Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Schistosoma mansoni Infection

Abstract

Eosinophils have been long associated with helminthic infections, although their functions in these diseases remain unclear. During schistosomiasis caused by the trematode Schistosoma mansoni, eosinophils are specifically recruited and migrate to sites of granulomatous responses where they degranulate. However, little is known about the mechanisms of eosinophil secretion during this disease. Here, we investigated the degranulation patterns, including the cellular mechanisms of major basic protein-1 (MBP-1) release, from inflammatory eosinophils in a mouse model of S. mansoni infection (acute phase). Fragments of the liver, a major target organ of this disease, were processed for histologic analyses (whole slide imaging), conventional transmission electron microscopy (TEM), and immunonanogold EM using a pre-embedding approach for precise localization of major basic protein 1 (MBP-1), a typical cationic protein stored pre-synthesized in eosinophil secretory (specific) granules. A well-characterized granulomatous inflammatory response with a high number of infiltrating eosinophils surrounding S. mansoni eggs was observed in the livers of infected mice. Moreover, significant elevations in the levels of plasma Th2 cytokines (IL-4, IL-13, and IL-10) and serum enzymes (alanine aminotransferase and aspartate aminotransferase) reflecting altered liver function were detected in response to the infection. TEM quantitative analyses revealed that while 19.1% of eosinophils were intact, most of them showed distinct degranulation processes: cytolysis (13.0%), classical and/or compound exocytosis identified by granule fusions (1.5%), and mainly piecemeal degranulation (PMD) (66.4%), which is mediated by vesicular trafficking. Immunonanogold EM showed a consistent labeling for MBP-1 associated with secretory granules. Most MBP-1-positive granules had PMD features (79.0 ± 4.8%). MBP-1 was also present extracellularly and on vesicles distributed in the cytoplasm and attached to/surrounding the surface of emptying granules. Our data demonstrated that liver-infiltrating mouse eosinophils are able to degranulate through different secretory processes during acute experimental S. mansoni infections with PMD being the predominant mechanism of eosinophil secretion. This means that a selective secretion of MBP-1 is occurring. Moreover, our study demonstrates, for the first time, a vesicular trafficking of MBP-1 within mouse eosinophils elicited by a helminth infection. Vesicle-mediated secretion of MBP-1 may be relevant for the rapid release of small concentrations of MBP-1 under cell activation.

Keywords: eosinophil degranulation; granuloma; immunonanogold electron microscopy; inflammation; liver; major basic protein-1; piecemeal degranulation; schistosomiasis.

Figure 1

Representative types of granulomas and their frequencies in the livers of mice infected with S. mansoni. (A) Histological analyses on entire tissue sections identified three types of granulomas: (B) Pre-granulomatous exudative (PE), characterized by an infiltrate of inflammatory cells in process of organization around the parasite egg; (C) necrotic-exudative (NE), identified by a central halo of necrosis and numerous inflammatory cells distributed irregularly on subsequent layers; and (D) exudative-productive (EP), characterized by a rich structure of collagen fibers and inflammatory cells concentrated in the periphery and by a more organized and circumferential aspect. The numbers of granulomas and their areas in the hepatic tissue are shown in (E,F), respectively. Morphometric analyses were performed using Pannoramic Viewer software after whole slide scanning. Data represent mean ± SEM. ****P < 0.0001 vs. numbers of granulomas PE and EP; ^####P < 0.0001 area of granuloma PE; ⁺P = 0.04 vs. area of granuloma PE. Images are representative of 3 independent experiments.

Figure 2

Detection of eosinophils in the peritoneal cavity (A) and hepatic granulomas (B–C) of mice infected with S. mansoni. (A) Eosinophil numbers quantitated in the peritoneal lavage. (B) Eosinophil numbers quantitated per area of granuloma considering all granulomas, and in the most frequent type of hepatic granuloma (NE type). In (C), a representative NE granuloma. The boxed area in (C) is shown in (Ci). Arrowheads indicate examples of eosinophils with characteristic acidophilic cytoplasm. Data represent mean ± SEM. **P = 0.003 vs. control uninfected mice; ****P < 0.0001 vs. other immune cells within granulomas of infected mice. Morphometric evaluations were done with the use of Histoquant software. Cytocentrifuged preparations were stained and analyzed at magnification of 1000x. A total of 34,432 eosinophils were counted in 203 granulomas at a magnification of 20x.

