N-acetylation by N-acetyltransferase 1 (NAT1) is an important biotransformation pathway of the human skin and it is involved in the deactivation of the arylamine and well-known contact allergen para-phenylenediamine (PPD). Here, NAT1 expression and activity were analyzed in antigen presenting cells (monocyte-derived dendritic cells, MoDCs, a model for epidermal Langerhans cells) and human keratinocytes. The latter were used to study exogenous and endogenous NAT1 activity modulations. Within this thesis, MoDCs were found to express metabolically active NAT1. Activities were between 23.4 and 26.6 nmol/mg/min and thus comparable to peripheral blood mononuclear cells. These data suggest that epidermal Langerhans cells contribute to the cutaneous N-acetylation capacity. Keratinocytes, which are known for their efficient N-acetylation, were analyzed in a comparative study using primary keratinocytes (NHEK) and different shipments of the immortalized keratinocyte cell line HaCaT, in order to investigate the ability of the cell line to model epidermal biotransformation. N-acetylation of the substrate para-aminobenzoic acid (PABA) was 3.4-fold higher in HaCaT compared to NHEK and varied between the HaCaT shipments (range 12.0"44.5 nmol/mg/min). Since B[a]P induced cytochrome p450 1 (CYP1) activities were also higher in HaCaT compared to NHEK, the cell line can be considered as an in vitro tool to qualitatively model epidermal metabolism, regarding NAT1 and CYP1. The HaCaT shipment with the highest NAT1 activity showed only minimal reduction of cell viability after treatment with PPD and was subsequently used to study interactions between NAT1 and PPD in keratinocytes. Treatment with PPD induced expression of cyclooxygenases (COX) in HaCaT, but in parallel, PPD N-acetylation was found to saturate with increasing PPD concentration. This saturation explains the presence of the PPD induced COX induction despite the high N-acetylation capacities. A detailed analysis of the effect of PPD on NAT1 revealed that the saturation of PPD N-acetylation was caused by a PPD-induced decrease of NAT1 activity. This inhibition was found in HaCaT as well as in primary keratinocytes after treatment with PPD and PABA. Regarding the mechanism, reduced NAT1 protein level and unaffected NAT1 mRNA expression after PPD treatment adduced clear evidences for substrate-dependent NAT1 downregulation. These results expand the existing knowledge about substrate-dependent NAT1 downregulation to human epithelial skin cells and demonstrate that NAT1 activity in keratinocytes can be modulated by exogenous factors. Further analysis of HaCaT cells from different shipments revealed an accelerated progression through the cell cycle in HaCaT cells with high NAT1 activities. These findings suggest an association between NAT1 and proliferation in keratinocytes as it has been proposed earlier for tumor cells. In conclusion, N-acetylation capacity of MoDCs as well as keratinocytes contribute to the overall N-acetylation capacity of human skin. NAT1 activity of keratinocytes and consequently the detoxification capacities of human skin can be modulated by the presence of exogenous NAT1 substrates and endogenous by the cell proliferation status of keratinocytes.
The allergic contact dermatitis (ACD) to small molecular weight compounds is a common inflammatory skin reaction. ACD is restricted to industrialized countries, has an enormous sociomedical and socioeconomic impact. About 2,800 compounds from the six million chemicals known in our environment are believed to have allergic, and to a lesser degree also contact-sensitizing or immunogenic properties causing allergic contact dermatitis. ACD results from T cell responses to harmless, low molecular weight chemicals (haptens) applied to the skin. Haptens are not directly recognized by the cells of the immune system. They need to be presented by subsets of antigen presenting cells to the cells of the immune system. In this regard, epidermal Langerhans cells (LC) and the cells into which they mature (dendritic cells) are believed to play a pivotal role in the sensitization process for ACD. LC are able to bind the haptens, internalize them, and present them to naive T cells and induce thereby the development of effector T cells. They are so-called professional antigen presenting cells. This process is initiated and maintained by the release of several mediators, which are released by various cells after their contact with the haptens. One of the first proteins secreted into the environment is interleukin (IL)-1ß. This cytokine is produced and secreted minutes after an antigen enters the cell. It is commonly believed that the large amounts of this protein and other cytokines such as granulocyte-colony stimulation factor (GM-CSF) and tumor necrosis factor alpha (TNF-ï¡) needed for the initiation and activation of ACD are coming first from other cells residing in the skin, e.g., keratinocytes, monocytes and macrophages. These cytokines provide the danger signals needed for the activation of the Langerhans cell (LC), which then produce via a positive feedback loop various cytokines themselves. In addition, other proteins such as chemokines influence the generation of danger signals, migration, homing of T cells in the local lymph nodes as well as the recruitment of T cells into the skin. Thus, a small molecular compounds or hapten needs to be able to induce danger signals in order to become immunogenic. In this study, we investigated whether para-phenylenediamine (PPD), an arylamine and common contact allergen, is able to induce danger signals and likely provide the signals needed for an initiation of an immune response[162, 163]. PPD is used as an antioxidant, an ingredient of hair dyes, intermediate of dyestuff, and PPD is found in chemicals used for photographic processing. But up to date, it has not been clearly demonstrated if PPD itself is a sensitizing agent. Thus, this study aimed on the potential of PPD to provide the danger signals by studying IL-1β, TNF-ï¡, and monocyte chemoattractant proteins (MCP-1) in human monocytes, peripheral blood mononuclear cells (PBMC) from healthy volunteers, and also in two human monocyte cell lines namely U937, and THP-1. This study found that PPD decreased dose- and time-dependently the expression and release of three relevant mediators involved in the generation of danger signals. Namely, PPD reduced the mRNA and protein levels for IL-1ß, TNF-ï¡, and MCP-1 in primary human monocytes from various donors. These findings were extended and validated by investigations using the cell line U937. The data were highly specific for PPD, and no such results were gained for its known auto oxidation product called Bandrowski- base or for meta-phenylenediamine (MPD), and ortho-phenylenediamine (OPD). Therefore, we can speculate that this effect is likely to be dependent on the para-substitution. Based on these results we conclude that PPD itself is not able to mount a cascade for the induction of danger signals. It should be mentioned that it is still possible that PPD induces danger signals for sensitization by other unknown processes. Therefore, more research is still needed focusing on this subject especially in professional antigen presenting cells in order to solve the still open question whether PPD itself sensitizes naive T cells or if PPD is solely an allergen. Independently we found unexpectedly that PPD as well as other haptens such as 2, 4-Dinitrochlorobenzene, nickelsulfate, as well as some terpenoide increased clearly the expression of CC chemokin receptor 2 (CCR2), the receptor for the chemokine MCP-1. Up to date, the main importance for the CCR2 receptor comes from results demonstrating that CCR2 is critical for the migration of monocytes after encounter with bacterial lipopolysaccharides. Under these circumstances the receptor disappears from the cell surface and is down regulated. An up regulation of CCR2 has not been reported for haptens, and deserves further investigations.