Article Figures & Data
Figures
- FIGURE 1.
Induction of HBD-2 peptide expression in epidermal cultures. Epidermal cultures were stained for HBD-2 2 days after stimulation with IL-1α (50 ng/ml) or CM. Tissue sections with or without stimulus stained with preimmune serum were used as negative controls. The HBD-2 peptide is immunostained and developed with fast red chromogen and counterstained with Harris hematoxylin. Similar results were obtained in three independent experiments.
- FIGURE 2.
Western analysis of time course of HBD-2 induction. HBD-2 expression was analyzed at 2, 6, 12, 24, 36, and 48 h after stimulation with 1/8 dilution of CM. Groups of normal epidermal cultures not exposed to the stimulus were harvested at 0 and 48 h for controls. HBD-2 peptide was detectable at 12 h after CM stimulation and accumulated in keratinocytes thereafter.
- FIGURE 3.
Northern blot analysis of HBD-2 expression in stimulated epidermal cultures. Epidermis stimulated by dilute (1/8) CM was analyzed for HBD-2 mRNA expression at 2, 6, 12, 24, 36, and 48 h after stimulation. Unstimulated epidermal cultures (−) were harvested at 0 and 48 h. Total RNA samples were loaded at 10 μg/lane. The blot was hybridized with 32P-labeled HBD-2 and G3PD cDNAs.
- FIGURE 4.
Effects of IL-1Ra on LPS-induced HBD-2 mRNA expression. CM-induced HBD-2 mRNA expression. Northern blot, film exposure time of 3 h. Epidermis stimulated for 48 h by diluted CM with or without IL-1Ra (200 ng/ml) was analyzed for HBD-2 mRNA expression. For comparison, epidermis exposed to LPS and culture medium for monocytes, but without monocytes, was harvested at 48 h. RNA from unstimulated epidermis cultures is shown as a control. HBD-2 mRNA was strongly induced by CM, and the induction was blocked by adding IL-1Ra.
- FIGURE 5.
Western analysis of the effects of IL-1Ra, CM, and LPS on HBD-2 expression. Epidermis was harvested after 48 h of exposure to the stimuli. Unstimulated epidermal cultures (−) were harvested with the stimulated groups and used as controls. HBD-2 peptide expression was detected only in the epidermis stimulated by CM and completely inhibited by adding IL-1Ra.
- FIGURE 6.
Gene expression changes induced by CM, IL-1α, CM plus IL-1Ra, or LPS stimulation for 24 h. Each dot on the linear scatter plot compares the expression of a single gene in stimulated epidermis (vertical axis) to the baseline expression in unstimulated epidermis (horizontal axis). The signal from HBD-2 is marked by a circle. Signals from genes whose expression is unchanged line up on or near the diagonal (solid line). Two-fold increase or decrease from control is shown by dashed lines.
- FIGURE 7.
Effects of stimuli and inhibitors on the expression of antimicrobial peptide mRNAs. HBD-2 mRNA is the only antimicrobial peptide induced substantially by CM. The mean signal for all genes on the Affymetrix HG-U133A is also shown for comparison. Antimicrobial peptides include the α-defensins: human neutrophil peptides (HNP)-1 and -4, human defensins (HD)-5 and -6; the β-defensins: HBD-1, -2, and -3; and the cathelicidin, LL-37. Similar results were obtained in a second experiment with CM from a second donor and another sample of epidermis.
- FIGURE 8.
Induction of HBD-2 mRNA by LPS in whole human skin. Fresh whole human skin mRNA fragments were treated with solvent, LPS alone (100 ng/ml), or LPS with IL-1Ra (200 ng/ml). The Northern blot was probed with HBD-2 cDNA and G3PD cDNA. Similar results were obtained with skin samples from two other donors.
Tables
Stimulus, Expt. No. HBD-2 Fold Induction HBD-2 Signal Rank Psoriasin Fold Induction Psoriasin Signal Rank SPRR-2B Fold Induction SPRR-2B Signal Rank IL-1α, no. 1 42 2 30 1 2.2 3 IL-1α, no. 2 24.6 3 12.4 2 2.9 1 CM, no. 1 10.6 3 4.4 4 Suppressed − CM, no. 2 23.8 4 11.7 2 2.4 1 -
a Signal rank is determined relative to all mRNAs induced >2-fold.
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