Marcel C. Pasch Journal of Investigative Dermatology

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Effects of UVB on the Synthesis of Complement Proteins by Keratinocytes Marcel C. Pasch Journal of Investigative Dermatology Volume 111, Issue 4, Pages 683-688 (October 1998) DOI: 10.1046/j.1523-1747.1998.00358.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Increasing doses of UVB cause progressive loss of cell viability. Subconfluent keratinocyte monolayers were exposed to UVB (0–64 J per m2) and were cultured for 48 h and 72 h. Cells were counted with the hemacytometer and cell survival was analyzed with the flow cytometer after propidium iodide staining. Cells living after exposure with each dose are presented as a percentage of unexposed living cells. Data are averages ± SD of three experiments. Journal of Investigative Dermatology 1998 111, 683-688DOI: (10.1046/j.1523-1747.1998.00358.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 UVB exposure has no influence on the synthesis of C3 and Factor B by human keratinocytes. Subconfluent keratinocyte cultures were exposed to indicated doses of UVB. Supernatants were harvested at 72 h and concentrations of C3 and Factor B determined by ELISA. IL-8 was included as a positive control. The figure shows C3, Factor B, and IL-8 protein concentrations in supernatants of three independent experiments. In each experiment three wells were exposed to each dose and the contents of each well were analyzed in quadruplicate. Mean values and SD are shown. Journal of Investigative Dermatology 1998 111, 683-688DOI: (10.1046/j.1523-1747.1998.00358.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cultured human keratinocytes express DAF, MCP, and CD59. Cultured keratinocytes were washed and 104 cells were analyzed for the expression of C-regulatory proteins by FACS. Continuous lines show keratinocyte staining of MCP, DAF, or CD59. Broken lines show staining with isotype controls. For all parts the data are shown as cell number versus the log of fluorescence and are representative of three staining experiments each carried out in triplicate. Journal of Investigative Dermatology 1998 111, 683-688DOI: (10.1046/j.1523-1747.1998.00358.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 A single dose of UVB causes prolonged increase in the surface expression of DAF, MCP, and CD59. Subconfluent keratinocyte cultures were exposed to 32 J per m2 and expression of DAF, MCP, and CD59 was monitored by flow cytometer at the indicated time points. MFI, corrected for isotype control, is shown. Data represent one of two independent experiments. In each experiment three wells were exposed to UVB for each time point and cells in each well were analyzed in duplicate. Mean values and SD are shown. Journal of Investigative Dermatology 1998 111, 683-688DOI: (10.1046/j.1523-1747.1998.00358.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Increasing doses of UVB cause a progressive increase in surface expression of DAF, MCP, and CD59. Subconfluent keratinocyte cultures were exposed to a moderate dose (32 J per m2) and a high dose (64 J per m2) of UVB. Expression of DAF, MCP, and CD59 was analyzed by flow cytometer at the time points at which a maximum increase in upregulation was seen in previous experiments: 48 h for DAF and 72 h for MCP and CD59 with 32 J per m2 and at 48 h for all three proteins at 64 J per m2 (see Figure 4. Single parameter histograms for DAF, MCP, and CD59 expression are shown. Thin lines show the expression of DAF, MCP, and CD59 on unexposed keratinocytes and bold lines show that on UVB-exposed keratinocytes. Broken lines show the expression obtained with isotype controls. The data are representative of at least two independent experiments carried out in triplicate. MFI after exposure to 64 J per m2 was significantly higher for DAF (p < 0.01) and CD59 (p < 0.001), but not for MCP (p = 0.18), than after exposure to 32 J per m2. Journal of Investigative Dermatology 1998 111, 683-688DOI: (10.1046/j.1523-1747.1998.00358.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions