Charcot-Leyden crystal formation is closely associated with eosinophil extracellular trap cell death

Abstract

Protein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.

Graphical abstract

Figure 1.

CLCs are associated with EETosis in human tissues. (A-B) Tissue CLCs in biopsies of frontal sinus (A, allergic patient) and bacterially infected colon (B, ulcerative colitis) with a large number of FEGs released by infiltrating lytic eosinophils. Note hexagonal crystals and chromatolytic nuclei (N). Samples were prepared for conventional TEM. (C) Evaluation of CLCs in nasal polyps from ECRS patients. (i) Arrows indicate typical CLCs (40× objective). Note the abundant eosinophils with chromatolysis and FEGs. (ii) The percentage of CLC-positive patients was assessed by hematoxylin and eosin staining according to clinical severities. Assessment of CLCs and detailed study subject information are provided in the supplemental materials. Numbers represent CLC-positive patients/total patients in each group. (D) Maximal projection of 3-dimensional z-stack images of galectin-10 (green) and MBP (red) staining of nasal polyps from ECRS patients. (i) A region of lesser eosinophil infiltration exhibiting intact eosinophils; (ii) a highly inflamed lesion with abundant CLCs. In panel Di, eosinophils with bilobed nuclei (blue) showed cytoplasmic/perinuclear galectin-10 staining. In contrast, small punctate galectin-10 and loss of cytoplasmic CLC with extracellular MBP were observed in panel Dii. CLCs with a bipyramidal structure (arrows) were stained with galectin-10. Images were obtained with a Carl Zeiss LSM780 confocal microscope (100× objective). The scale shows each 10 µm.

Figure 2.

CLC formation in EETosis in vitro. (A) Cytoplasmic CLC formation in an eosinophil undergoing ETosis (arrow). Eosinophils were observed with time-lapse, phase-contrast imaging following IL-5 and platelet activating factor stimulation. Images were captured from supplemental video 1. (B) CLCs were associated with EETosis. Eosinophils were stimulated with PMA (10 ng/mL) with/without DPI for 120 minutes. CLCs were counted using an inverted microscope (40× objective, Eclipse TE300, Nikon). A total of 240 fields from 4 independent donors were studied. The bar graph represents the mean ± standard deviation. (C) Cellular galectin-10 localization during EETosis. PMA stimulated cells were fixed at 15, 45, and 120 minutes and stained with anti-galectin-10 Ab (green). At 15 minutes (i), galectin-10 was eccentrically located in adherent eosinophils. Galectin-10 was homogeneously distributed in cytoplasm at 45 minutes (ii). Note the loss of the typical bilobed nuclear shape. At 120 minutes (iii), cytoplasmic galectin-10 had disappeared and CLCs (arrows) could be recognized. ETs (filamentous DNA in blue) were also evident. EVs (supplemental Figure 7B) were out of focus. (D) Extracellular CLC formation. (i) Stacked EETosis cells form varied sizes of CLCs. Eosinophils (5 × 10⁶ cells in 1.5 mL) were stimulated with PMA (10 ng/mL) in round-bottomed microtubes for 3 hours, followed by fixation and processing for TEM. Sectioned CLCs of variable size (arrowheads) were observed. (ii) Free galectin-10 levels in culture supernatants. Eosinophils were stimulated with PMA with/without DPI for 3 hours, and culture supernatants were recovered by centrifugation at 10 000g for 10 minutes to remove vesicles, free granules, and CLCs. The graph represents the mean ± standard deviation from 6 different donors. ***P < .001. cont, nonstimulated control.