Expression and activation of protein kinase C-ζ in eosinophils after allergen challenge

Protein kinase (PK) C is an increasingly diverse family of enzymes that has been implicated in a range of cellular functions within the eosinophil. Using isoform-specific polyclonal antibodies, we have explored the expression of PKC isoforms in circulating eosinophils. Initial studies demonstrated the presence of the α, βI, βII, and ζ and the low-level expression of the δ, ε, ι, and μ isoforms but no detectable expression of the γ, η, and θ isoforms in both normal and asthmatic subjects. There was no difference in the total protein expression between these two groups. Subsequent studies examined the expression and activation of PKC isoforms in circulating eosinophils from asthmatic patients before and 24 h after a late asthmatic response to an inhaled allergen. Cellular fractionation showed PKC-α and PKC-βII to be mainly located in the cytosol, whereas PKC-βI was constitutively more expressed in the membrane. No changes in expression or subcellular localization of these isoforms were seen after allergen challenge. In contrast, PKC-ζ expression was increased after allergen challenge, and we demonstrated a significant PKC-ζ translocation to the membrane, in keeping with activation of the enzyme. Our results suggest that 24 h after allergen exposure of asthmatic patients, there is increased expression and activation of eosinophil PKC-ζ that correlates with late asthmatic responses recorded between 4 and 10 h postallergen challenge.