Figure 3

Conventional TEM shows infiltrating eosinophils in the liver of S. mansoni-infected mice. (A) A representative electron micrograph of the hepatic tissue in low magnification shows a group of eosinophils (colored in pink) in close contact with each other and with neutrophils (brown) and plasma cells (blue). In (B), eosinophils exhibit their typical ultrastructure with a lobulated nucleus (N) and a robust cytoplasmic population of specific granules with a unique morphology-an internal well-defined electron-dense crystalline core and an outer electron-lucent matrix (seen in higher magnification in Bi).

Figure 4

Ultrastructural features of eosinophil degranulation in inflammatory sites of the liver of S. mansoni-infected mice. (A) A representative eosinophil shows PMD, characterized by the presence of emptying, non-fused secretory granules. The population of eosinophil specific granules is colored in yellow while large vesicles are highlighted in pink. The boxed areas in (A) are shown in (Ai–Aiii) in higher magnification. (Ai–Aiii) Note structural signs of PMD such as granule enlargement and disarrangement of granule cores and matrices. (B) Quantification of the secretory patterns shown in vivo by hepatic eosinophils in response to the acute infection. In (C), an eosinophil in advanced stage of cytolysis shows extracellular free secretory granules (Gr). (D) Most eosinophil secretory granules undergo structural changes indicative of PMD compared to that in uninfected mice. Data represent mean ± SEM. One hundred eight electron micrographs showing the entire cell profile and nucleus were analyzed and 2868 secretory granules were counted. ***P = 0.001 vs. intact granules, ⁺⁺P = 0.003 vs. emptying granules, ^#P = 0.03 vs. fused granules of uninfected mice. Gr, secretory granule. N, nucleus. Fragments of the liver of animals experimentally infected (acute phase) and intestines (for uninfected controls) were prepared for conventional TEM.

Figure 5

Ultrastructure of eosinophils undergoing compound exocytosis in inflammatory sites of the liver of S. mansoni-infected mice. (A) A representative electron micrograph shows an eosinophil with granules in process of fusion. In (B), secretory granules show several crystalline cores, a feature also considered as an evidence of fusion. The boxed areas in (A,B) are shown in (Ai,Bi) in higher magnification. (*) Denotes some of the secretory granules. Arrows indicates a fusion area. N, nucleus. Fragments of the liver of animals experimentally infected (acute phase) were prepared for conventional TEM.

Figure 6

Ultrastructural immunolocalization of MBP-1 in the liver from a S. mansoni-infected mouse. (A,B) Positivity is seen within representative infiltrating eosinophils while other inflammatory cells such as lymphocyte (A) and neutrophil (B) are negative. Labeling was associated with secretory (specific) granules, as observed in higher magnification (Bi). A close apposition between an eosinophil and a lymphocyte is noted in (A). Liver fragments were prepared for pre-embedding immunonanogold electron microscopy. N, nucleus.

Figure 7

Immunolocalization of MBP-1 on liver-infiltrating eosinophils from S. mansoni-infected mice. (A) Most MPB-1-positive secretory granules (~80%) showed characteristics of piecemeal degranulation (PMD). (B–E) Single-cell analyses at high-resolution reveal robust labeling of MBP-1 within secretory granules (Gr) of activated eosinophils. Note the typical signs of PMD such as enlargement and disarrangement of granule cores and matrices. N, nucleus. Data represent mean ± SEM. The numbers of labeled and not labeled granules (n = 218 granules) were counted in electron micrographs (n = 9). **P = 0.005 vs. intact granules; ****P < 0.0001 vs. fused granules. Liver fragments were prepared for pre-embedding immunonanogold electron microscopy.

Figure 8

Vesicular trafficking of MBP-1 in the cytoplasm of inflammatory eosinophils in the liver of S. mansoni-infected mice. (A) The acute infection induces a prominent formation of cytoplasmic, large (80–150 nm) round vesicles (highlighted in pink in B. Note in Bi) that immunolabeling for MBP-1 is clearly associated with several of these vesicles in addition to secretory granules (Gr, highlighted in yellow). Arrows indicate extracellular deposition of MBP-1. Data represent mean ± SEM. The numbers of vesicles (n = 755) were counted in a total of 19 electron micrographs, after conventional processing for TEM. ****P < 0.0001 vs. control group. For immunolabeling of MBP-1, liver fragments were prepared for pre-embedding immunonanogold electron microscopy.