Background:  House dust mites produce serine and cysteine proteases. Mite‐derived proteases have been suggested to be involved in the pathogenesis of allergies; however, whether mite‐derived serine protease activity can stimulate keratinocytes remains unknown.

Methods:  We examined the activation of primary human keratinocytes by serine protease‐rich extract of whole mite culture and compared with that by recombinant group 1 allergens (rDer f 1 and rDer p 1), which exclusively exhibit cysteine protease activity.

Results:  Protease activity of whole mite culture extract (WCE), rDer f 1 and rDer p 1 induced the release of IL‐8 and granulocyte‐macrophage colony‐stimulating factor. Protease activity of WCEs induced a significant upregulation of their mRNA expression but rDer f 1 had much less effect. Protease activity of the WCE stimulated intracellular Ca²⁺ mobilization but rDer f 1 and rDer p 1 did not. The mobilization induced by agonists for the human protease‐activated receptor (PAR)‐2, an agonist peptide or trypsin, was diminished by pre‐incubation of keratinocytes with WCE. rDer f 1 inefficiently cleaved a synthetic N‐terminal peptide of PAR‐2 at different sites from trypsin, but the resultant peptides did not stimulate the release of interleukin‐8.

Conclusions:  The results suggest that mite‐derived serine protease activity may contribute to the pathogenesis of atopic dermatitis by activating keratinocytes via PAR‐2 activation but cysteine protease activity of Der f 1 and Der p 1 acts via another mechanism.