Macrophage functions measured by magnetic microparticles in vivo and in vitro

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Abstract

Monodisperse ferrimagnetic iron-oxide particles of 1.4 μm geometric diameter were used to study alveolar macrophage functions (phagocytosis, phagosome transport) and cytoskeletal integrity in healthy subjects and in patients with idiopathic pulmonary fibrosis as well as in cultured macrophages. Dysfunctions in phagocytosis, in phagosome transport and cytoskeletal integrity correlated with an impaired alveolar clearance and could be induced in vitro by cytoskeletal drugs.

Introduction

The lungs with about 140 m2 inner surface area [1] represents the organ of the human body with the biggest contact area with the environment. Every breath transports many environmental particles into the lungs, where they are partially removed and deposited. The different areas of the lung have different mechanisms of deposition and clearance [2]. Clearance from the airways is primarily due to mucociliary transport, that removes most particles from the lungs within 24 h. The alveolar region is not covered by a mucus layer. Alveolar macrophages (AM) are resident in each of the alveoli and phagocytize foreign materials. Phagocytosis transfers the microparticles into phagosomes of the macrophages.

Magnetopneumography (MPG) uses ferrimagnetic iron-oxide particles as a tracer to investigate macrophage functions (phagocytosis, phagosome motions), cytoskeletal integrity, and the long-term clearance from the alveolar region of the human lungs [3], [4]. These particles are non-toxic and non-carcinogenic [5] and can be inhaled and detected by sensitive magnetic field sensors. Because of the chemical stability, these particles can be used as a probe for intracellular phagosome motions. Permanent cytoskeletal reorganizations and the intracellular transport cause a randomization of the magnetic dipole particles, which is reflected in a decay of the magnetic lung field and is called relaxation. Directed (external) phagosome motions are induced in a weak magnetic twisting field in order to investigate the mechanical properties of the cytoskeleton.

MPG was used to perform this study, aim of which was first, to investigate phagocytosis, stochastic and directed phagosome motions in healthy subjects with respect to the effects of chronic cigarette smoke exposure, previously investigated by in vitro tests [6], [7]. Secondly, to investigate phagocytosis and phagosome motions in patients with idiopathic lung fibrosis (FIB), whose etiology is not known. Thirdly, in order to understand pathophysiological results in patients, to perform in vitro studies, where cytoskeletal filaments were selectively disrupted by cytoskeletal drugs.

Section snippets

In vitro macrophage assay and phagocytosis

J774A.1 cell line was obtained from European Collection of Animal Cell Cultures. J774A.1 is derived from a tumor of a female BALB/c/NIH mouse [8]. This mouse monocyte macrophage cell line grows at 37°C in RPMI 1640 medium containing 5% fetal calf serum (FCS) in 5% CO2. Magnetic particles binding assay was performed as follows: one million cells were suspended in 2 ml medium together with 15 μg of magnetic particles and plated in plastic Petri dishes (3 cm diameter, Nunc Inc.) for 24 h. After this

Phagocytosis in vitro and in vivo

Fig. 3 shows the course of phagocytosis of iron-oxide microparticles by J774A.1 macrophages. Both graphs demonstrate that the phagocytosis performance (engulfed particles/cell/hour) was constant for different particles to cell ratios. For the assay used in this study using J774A.1 macrophages and 1.4 μm particles, the phagocytosis performance was 2.2±0.2 particles/cell/h for control probes. Cytochalasin D disrupts the microfilaments and completely inhibits phagocytosis, while Nocodazole, which

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