Cystatin A inhibits IL-8 production by keratinocytes stimulated with Der p 1 and Der f 1: biochemical skin barrier against mite cysteine proteases.
BACKGROUND
Der p 1 and Der f 1 are the most immunodominant allergens produced by house dust mites and are suspected to be involved in the pathogenesis of allergy through their cysteine protease activity. However, stimulation of keratinocytes by these protease allergens and protective systems in the skin against them have not been well investigated.


OBJECTIVE
We purified and identified the dominant skin-derived inhibitor against the proteolytic activities of these allergens and analyzed its effect on keratinocyte activation.


METHODS
Recombinant allergens were used for the experiments. We analyzed whether human sweat inhibits the enzymatic activities of Der p 1 and Der f 1 and used sweat as the skin-derived material to isolate the inhibitor. The inhibitor was purified by means of column chromatography and subsequently identified by means of protein sequencing and immunoblotting. Keratinocytes were stimulated with the allergens in the absence or presence of the inhibitor, and the concentration of secreted IL-8 was measured.


RESULTS
Sweat inhibited the proteolytic activities of Der p 1 and Der f 1. The sweat inhibitor was identified as cystatin A. The stimulation of normal human keratinocytes and the human keratinocyte cell line HaCaT with these protease allergens upregulated IL-8 secretion, and addition of cystatin A blocked this upregulation. Normal human keratinocytes secreted cystatin A into the medium.


CONCLUSIONS
The proteolytic activity of Der p 1 and Der f 1 stimulates human keratinocytes in vitro. Cystatin A produced by keratinocytes is the dominant biochemical skin barrier that eliminates the enzymatic activity of these mite cysteine proteases and prevents them from stimulating keratinocytes.
Kato
Takeshi
T
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
Takai
Toshiro
T
Mitsuishi
Kouichi
K
Okumura
Ko
K
Ogawa
Hideoki
H
D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology*; D002460:Cell Line; D015891:Cystatins / Q000032:analysis / Q000276:immunology* / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D016209:Interleukin-8 / Q000096:biosynthesis*; D015603:Keratinocytes / Q000276:immunology / Q000378:metabolism*; D039981:Pyroglyphidae / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D012867:Skin / Q000276:immunology*; D013542:Sweat / Q000737:chemistry / Q000276:immunology
D039741:Antigens, Dermatophagoides; D015891:Cystatins; D016209:Interleukin-8; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases