ATPγS Enhances the Production of Inflammatory Mediators by a Human Dermal Endothelial Cell Line via Purinergic Receptor Signaling  Kristina Seiffert, W. Ding, John A. Wagner, Richard D. Granstein  Journal of Investigative Dermatology  Volume 126, Issue 5, Pages 1017-1027 (May 2006) DOI: 10.1038/sj.jid.5700135 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 (a) IL-6 and (b) IL-8 accumulate in HMEC-1 supernatants over time and are increased by ATPγS. HMEC-1 cells were incubated with and without ATPγS 100μm for 4, 8, 12, or 24hours and their IL-6 and IL-8 secretion into the supernatant was measured by ELISA (mean±standard deviation; *P<0.05; **P<0.01 for ATPγS vs baseline); values represent one experiment performed in triplicate. Journal of Investigative Dermatology 2006 126, 1017-1027DOI: (10.1038/sj.jid.5700135) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 ATPγS induces IL-6, IL-8, GROα, and MCP-1 secretion in HMEC-1 cells, and purinergic antagonists inhibit both basal and ATPγS-induced production of these cyto/chemokines. HMEC-1 cells were maintained in depleted medium throughout the experiment. After reaching subconfluence, HMEC-1 cells were incubated with (a) 100μm or (b) 1mm purinergic antagonists for 30minutes before treatment with 10 or 100μm ATPγS, respectively. Some wells received (a) 10 or (b) 100μm ATPγS without antagonists. Some wells were cultured without addition of ATPγS or inhibitors. Conditioned supernatants were collected after 24hours and cytokine/chemokine secretion measured by ELISA (mean±standard deviation; **P<0.01; *P<0.05). Data shown in (a) are one experiment representative of four independent experiments, each of which was run in triplicate. Data shown in (b) are one experiment representative of three independent experiments. The experiment shown was run in quintuplicate. Statistics are based on comparisons of the mean values of the individual experiments. Journal of Investigative Dermatology 2006 126, 1017-1027DOI: (10.1038/sj.jid.5700135) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 ATPγS induces ICAM-1 expression in HMEC-1, and the antagonists suramin and PPADS inhibit both basal and ATPγS-induced ICAM-1 expression. HMEC-1 cells were maintained in depleted medium, and were incubated with purinergic antagonists (1mm) 1hour before treatment with ATPγS 100μm. After 24hours the cells were collected, stained with anti-ICAM-1 antibodies, and ICAM-1 expression was measured by FACS analysis. (a) Histograms depicting the ICAM-1 staining profiles for the conditions indicated; (b) arithmetic mean of ICAM-1 fluorescence intensity (MnX)±standard deviation recorded for all conditions (**P<0.01); experiment representative of two independent experiments, each of which was run in triplicate. Journal of Investigative Dermatology 2006 126, 1017-1027DOI: (10.1038/sj.jid.5700135) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 HMEC-1 cells express mRNA for the purinergic receptors P2X1, P2X3, P2X4, P2X5, P2X7, P2Y2, and P2Y11. Poly(A)+RNA was extracted from unstimulated HMEC-1 and DNase-treated to remove genomic DNA contamination (designated as +). RNA extracted from HMEC-1 without DNase digestion, and confirmed to contain genomic DNA, served to demonstrate that the primer pairs could detect the relevant sequence (designated as ++). RT-PCR was performed using primers designed from sequences in GenBank (see Table 1). To demonstrate that PCR products were derived from RNA rather than contaminating DNA, reverse transciptase was omitted from the reaction mixture containing DNase digested HMEC-1 mRNA (designated as −). Receptor expression was confirmed with at least three different experiments, each of which had a separate RNA isolation. Journal of Investigative Dermatology 2006 126, 1017-1027DOI: (10.1038/sj.jid.5700135) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Purinergic agonists differentially affect IL-6 production in HMEC-1 cells. HMEC-1 cells were incubated with ATP, ATPγS, UTP, BzATP, UTP, α,β-meATP, and 2-MeSATP for 24hours at the concentrations indicated. Conditioned supernatants were collected and IL-6 secretion measured by ELISA. Combined data from three independent experiments, each of which was performed in triplicate, are presented (mean±standard deviation). Journal of Investigative Dermatology 2006 126, 1017-1027DOI: (10.1038/sj.jid.5700135) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions