Phagocytosis of chitin particles induced IL-1α release after cell death and acute neutrophilic airway inflammation.

(A) Flow cytometric analysis of chitin particle size. The yellow peak indicates chitin particles. Gray peaks indicate 0.6-, 1-, and 5.9-μm latex beads. (B, C) Phagocytosis assay of chitin particles. Bone marrow-derived dendritic cells (BMDCs) derived from C57BL/6 mice were incubated with 50 μg/mL of calcofluor white (CFW)-labeled chitin particles (blue). After washing with phosphate-buffered saline (PBS), cells were stained with Alexa Fluor 555 Phalloidin (yellow) and nuclei were stained with NucRed Live 647 ReadyProbes Reagent (red), and observed by confocal laser scanning microscopy (B). The mean fluorescence intensity (MFI) of cells that had phagocytosed chitin particles was measured by flow cytometry (C). (D, E) BMDCs were incubated with chitin particles (2–250 μg/mL), rLdpA (25 μg/mL), or PBS (vehicle control), and TNF-α, KC/CXCL1, and IL-1α levels in the culture supernatant and IL-1α in the cell lysate were measured by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) (E). (F, G) BMDCs were incubated with 1 μM cytochalasin D for 30 min, followed by the addition of chitin particles (250 μg/mL). After 24-h incubation, cytotoxicity was assessed by measuring the release of LDH (F), and IL-1α protein levels in the culture supernatant were measured by ELISA (G). (H) C57BL/6 mice were administered chitin particles (100 μg), rLdpA (10 μg), or PBS (vehicle control) in a single intranasal dose. Bronchoalveolar lavage (BAL) fluid samples were obtained after 3, 6, and 12 h, and the numbers of neutrophils were measured by flow cytometry. (I) Chitin particles (100 μg) and rLdpA (10 μg) were administered to C57BL/6 mice in a single intranasal dose. The number of neutrophils, IL-1α level, and LDH level in the BAL fluid were measured after 24 h. (J) Anti-IL-1α neutralizing antibody or isotype control antibody was injected into the peritoneal cavity of C57BL/6 mice at a dose of 80 μg/mouse 1 h before intranasal chitin particle administration. The number of neutrophils in the BAL fluid was determined after 24 h. Data are shown as the mean ± standard deviation (SD). (H, n = 3 mice/group; I, n = 6 mice/group; J, n = 7–8 mice/group). Each symbol represents an individual sample (I and J). *P P t test (I and J).