Cells at air-liquid interfaces

Pulmonary epithelial cells are submerged on the apical side by a nanometer thin layer of water. Such nanoscaled environment is necessary to guarantee the least resistance to gasses diffusion and to preserve a "wet" environment to pulmonary cells. The thinness of the layer represents a threat for the cells themselves as well as for the entire body: failing in regulating the volume of the hypophase can cause a deficiency in the respiratory capability (i.e. when the liquid fluid is too high; oedema) and, in the other direction (i.e. decrease in the hypophase volume), risks to expose the cells to the lethal interfacial forces. We hypothesize that (I) Alveolar Type II cells (ATII, elsewhere also called as "the alveolar defender") are able to sense the presence of the air-liquid interface and, therefore, are responsible for the regulation of the hypophase volume and (II) that the ATII exocytosis product (surfactant) serves to protect lung epithelium against the potential harmful nature of surface forces. In favor of these hypotheses we have already collected a large number of strong evidences (listed in chapter 2). Aims of the project are to identify (I) the biophysical nature of the cells-interface sensing, (II) the sensing mechanism and its specificity for the lung epithelium and (III) the biological consequences of cell-interface interaction. Importantly, the present project has a great originality in contents and experimental designs. Therefore, the outcome should be of considerable basic biological as well as medical interest. In addition to these specific goals, which aim to bring a new light into the physiology of the lung, this project will open the opportunity to develop new technological strategies suited for important translational studies (planed in our laboratory) of great relevance to Human Health (such as cell reactions to volatile compounds; e.g. anesthetics and environmental pollutants or ultra-fine particulate materials).

Alevolar cells are located at the branching endpoints of the lungs conducting airways, at the site of gas diffusion into (O2) and out (CO2) of our body. Normally, this gas transfer is so efficient that it is not rate limiting, not even at high respiratory demands during intensive exercise. This is mainly due to the high surface area - in the range of that of a tennis court - formed by the hundreds of millions of alveoli, small bubble-like invaginations with a tissue thickness below that what can be seen by naked eyes (~1 m). However, alveoli are intrinsically instable. Surface forces, arising from the wet and bended surface structures, tend to collapse them, if there would not be pulmonary surfactant (an acronym of surface active agent). This complex of different substances, a product of the alveolar (AT II) cells, behaves like a compressed molecular spring that is able to counteract these surface forces, but in a very dynamic and still not fully understood way. According to our new data, it is also very likely that surfactant serves as a general protective shield for air exposed epithelia. Surfactant is produced by the AT II cells continuously, but also on demand. A major stimulus seems to be lung distension, and another one the presence of an air-liquid phase boundary. The closer it is to the cells, the higher the intracellular amount of free Ca2+-ions, entering the cells through mechanosensitive ion channels. These ions generally serve as cellular messengers, or molecular switches. In the particular case, Ca2+-ions trigger the release of surfactant into the extracellular space, astonishingly by a unique, active squeeze-out mechanism. Outside the cell, surfactant becomes activated by physical means and reduces surface tension. Due to that, the strength of the initial stimulus is then largely alleviated. Thus, we observed a feedback loop, consisting of a physical force that initiates a biological process, which, in turn, leads to a change in the physical environment. But furthermore, we found that surface contact leads to even more complex phenomena. The cells respond with an enhanced or attenuated expression of specific genes and many of them are associated with lung diseases. This issue has a growing relevance in lung physiology and in a clinical context. Different ventilation strategies used in emergency units, for example, may have different clinical outcomes but the explanation of the underlying mechanisms is still a rather hypothetical one. It is the complexity of the alveolar system that makes in vitro investigations very intricate. In the course of our project, we therefore had to design new strategies to make this system accessible for experimentation. Particularly the study of interactions of epithelial cells with an air-liquid phase boundary, just at a micrometer scaled distance to it, and the highly dynamic chemical and physical processes that take place on that interface, necessitates an interdisciplinary approach between cell biologists, physicists and chemists. Thus, we worked in close collaboration with biophysical institutions mainly in Austria and Spain whose expertise in optical methods and surface chemistry has been very helpful.