Sweat stimulated signal activation and induced cytokine production in keratinocytes.

<p>(A) Sweats obtained from three volunteers were respectively concentrated and added into cultures (50 ng/ml), the levels of IκBα, phospho-IκBα, ERK, phospho-ERK, JNK and phospho-JNK in keratinocytes were examined by Western blotting. (B) Keratinocytes were stimulated with concentrated sweat, and the expression of IL-8 and IL-1β mRNA was detected by real time PCR. The relative mRNA levels are expressed as means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of 0 h.</p

IL-1α, IL-1β and IL-31 were quantified in the sweats obtained from volunteers.

<p>(A) Sweat samples obtained from 11 healthy volunteers were subjected to ELISA, and the concentrations of IL-1α, IL-1β and IL-31 were detected and quantified against the levels of total sweat protein, respectively. Concentrations (expressed as ng/mg sweat protein) represent means±SD (<i>n</i> = 11). (B) Immunohistochemistry showed the specific expression of IL-31 protein in eccrinne gland in normal skin. (a) The specific staining of IL-31 protein in the eccrine gland apparatus (original magnification: 40X) (b) IL-31 staining in eccrine gland and duct (original magnification: 200X) (c) IL-31 staining in eccrine gland and duct (original magnification: 400X) (d) the marked IL-31 staining in eccrine straight duct (original magnification: 400X) (e) the marked IL-31 staining in the coiled eccrine ducts (original magnification: 200X) (f) negative staining with mouse IgG and then nuclear staining with haematoxylin (original magnification: 40X) (small arrows indicate eccrine glands, and big arrows indicate eccrine ducts).</p

The bioactivity of sweat IL-1 is required for sweat-mediated keratinocyte activation.

<p>(A) To block the activation of IL-1 signaling, different concentration of IL-1Ra was applied into cultures for 1 h before stimulation with IL-1β for 24 h, the secretion of IL-8 was detected by ELISA of supernatants. Concentrations represent means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of IL-1β stimulation only. (B) 100 ng/ml IL-1Ra was applied into cultures for 1 h before the addition of concentrated sweat obtained from volunteer 3, keratinocytes were collected after 10 min of stimulation and the phosphorylation of IκBα, ERK and JNK was investigated by Western blotting. Keratinocytes were primed with IL-1Ra for 1 h then stimulated with concentrated sweat obtained from volunteers for 6 h, the mRNA expression of IL-8, IL-1β, RIG-I, NOD2 (C), and CCL2 (E) was detected by real time PCR. Relative mRNA levels were expressed as means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of sweat stimulation without IL-1Ra. (D) Keratinocytes were primed with IL-1Ra for 1h then stimulated with concentrated sweat obtained from volunteers for 24 h, IL-8 secretion from cultures was detected by ELISA of supernatants. Concentrations represent means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of sweat stimulation without IL-1Ra.</p

The activation of IL-31 signal in keratinocytes.

<p>(A) Keratinocytes were treated with IL-31 (0∼10 ng/ml) for 8 h, the mRNA expression of CCL2, IL-8 and IL-1β were examined by real time PCR. Relative mRNA levels are expressed as means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of control. (B) Keratinocytes were treated with IL-31 (10 ng/ml) for the indicated times, the levels of phospho-ERK, ERK, phospho-STAT3 and STAT3 were decided by western blotting. (The bands for phospho-STAT1 and phospho-AKT were not detected) (C) Keratinocytes were primed with IL-1Ra for 1h before stimulated with IL-31 (10 ng/ml) or IL-1α (10 ng/ml) for 8 h, the mRNA expression of CCL2, IL-1β and IL-8 was detected by real time PCR of total RNA and their relative mRNA levels were expressed as means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that without IL-1Ra (D) Keratinocytes were treated with IFN-γ (100 mg/ml), IL-4 (10 ng/ml), or IL-13 (50 ng/ml) for 6 h, the mRNA expression of IL-31RA and OSMR was examined by RT-PCR. (E) Keratinocytes were pretreated with IFN-γ for 24 h, then stimulated with or without IL-31 for 8 h, the mRNA expression of CCL2 were examined by real time PCR. Relative mRNA levels are expressed as means SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of IFN-γ stimulation only.</p

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