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Vesicular LL-37 contributes to inflammation of the lesional skin of palmoplantar pustulosis.

Murakami M, Kaneko T, Nakatsuji T, Kameda K, Okazaki H, Dai X, Hanakawa Y, Tohyama M, Ishida-Yamamoto A, Sayama K - PLoS ONE (2014)

Bottom Line: Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM).Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF.In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Ehime University Graduate School of Medicine, Ehime, Japan.

ABSTRACT
"Pustulosis palmaris et plantaris", or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

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PPP vesicles stimulate expression of cytokine-encoding mRNAs in LSEs.PPP vesicles were suspended in 1% (w/v) agar and mRNAs measured using qRT-PCR. A) 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) 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). C) mRNA expression levels in LSEs stimulated by original and depleted PPP-VF, as calculated via qRT-PCR. *p<0.05. D) The illustration of structure of hCAP-18/LL-37.
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pone-0110677-g001: PPP vesicles stimulate expression of cytokine-encoding mRNAs in LSEs.PPP vesicles were suspended in 1% (w/v) agar and mRNAs measured using qRT-PCR. A) 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) 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). C) mRNA expression levels in LSEs stimulated by original and depleted PPP-VF, as calculated via qRT-PCR. *p<0.05. D) The illustration of structure of hCAP-18/LL-37.

Mentions: After incubation with PPP-VF, the expression levels of cytokine mRNAs in LSEs were evaluated via qRT-PCR (Fig. 1A). 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 non-treated LSEs (control). None of IL-22, IL-17A, or IL-17F was detected. For IL-8, IL-1α, and IL-1β, the increases were statistically significant (Fig. 1A).


Vesicular LL-37 contributes to inflammation of the lesional skin of palmoplantar pustulosis.

Murakami M, Kaneko T, Nakatsuji T, Kameda K, Okazaki H, Dai X, Hanakawa Y, Tohyama M, Ishida-Yamamoto A, Sayama K - PLoS ONE (2014)

PPP vesicles stimulate expression of cytokine-encoding mRNAs in LSEs.PPP vesicles were suspended in 1% (w/v) agar and mRNAs measured using qRT-PCR. A) 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) 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). C) mRNA expression levels in LSEs stimulated by original and depleted PPP-VF, as calculated via qRT-PCR. *p<0.05. D) The illustration of structure of hCAP-18/LL-37.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199729&req=5

pone-0110677-g001: PPP vesicles stimulate expression of cytokine-encoding mRNAs in LSEs.PPP vesicles were suspended in 1% (w/v) agar and mRNAs measured using qRT-PCR. A) 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) 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). C) mRNA expression levels in LSEs stimulated by original and depleted PPP-VF, as calculated via qRT-PCR. *p<0.05. D) The illustration of structure of hCAP-18/LL-37.
Mentions: After incubation with PPP-VF, the expression levels of cytokine mRNAs in LSEs were evaluated via qRT-PCR (Fig. 1A). 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 non-treated LSEs (control). None of IL-22, IL-17A, or IL-17F was detected. For IL-8, IL-1α, and IL-1β, the increases were statistically significant (Fig. 1A).

Bottom Line: Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM).Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF.In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Ehime University Graduate School of Medicine, Ehime, Japan.

ABSTRACT
"Pustulosis palmaris et plantaris", or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

Show MeSH
Related in: MedlinePlus