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). This suggests that the cutaneous defense system has the capacity to effectively control the growth of S. aureus. There is increasing evidence that antimicrobial proteins are important effectors of the cutaneous defense system (
). In addition to human β-defensin-3,, other antimicrobial proteins may also participate in cutaneous defense against S. aureus. One candidate is RNase 7, a potent antimicrobial ribonuclease that is highly expressed in healthy skin (
). To assess the induction of RNase 7 by S. aureus in the context of more physiological conditions, we used human skin explants and incubated them for 2, 6, and 20hours with living S. aureus (the use of skin material for this study was approved by the ethical committee of the University Kiel (A 104/06) in accordance with the Declaration of Helsinki Principles). As shown in Figure 1, only 2hours of incubation with living S. aureus significantly induced the secretion of RNase 7 on the skin surface as measured by an RNase 7-specific ELISA (
). This indicates a fast release of preformed material. The reduced secretion after 20hours could be a result of cytotoxic effects from S. aureus, as well as of a decreased viability of skin explants. Secretion of RNase 7 after 2hours was enhanced by increasing concentrations of S. aureus (Supplementary Figure S1 online).
To further assess the physiological relevance of RNase 7 in cutaneous defense against S. aureus, we investigated whether the killing activity of skin extracts derived from stratum corneum was inhibited through blocking of the antimicrobial activity of RNase 7 by RNase 7-specific antibodies as previously described (
). First, we analyzed whether RNase 7 antibodies neutralized the antibacterial activity of RNase 7 against S. aureus. For this purpose, we tested the activity of RNase 7 against S. aureus in an antibacterial microdilution assay in the presence of RNase 7 antibodies. This approach revealed that the application of RNase 7 antibodies completely blocked the S. aureus-killing activity of RNase 7. In contrast, the antimicrobial activity was not inhibited when equivalent concentrations of irrelevant antibodies (derived from goat preimmune serum;
) were used (data not shown). Having established that RNase 7-specific antibodies neutralized the antimicrobial effect of RNase 7 against S. aureus, we used this approach to test the functional role of RNase 7 in cutaneous defense against S. aureus. To this end, we first incubated a stratum corneum skin extract with S. aureus. The skin extract was diluted to a ratio of 1:200 in 25μl of sodium phosphate buffer (pH 7.4) without or with RNase 7-blocking antibody (4mgml−1) or an equivalent concentration of an irrelevant antibody. This dilution leads to a final concentration of 6μgml−1 RNase 7 in the assay system, which corresponds to the determined range of the lethal dose90 of RNase 7 against S. aureus (see above). After 45minutes, 25μl of S. aureus (ATCC 6538) in sodium phosphate buffer (pH 7.4) containing 2% tryptone soya broth was added and incubated for 3hours. Killing activity was analyzed by plating serial dilutions of the incubation mixture and determining colony-forming units the following day. The skin extract exhibited a potent S. aureus-killing activity, as incubation of S. aureus with the skin extract resulted in a complete killing of S. aureus (Figure 2a). The application of RNase 7-blocking antibodies to the skin extract before inoculation with S. aureus resulted in an outgrowth of S. aureus of approximately 35% as compared with the growth control (Figure 2a). In contrast, an irrelevant antibody did not inhibit the killing activity of the skin extract (Figure 2a). These data indicate an important contribution of RNase 7 to the S. aureus-killing activity of the skin extracts. Other skin-derived antimicrobial proteins may be responsible for the S. aureus-killing activity, which remained after blocking the antimicrobial activity of RNase 7.
To obtain additional insight into the functional role of RNase 7 in cutaneous defense against S. aureus, we studied the role of RNase 7 using human skin explants. In this experimental ex vivo model, S. aureus was applied to defined areas (0.8cm2) of the surface of skin explants. Preincubation of skin samples with 1.5mgml−1 RNase 7-blocking antibody in 100μl of sodium phosphate buffer (pH 7.4) led to a significant outgrowth of S. aureus as compared with pretreatment of skin with an equivalent concentration of irrelevant antibody (Figure 2b). These data further indicate a physiological relevance of RNase 7 for the control of S. aureus growth on the skin surface.
In summary, data present herein show that skin infected with living S. aureus responds with an increased release of RNase 7, which contributes to limit the growth of S. aureus. These data are in concordance with a recently published study reporting that lower RNase 7 expression in healthy skin is associated with a higher risk of S. aureus skin infection (
). Future studies have to evaluate whether skin infections caused by S. aureus may be associated with an impaired expression or function of RNase 7.
We thank H Hinrichs, E Jeske, C Martensen-Kerl, and C Wilgus for excellent technical assistance. This study was supported by a grant from the Federal Ministry of Education and Research (BMBF, Skin Staph) to R Gläser and J Harder and by a Heisenberg program of the Deutsche Forschungsgemeinschaft (DFG) to J Harder.