et al, 2003}) significantly upregulated the expression of VDR and hCAP18. These results are in line with our recent data (^{Weber et al, 2005}) and indicate that the upregulation of hCAP18 mRNA is associated with UVB-induced production of vitamin D. The upregulation of VDR after UVB irradiation is in accordance with previous findings demonstrating that vitamin D induces the expression of VDR in keratinocytes (^{Solvsten et al, 1997}). The regulation of VDR expression, however, seems complex and it has been reported that UVB transiently downregulates the expression of VDR in keratinocytes in vitro up to 24 h following irradiation (^{Courtois et al, 1998}).

In humans, skin is the major source for vitamin D and interestingly its production in response to UVB is conserved through evolution. The role of UVB-induced vitamin D production, which has been demonstrated in earlier life forms such as plants and phytoplankton, is unclear. In view of the data presented here, an important physiologic function for vitamin D in regulating components of the innate immune system is emerging.

Thus, taken together, these findings suggest the evolution of an intriguing biosystem wherein the skin interacts with the environment to enhance its antimicrobial protection and repair.