Occludin-labels are associated with TJ and non-TJ structures.

<p>(A) A projection image of deeper layers of the same area of the epidermal sheet shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031641#pone-0031641-g001" target="_blank">Figure 1D</a>. (A′-A″″) 3D images of the third (3), fourth (4) and fifth (5) cells from the uppermost nucleated cell layers of the square in A. (B, C) Occludin immunoelectron microscopy. The labels are associated with TJs (brackets) in SG1 and SG2, but with small dots on the cell membrane in the lower epidermis (C″) (arrow). Silver intensified nanogold labels (B) and 5 nm-sized gold labels (C). B′, C′ and C″ are high-magnification views of B and C. Bars = 10 µm (A), 500 nm (B, C) and 200 nm (B′, C′, and C″).</p

Secreted LGs are frequently found in the lanthanum-positive spaces.

<p>Lower (A, B) and higher (C) magnification views of a lanthanum penetration assay performed on normal human skin. LGs secreted into the lanthanum-positive extracellular spaces are outlined in red and those in the cytoplasm are outlined in green. In the 5 µm-width (blue arrows) area of a SG2 cell, secreted LG-areas are 0.510 µm² and cytoplasmic ones are 0.376 µm². The point where lanthanum stops is indicated with a yellow arrow. Lanthanum is lost during tissue preparation in the dilated intercellular spaces in the layers below SG3.</p

LGs start being secreted before establishment of paracellular permeability barrier.

<p>(A, B) TJ permeability assay using a surface biotinylation technique. LG molecules, CDSN and glucosylceramides (GlcCer) are co-localized with biotin (areas between arrowheads). A part of CDSN/biotin image in A is magnified in A′ and the points where the tracer stops are indicated with yellow arrows. The basement membrane zone is highlighted by the broken line. (C, D) Lanthanum penetration assay on normal human skin. (C) Lanthanum penetration is blocked at the TJ (bracket) in the SG1. (D) Secreted LGs (red arrows) can be found in the tracer permeable intercellular space. Green arrows indicate LGs in the cytoplasm. The area in the rectangle with broken lines in D is magnified in D′. D, desmosome. Bars = 10 µm (A, A′, B), 200 nm (C, D′) and 500 nm (D).</p

Multilayered TJ-protein immunoreactive networks in the human epidermis.

<p>(A, A′) Biotin-tracer passed through some occludin-positive dots (arrows), but stopped at the most superficial dots (arrowhead). (B, B′) The CDSN-positive SG1 and SG2 have occludin-positive dots (arrows). (C, D) Occludin immunostaining of a sweat duct (C) and an epidermal sheet (D). D and D″ show a projection image of the upper three nucleated cell layers. D″ shows a 3D image from the corner (arrow). D′″ shows slice views dissecting uppermost (1), the second (2) and the third (3) cells. (E, F) EM of TJs (brackets) in the sweat duct (E) and the epidermis (F). A′, B′ and D′ are high-magnification views of A, B and D respectively. Bars = 10 µm (A–D′) and 200 nm (E, F).</p

CDSN is found in the intercellular space (ICS) where a biotin tracer is detected.

<p>This figure is the result of the paracellular permeability assay using a biotin tracer performed on normal human skin. The areas in the rectangles in panel A are magnified in panels B and C. CDNS labels (red arrows) are seen in the biotin tracer negative ICS between SC and SG1 associating with corneodesmosomes (CD) (B), but they are in the tracer (blue arrows) positive ICS between SG1 and SG2 (C). Larger dots are silver intensified streptavidin/gold particles reacted with biotin. Smaller dots are 10 nm gold particles labelling CDSN. CDSN labels associated with LGs in the cytoplasm are indicated with green arrows. D, desmosome. Bars = 1 µm (A) and 200 nm (B, C).</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