Original articles: Basic and clinical immunology
Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production

https://doi.org/10.1067/mai.2000.106210Get rights and content

Abstract

Background: Proteases in extracts of Aspergillus fumigatus cause epithelial cell desquamation and release of proinflammatory cytokines. Objective: We sought to assess protease activity in Alternaria alternata , Cladosporium herbarum , and Aspergillus fumigatus extracts and study the ability of these extracts to cause desquamation and release of proinflammatory cytokines from epithelial cells. Methods: Protease activities of the fungal extracts were quantified. Changes with respect to cell morphology, cell desquamation, and cytokine production (IL-6 and IL-8) were measured in the absence and presence of the fungal extracts in an airway-derived epithelial cell line (A549) and primary epithelial nasal cells. Results: Fungal proteases differentially induced morphologic changes, cell desquamation, and production of IL-6 and IL-8 in a dose- and time-dependent fashion. Alternaria alternata extracts induced cell shrinking and cell desquamation and strongly enhanced the production of IL-6 and IL-8 at higher concentrations. Aspergillus fumigatus extracts caused cell shrinking, cell desquamation, and production of IL-6 and IL-8, even at low concentrations. The Aspergillus fumigatus –derived extract grown on collagen medium induced a strong dose-dependent decline in cytokine production at higher concentrations. Cladosporium herbarum extracts did not induce morphologic changes or cell desquamation but enhanced IL-6 and IL-8 productions at higher concentrations. The dependence of these effects on intact protease activity was shown by their abrogation by protease inhibitors. Conclusion: Proteases present in fungal extracts interact with epithelial cells, leading to morphologic changes, cell desquamation, and induction of proinflammatory cytokines. It is proposed that these fungal proteases may activate epithelial cells through a protease-activated receptor type 2–driven mechanism. (J Allergy Clin Immunol 2000;105:1185-93.)

Section snippets

Fungal extracts and quantification of protease activities

Biologically standardized lyophilized fungal allergen extracts from fungal hyphae and spores obtained from stationary cultures (Cladosporium herbarum , Alternaria alternata , and Aspergillus fumigatus ) were kindly provided by Dr Lars Jacobsen (ALKAbelló). Crude extracts were obtained by using aqueous extraction for 24 hours at 4°C to 8°C and purified by using microdialysis with a cut-off point of 10,000 d, which is similar to that of extracts used for diagnostic purposes. Culture filtrate of

Protease activity in fungal extracts

Protein content and gelatinase, elastase, and total protease activities are shown in Table I.

. Protein (percentage of total weight) and protease activities in fungal extracts (n = 4)

Fungal extractProtein (%)Total protease activity (U/mg)Elastase activity (Vmax)Gelatinase activity (U/mg)
Alternaria alternata23.02.1800.40
Cladosporium herbarum11.51.5025.00.14
Aspergillus fumigatus33.71.9317.70.57
E-ColCF36.33.8955.13.15
The two Aspergillus fumigatus extracts contain over 30% proteins. The protein

DISCUSSION

Epithelial cells are important in innate immunity. Aside from their mechanical barrier function, they may also express surface receptors that are able to recognize microorganisms or their soluble components. Our data show that airway epithelial cells interact with proteases from allergenic fungi, resulting in IL-6 and IL-8 release. Earlier observations have shown that proteases from Aspergillus fumigatus induce cytokines known to be important in the recruitment of inflammatory cells (IL-6,

Acknowledgements

We thank Dr A. E. J. Dubois, MD, PhD, for critical review of the manuscript and Dr R. Weissenbruch for delivery of the nasal resections.

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    Reprint requests: Henk F. Kauffman, PhD, Laboratory of Allergology and Pulmonology, Clinic for Internal Medicine, University Hospital Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands.

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