Structure and Development of Alveolar Epithelial Cells

Introduction

The ultimate temporal and morphologic stage of lung development is the formation of millions of gas exchange units, the alveoli. Life is possible without the alveolus; there are few in very premature infants. However, without expansion and maturation of the alveolar population, respiratory function and even survival are likely to be compromised.

In this chapter, the development and unique features of AT1 and AT2 cells are reviewed. The structure of the alveolus is fairly simple. In general, there are type I (AT1) and type II (AT2) alveolar epithelial cells, alveolar macrophages, endothelial cells, and pericytes. The last two are part of the vascular system. Macrophages act to intercept and process foreign material and to initiate and control local inflammatory responses. The AT2, a cuboidal cell located in the alveolar corners, has critical roles in surfactant formation, secretion, and recirculation as well as in innate immunity and alveolar repair. It constitutes 10% to 15% of lung cells and 60% of the cells within the alveolus but covers only some 4% of the alveolar surface. The AT1, which only constitutes about 10% of the total alveolar cells, covers more than 95% of the surface area. It is very large but very flat, resembling a snake after a recent large meal where the nucleus bulges out of the otherwise very thin cell profile. Based on its location, appearance, components, and the changes that occur when it is absent or damaged, the AT1 is believed to be essential for gas exchange as well as for the water and ion homeostasis that helps create the air–liquid interface within the alveolus. The AT1 cell may also have innate immune function.

A cautionary note is warranted regarding the extrapolation of what is observed in the pulmonary development of animals to human lung development, because there are a variety of maturational and perhaps functional differences between species. For instance, in sheep and rabbits, alveolarization is complete at birth. In contrast, marsupials such as the quokka wallaby are born with lungs equivalent to the canalicular stage. Lung development at birth coincides with the saccular stage of development in mice and rats. During human lung development, alveolarization starts 4 weeks before term (40 weeks postmenstrual age) and continues for as long as 2 years after birth, with the ultimate development of 300 to 480 million alveolar subunits.

Whatever the species, the theme of alveolar development is one of thinning of the basement membrane and approximation of the epithelial and endothelial cell layers, with exponential growth of the surface area. This process optimizes gas diffusion and the movement of solutes and water within the air–liquid interface. This structural alveolar interface, in turn, provides the necessary environment for the function of AT2 cell surfactant. It therefore follows that if alveoli are underdeveloped because an individual is born before sufficient thinning and expansion of the surface area has occurred or if subsequent normal development is interrupted by injury from mechanical ventilation and high oxygen levels, significant compromise of normal pulmonary function may occur.

Basement Membrane

Juxtaposed between the airspace and the capillary lumen are the very thin AT1 cells, the endothelial cells, and the basement membrane. Within the alveolus is a thicker basement membrane made up primarily of capillary and alveolar basement membranes and a thinner portion where the membranes essentially fuse into one. Each type covers about 50% of the alveolar walls. Morphologists have also noted interruption of the continuous basement membrane beneath the AT2 cells, permitting direct contact with the endothelium. It is speculated that this structural morphology facilitates the synthetic function and development AT2 cells.

The basement membrane is made up of collagen, heparan sulfate proteoglycans, and laminins. This is not uniform throughout the alveolus. The basement membrane associated with AT2 cells is quantitatively less sulfated than the membrane associated with AT1 cells. Histologically, the basement membrane progressively thins during lung development. It is unclear when the final fused endothelial/epithelial membrane becomes well established. The membrane components also change in the course of development. For example, laminin α-3, -4, and -5 are absent early in gestation, appearing only later, as distal and alveolar structures begin to form.

These modifications may play a role in alveolar epithelial cell differentiation. For instance, in mice lacking laminin α-5, AT2 cells were diminished and AT1 cells absent, whereas loss of type IV collagen had the opposite effect. The addition of heparin (to mimic sulfated proteoglycans) to human AT2 cells in culture increased the expression of FoxA1 and Wnt7A, components of developmental pathways involved in the transdifferentiation of the AT2-to-AT1 cell phenotype, implying that these proteoglycans may have a regulatory role in the process of epithelial differentiation.

The Type I Cell

Before Frank Low’s first detailed description of the AT1 cell, histologists generally felt that the capillary endothelium was “naked,” separated from the air space only by a noncellular basement extracellular matrix and by noncellular “plaques.” Low demonstrated by electron microscopy that these plaques were, in fact, the thin cytoplasm of a large epithelial cell, subsequently called the type I alveolar epithelial cell. Along with its size and shape, it was easily distinguishable from other nearby cell types because of the relative paucity of cytoplasmic organelles away from the nuclear bulge.

Based on its appearance by light microscopy, the recognizable flat AT1 cell is not seen with any consistency before the second half of the canalicular phase, when the cuboidal airway cells at the distal tip of the lung branches begin to flatten and take on the appearance of AT1 cells. This occurs as the extracellular matrix thins, corresponding to the gestational age of clinical viability, which is around 22 to 24 weeks in humans.

In most descriptions of this temporal histologic progression, AT2 cells appear just before the point at which AT1cells can reliably be identified. This was one piece of evidence in support of the model in which AT1 cells are derived from AT2 cells. Another was that in assessments of normal cell proliferation or turnover, it was difficult to see such activity in AT1 cells in contrast to AT2 cells. In 1989, Cheek and colleagues described how freshly isolated adult rat AT2 cells transdifferentiated into AT1 cells ; this model has produced significant insights into the molecular mechanisms needed for this differentiation. Perhaps the strongest evidence for the AT2-to-AT1 cell paradigm was the repeated observation that, after lung injury affecting AT1 cells, as by NO ₂ or hyperoxia, ^, only AT2 cells demonstrate proliferation, and AT2 lineage markers are seen in the reconstituted AT1 cells.

The discovery of cell-specific markers greatly improved our understanding of AT1 biology. Using these cellular markers, cells with AT1 characteristics have been shown to appear in the lung earlier in gestation than previously thought. More recently, techniques such as single-cell RNA sequence analysis and lineage mapping have produced corroborating evidence to refute the concept that the AT1 cell is terminally differentiated, with functions limited to gas and water/solute exchange and that the sole source of the AT1 cell is the AT2 cell.

Sources of AT1-Cell Markers

Desai and colleagues demonstrated that before birth there is a bipotent progenitor that expresses AT1 proteins such as T1α and RAGE as well as AT2 markers (SFTPC, MUC1, or CTSH). Subsequently, AT1 cells progressively lose the AT2 markers and start expressing AQP5, whereas the cells leading to the AT2 phenotype stop expressing RAGE and T1α and start expressing proteins like SFTPB and ABCA3. These bipotent progenitors may be similar to the cells described in electron microscopic evaluations of fetal sheep, where cells that had both AT1 and AT2 characteristics were seen during development ( Fig. 76.1 ).