PLAN DE ACCIÓN

All along the respiratory tract there is an uninterrupted layer of epithelial cells. The structural properties of these cells differ considerably between the conducting zone and the gas-exchange zone. The pseudo-stratified epithelium of the trachea and bronchi is composed of

a number of different cell types; among which are secretory goblet cells and ciliated cells.

The former produce mucus, whereas the latter enable mucociliary clearance via the

“mucociliary escalator” to the pharynx, where components are then swallowed or expectorated. In the bronchioles, the epithelium becomes a simple columnar epithelium with shorter cilia and secretory Clara cells. At the level of the alveoli, where the mucociliary escalator is absent, there is a transition to a thin, squamous epithelium, occupied by alveolar epithelial type 1 (AT1) and cuboidal alveolar epithelial type 2 (AT2) cells [31] (Figure 1.7).

As mentioned above, the alveolus constitutes the gas exchange region of the lung. The air-blood barrier is made of three tissue compartments: the alveolar epithelium, the interstitium (including cellular elements and connective tissue), and the capillary endothelium. This highly specialised structure provides a thin barrier to gas exchange between the airspace and the underlying vasculature. The total surface area available for gas exchange in the human lung is about 140m², equivalent to almost the size of a single tennis court [31]. The alveolar epithelium is formed of two different cell types: AT1 and AT2 cells. Within the alveolar unit, the first line of cellular defence is formed by alveolar macrophages (AMs) (Figure 1.7).

1.2.2.1 Alveolar type 1 cells (AT1)

AT1 are large, thin, squamous cells (depth, 0.2-0.5μm; diameter, 80-100μm) that cover approximately 95% of the alveolar surface (Figure 1.7 and Table 1.1). These cells provide a physical barrier with minimal diffusion distance between the alveolar air space and the underlying capillary network, thus maximising gas exchange [32]. AT1 cells are also important in regulating the alveolar fluid balance by expressing membrane transport proteins, such as epithelial sodium channels and aquaporin water channels [33,34]. Rich in caveolae and vesicles, monocultures of AT1 cells have been demonstrated to internalise significantly more NPs than monocultures of AT2 cells (75% of the initial amount of NPs are internalised by the former, 10% by the latter) [35]. In addition, the process of uptake is rapid:

one-third of the total NPs internalised over 24h was taken up during the first hour [35].

Therefore, AT1 cells could be fundamental in the understanding of NP behaviour at their primary site of deposition. Also, by demonstrating an avid uptake of NPs, AT1 cells might be crucial in translocation of inhaled nano-sized particles across the lung and in facilitating their subsequent possible effects in target organs (see Section 1.2.3.2). On the contrary, AT2 cells seem to have a relatively small role to play in this process, whereas macrophages are predominantly involved in particle clearance from the alveoli (see Section 1.2.2.3). However, the acquisition of a large amount of evidence relating to AT1 cells is hindered by their fragile nature, which makes AT1 cell isolation and subsequent culture extremely difficult [33,36]. In order to overcome this challenge, AT2 cells have been isolated and cultured on a plastic support for approximately 7 days, in order to drive the cells towards an AT1-like phenotype [37–39]. These terminally differentiated cells cannot be passaged though, and primary AT2 cells would need to be isolated regularly, complicating and prolonging NP studies in AT1 cells.

In our laboratory, human AT1-like cells have been successfully immortalised from freshly isolated and cultured primary human AT2 cells. AT2 cells were transduced using hTERT (i.e.

the catalytic subunit of human telomerase reverse transcriptase) and a temperature sensitive mutant of simian virus 40 large-tumor antigen [35]. These AT1-like, or transformed type-1-like (TT1) cells, represent the first ever human AT1-type-1-like cell line to be produced. They are negative for the typical AT2 cell markers pro-surfactant protein C, alkaline phosphatase and thyroid transcription factor-1, they lack lamellar bodies (characteristic of AT2 cells) and display a flattened morphology, containing vesicles and caveolae (refer to Table 1.1).

Furthermore, this unique cell line has been further validated by Swain and colleagues that showed, using the Raman microspectroscopy, that TT1 cells provide a suitable model for AT1 cells [39].

AT1 cells AT2 cells - Absence of microvilli, lamellar bodies

- Diameter 9µm - Cuboidal morphology

- Presence of microvilli, lamellar bodies - Absence of caveolae, vesicles microscope; structure and morphology of the cells have been obtained by using TEM [33];

typical cell markers were identified using Western Blotting [33].

1.2.2.2 Alveolar type 2 cells (AT2)

AT2 cells are cuboidal in shape, with a diameter of approximately 9μm, contain lamellar bodies and express apical microvilli (Figure 1.7 and Table 1.1). They represent, by number, 60% of the total alveolar epithelial cells, but only account for 5% of the alveolar surface area, as only a small part of the cell is exposed to the epithelial surface [32]. AT2 are secretory cells that synthesise, release and recycle pulmonary surfactant, which is a mixture of lipids (85-90%), proteins (10%) and carbohydrates (2%). The alveolar unit is covered in a thin aqueous hypophase, the alveolar lining fluid (a milieu for extracellular biochemical reactions) on top of which lays the pulmonary surfactant (Figure 1.7). The main function of the pulmonary surfactant is the reduction of the surface tension at the air-liquid interface and the

maintenance of the alveolar fluid balance [70]. AT2 cells also produce anti-proteases, antioxidants, defensins and other molecules that are important in lung defence and in maintaining pulmonary homeostasis. Importantly, they are also progenitor cells for AT1, and thus are essential for maintaining the integrity of the alveolar epithelial barrier. When an alveolar epithelial cell undergoes cell death, neighbouring AT2 cells will divide and differentiate to replace it and keep the epithelial layer intact [38,40,41].

1.2.2.3 Alveolar macrophages (AMs)

AMs represent the most prevalent mechanism for defence of the alveoli and for clearance of particles/debris. During this process, they release a broad spectrum of pro-inflammatory mediators such as cytokines and chemokines [42]. However, phagocytosis by AMs does not seem to be efficient for inhaled NPs. Indeed, Oberdorster et al. presented the results of several studies in which rats were exposed to different sized particles, from 10µm to 15nm.

After 24h, the lungs of the animals were lavaged and about 80% of the micron-sized particles were retrieved with the macrophages, whereas only 20% of nano-sized particles were lavaged with the macrophages [9]. The remainder of the nano-sized material was located within the parenchymal tissue. It has also been reported that the attempted clearance of long thin fibres (e.g. asbestos) in exposed AMs can induce “frustrated phagocytosis” [43], a phenomenon occurring when the target is physically too large for the AMs to engulf. This can lead to the release of potentially toxic pro-inflammatory mediators, thus amplifying the inflammatory response, with deleterious consequences on the surrounding cells. The fundamental mechanisms of NP clearance by AMs are still unclear.

Alveolar type II cell (AT2)

Alveolar type I cell (AT1)

Alveolar macrophage (AM)

Microvilli Lamellar body

Surfactant layer

Lung lining fluid (LLF)

Air space

Figure 1.7 Schematic representation of the human alveolus. Three cell types are shown:

AT1 cells (in yellow; large and thin), AT2 cells (in red; cuboidal, with microvilli and lamellar bodies) and AM (in green). Secretory AT2 release the surfactant that lies on top of the lung lining fluid (LLF; in light blue).