Abstract

Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their “cores”. After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB “cores” and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and during diverse diseases in which these organelles are functionally involved.

Highlights

  • Intracellular inclusions containing lipids are widely present in eukaryotic cells and bacteria

  • Our protocol for optimal preservation of purified eosinophils and identification of cytoplasmic lipid bodies (LBs) includes prompt aldehyde fixation while the cells are still in suspension followed by agar embedding for accurate sample handling and adequate osmium post-fixation in aqueous medium [10,13]

  • To characterize the LB two-dimensional ultrastructure in more detail, eosinophils freshly isolated from healthy donors were stimulated with different inflammatory stimuli, T cell expressed, and secreted (RANTES/CCL-5), tumor necrosis factor-alpha (TNF-a), interferon-gamma (IFN-c) or medium alone for 1 h and prepared for conventional transmission electron microscopy (TEM)

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Summary

Introduction

Intracellular inclusions containing lipids are widely present in eukaryotic cells (from protozoa to mammals) and bacteria. Proteins that interact with LDs do so only at the peripheral phospholipid monolayer [3] While this structural organization of LDs with a solely circumferential phospholipid monolayer underlies the roles of LDs as dynamic organelles pertinent to neutral lipid metabolism, whether all lipid inclusions, including those in leukocytes, have an identical structure has not been ascertained. LBs are critical to the functional capabilities of cells from the immune system, such as eosinophils, neutrophils and macrophages. In these cells, LBs are rapidly formed in response to a range of inflammatory diseases and are structural markers of inflammatory cells in innate immunity (reviewed in [9])

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