PPP vesicles stimulate expression of cytokine-encoding mRNAs in LSEs.

<p>PPP vesicles were suspended in 1% (w/v) agar and mRNAs measured using qRT-PCR. <b>A)</b> All of IL-17C (2.00±1.79-fold), IL-8 (1.8±1.7-fold), IL-1α (3.47±1.28-fold), and IL-1β (18.67±11.72-fold) were upregulated compared to the levels in non-treated LSEs (controls). The levels of cathelicidin, IL-8, IL-1α, and IL-1β differed significantly from those in control sweat. <b>B)</b> Western blotting showed that the hCAP-18/LL-37-depleted PPP-VF sample contained bands equivalent to hCAP-18 (18 kDa; full-length); an intermediate-sized fragment (∼14 kDa); mature LL-37 (4.5 kDa); and two additional bands (lane b). Lane a: the GST-hCAP18 peptide prior to incubation; Lane b: PPP-VF before depletion; Lane c: PPP-VF after depletion of endogenous hCAP-18/LL-37; Lane d: synthetic LL-37 peptide (3.2 pmol). <b>C)</b> mRNA expression levels in LSEs stimulated by original and depleted PPP-VF, as calculated via qRT-PCR. *<i>p</i><0.05. <b>D)</b> The illustration of structure of hCAP-18/LL-37.</p

Monocytes in PPP-VF.

<p>Hematoxylin-eosin staining revealed many mononuclear cells, but no polymorphonuclear cells, in vesicles (Fig. 5a). The mononuclear cells were positive for CD68 (Figs. 5c, d) but not CD56 (Figs. 5e, f) in all five instances. Pre-immune anti-mouse IgG did not stain the sections. (Fig. 5d). (Original magnifications: a, b, c, e: 100×, d, f: 400×).</p

Cytokine induction in NHKs by the synthetic LL-37 peptide.

<p>To assess the ability of LL-37 to induce cytokines, NHKs were incubated with 3 µM LL-37 for 0, 2, 4, 8, 20, and 24 h at 37°C. (A) Expression levels of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β mRNA, measured by qRT-PCR. The relative mRNA levels are expressed as means ±SDs (in -fold changes). Enzyme-linked immunoassays (ELISAs) were performed on culture media. All later values yielded by both qRT-PCR and ELISA were significantly different from those at 0 h (A, B, <i>p</i><0.05).</p

MCP-1 expression in PPP lesion skin and healthy skin.

<p>In lesion skin, strong expression of MCP-1 was detected around the PPP vesicle in the epidermis (Figs. 6a, b). In addition, acrosyringium in the lesions skin also showed the protein expression (Fig. 6c), but not in healthy skin (Fig. 6e, f). The eccrine pore at the surface of skin showed weak positive staining locating (Fig. 6f, arrowhead). (Original magnifications: a, c, d, e: 40×, b, f: 100×).</p

Quantification of hCAP-18/LL-37 in PPP-VF and eccrine sweat samples.

<p>Dot-blot analyses and densitometry were performed on PPP vesicles (15 samples), eccrine sweat samples (14 samples), a serially diluted LL-37 synthetic peptide solution, and a 10 µM solution of scrambled LL-37 synthetic peptide (negative control). hCAP-18/LL-37 was confirmed to be present in all PPP-VF and eccrine sweat samples, but not in the scrambled peptide control. The average concentrations of hCAP-18/LL-37 in PPP-VF and control sweat were 2.87±0.93 and 0.09±0.09 µM, respectively. *<i>p</i><0.05 compared to sweat.</p

LL-37 is synthesized from GST-rhCAP18 in depleted PPP-VF containing proteinase 3.

<p><b>A)</b> Several bands derived from GST-rhCAP18 were evident with dep-PPP-VF incubation. These were hCAP-18 (18 kDa; full length, indicated with ***), an intermediate-sized fragment (∼14 kDa, indicated with **), mature LL-37 (4.5 kDa, indicated with *), and two additional bands of ∼6 and 8 kDa (indicated with right arrow). In addition, 18-kDa bands reacting with anti-CATH Ab were present in PPP-VF tr-1 (F and R) and PPP-VF tr-2 (F). No mature LL-37 was detected in the sweat treated sample (lane R: sweat tr; anti-LL37 Ab staining). Abbreviations: α-LL37, anti-LL-37 antibody; α-CATH, anti-CATH antibody; F, peptide in flowthrough; R, peptide binding to resin; Sweat tr, eccrine sweat-treated peptide; PPP-VF tr, depleted PPP-VF component-treated peptide; crude, non-treated GST-hCAP-18 peptide (binding to resin); syn pep, LL-37 synthetic peptide (3.2 pmol). <b>B)</b> Proteinase 3 expression in concentrated PPP-VF was confirmed by Western blotting. Both depleted samples, PPP-VFs 1 and 2, exhibited single bands 29 kDa in size, thus that of PRTN3. Authentic PRTN3 (10 ng) served as a positive control. PPP-VF tr, depleted PPP-VF component-treated peptide; sweat, sweat sample 1; PRTN3, native proteinase 3. <b>C)</b> The illustration of structure of GST-rhCAP-18/LL-37.</p