Proteolytic activation of the interleukin-1beta precursor by Candida albicans

Abstract

Chronic inflammation rather than invasion is characteristic of some forms of superficial candidiasis such as denture stomatitis. We hypothesized that Candida albicans may play a critical role in the pathogenesis of inflammatory lesions observed in chronic candidiasis by activating the proinflammatory cytokine interleukin-1beta (IL-1beta) from epithelial stores of the precursor. The aim of this study was therefore to demonstrate the proteolytic cleavage and activation of the inactive precursor of IL-1beta (pro-IL-1beta) by C. albicans. After incubation of either blastospores or hyphae with the inactive precursor, proteolytic cleavage was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western immunoblotting analysis, and the biological activity of the cleavage products was tested in a bioassay. We report here that late-stationary-growth-phase blastospores as well as hyphae of C. albicans, but not exponentially growing cells, can efficiently cleave pro-IL-1beta to yield fragments of molecular masses compatible with mature biologically active IL-1beta (17 to 19 kDa). Assays conducted in the presence of selected proteinase inhibitors suggest that the cleavage of pro-IL-1beta involves the participation of one or more aspartyl proteinases. Cleavage products showed a dose-dependent IL-1beta-like activity in a thymocyte proliferation bioassay, which was inhibited by anti-IL-1beta neutralizing antibodies. The present data thus suggest a role for C. albicans proteinases in the activation and maintenance of the inflammatory response at epithelial surfaces.

FIG. 1

SDS-PAGE Western immunoblotting analysis of the cleavage of pro-IL-1β by C. albicans as a function of growth phase. Recombinant pro-IL-1β (24 ng) was incubated with cells or supernatants at 37°C for 8 h in RPMI-F12 medium (pH 6.0). (A) C. albicans (isolate 2) blastospores in exponential (8 h; lane 3) and late stationary (72, 96, and 120 h; lanes 4 through 5, 6) growth phases. Purified recombinant pro-IL-1β (33 kDa; lane 1) and mature IL-1β (17 kDa; lanes 2 and 7) are shown for reference. (B) Culture supernatants corresponding to the C. albicans blastospores used for panel A. Supernatants were harvested during the exponential (8 h; lane 3) and late stationary (72, 96, and 120 h; lanes 4 through 6) growth phases. Purified recombinant pro-IL-1β (33 kDa; lane 1) and mature IL-1β (17 kDa; lanes 2 and 7) are shown for reference. Bands a, b, and c are approximately 17, 18, and 21 kDa, respectively.

FIG. 2

SDS-PAGE Western immunoblotting analysis of the cleavage of pro-IL-1β by different clinical isolates of C. albicans. Blastospores from C. albicans isolates 3 through 6 (lanes 3 through 6) from four different patients with denture stomatitis were harvested in the stationary growth phase (96 h) and incubated with pro-IL-1β for 8 h at pH 6.0 in RPMI-F12. Purified recombinant pro-IL-1β (33 kDa) and mature IL-1β (17 kDa) (lanes 1 and 2) are shown for reference.

FIG. 3

IL-1β-like costimulatory activity from proteolytic cleavage of pro-IL-1β by C. albicans. A dose-response curve of mature recombinant IL-1β was generated by using the bioassay described in Materials and Methods. To titrate the biological activity of the supernatants of Candida incubated in the presence of 24 ng of pro-IL-1β, this standard curve was constructed in the presence of control supernatants (i.e., supernatants of C. albicans incubated in RPMI-F12 medium for 8 h at 37°C without pro-IL-1β). These adapted standard curves were required because of the inhibitory effect of Candida supernatants on this bioassay. The ConA concentration was increased to 2.0 μg/ml to overcome this inhibitory activity. Control supernatants were diluted (1/10) and added simultaneously with graded concentrations of recombinant mature IL-1β and ConA. The closed bar shows the proliferative response when the assay is conducted in the presence of pro-IL-1β cleavage products (assay supernatants) instead of mature recombinant IL-1β. At this dilution (1/10), Candida supernatants containing cleavage products generated from pro-IL-1β generate a proliferative response corresponding to the costimulatory activity of 20 to 30 pg of mature IL-1β per ml. This biological activity was neutralized by antibodies directed against mature IL-1β (open bar). Means ± standard deviations from three experiments are shown.