This is the first description of the time of first appearance and subsequent quantitative development of the RBM in humans without known respiratory disease. We have shown in our group of selected individuals that the RBM is difficult to discern by light microscopy before 30 weeks gestation, whereas between 30 weeks gestation and term, the RBM is visible in all of the cases studied. RBM thickness increases with age until term, subsequently increases rapidly until about 6 years of age and then continues to increase, but at a slower rate, until about 17 years. However, the large variability in RBM thickness between individuals after school age prevents the clear delineation of an age when its increase plateaus. In adulthood, RBM thickness appears to decrease gradually with age. We show for the first time a strong relationship between RBM thickness and epithelial height, and the change in epithelial height with age reflects the change in RBM thickness with age. These data support the proposed close interaction of the epithelium with its underlying RBM and their association as an EMTU.15
The first detection of the RBM by light microscopy ~10 weeks before term (with one subject having a visible RBM at 24 weeks gestation) is consistent with a previous report of it being visible only by electron microscopy after ~24 weeks gestation.16
It also highlights a key difference in RBM development between humans and primates, since in the latter it develops postnatally.2
The slope of the linear relationship between age and RBM thickness is steep from 30 weeks gestation up to 6 years of age (), indicating a relatively rapid rate of increasing RBM thickness during this period. This is consistent with the rate of total lung growth which is fastest during the first 2 years of life, due primarily to alveolar multiplication.17
Alveoli also first appear in utero at ~30 weeks gestation.18
Alveolar formation/maturation is controlled by interaction with the subepithelial elastin–collagen matrix, which acts as an anchoring fishnet through which alveoli protrude (ie, the ‘fishnet hypothesis’).19
The similar timing of onset of such a maturation process in the lung parenchyma and the appearance and rapid increase of bronchial RBM thickness suggests that both may be influenced by molecular changes in their adjacent lung connective tissue matrices.
We were unable to define clearly an age when maximal RBM thickness is reached. From our cross-sectional data it seems that the RBM increases in thickness, albeit at a slower rate after 6 years, without a definite plateau in thickness being reached until at least adolescence. The ‘soft plateau’ that we have seen in RBM thickening is also observed in normal alveolar development, whereby the exact time at which alveolar growth and multiplication ceases is unknown.20
This is the first report of a possible reduction in RBM thickness apparent during ageing, which interestingly is also mirrored in an age-associated reduction of alveolar surface area and wall tissue.20
This is the first description of change in epithelial height with age, and the first report of a positive relationship between RBM thickness and epithelial height. We speculate that as taller epithelial columnar cells develop, a function of the basement membrane may be to keep them anchored, and thus a thickening of the reticular component of the basement membrane may be required to help achieve this. Indeed the contact area between columnar cells and the basal lamina in subjects with asthma is significantly less than in healthy controls22
and is a proposed explanation for epithelial shedding in asthma. Increasing bronchial epithelial height with age has previously been reported from preterm until 8 months postnatal age,23
but subsequent changes in epithelial height with age were unknown. We have shown an increase in epithelial height until adolescence, with a subsequent reduction during adulthood. However, the number of adults included in our study is small, and the relationship between RBM thickness and age in adults is only weakly negative; therefore, it is difficult to draw definitive conclusions about change in RBM thickness and epithelial height with age in our adults. This relationship needs to be investigated further in future studies with a larger sample size. If confirmed, these features may be a reflection of the normal decline in lung function seen in adults. The lack of consensus about a relationship between RBM thickness and lung function may be explained by failure to take account of a natural reduction in RBM thickness in adulthood. Thus to allow accurate and meaningful interpretation of RBM data obtained in studies of endobronchial biopsies, it would seem important to include age-matched controls for children and adults.
One limitation of our current study is that we have not assessed changes in the components of the RBM with its development, which might provide clues to the important molecules and mechanisms involved. We have tried staining these formalin-fixed, paraffin wax-embedded sections for at least one RBM component, tenascin-C, but have been unsuccessful. While previous studies have reported RBM components, these have all used snap-frozen tissue, but as we only had access to paraffin sections we have been unable to explore this further. However, the importance of fibroblast growth factor in contributing to increased thickness and the role of the RBM in influencing trafficking of growth factors in the EMTU has been shown in primate airways.2
A further limitation is the use of light microscopy alone, and not additional electron microscopy to assess RBM ultrastructure. Although this may have shown the presence of a reticular layer earlier than 30 weeks gestation, such a study was not possible because all tissue was processed for clinical reasons, fixed and embedded in paraffin wax, not the resin embedding required for electron microscopy. However, we have previously reported on the ultrastructure of the RBM in non-wheezy infants, children without asthma and healthy adults, and shown that it is similar at all ages.24
We acknowledge that our postmortem lung specimens were not all fixed using the same technique, and this is why the data have been presented in three separate age groups, according to the origin of the tissue. Finally, we accept there is inequality in the number of subjects in each age group, with only small numbers available at some ages, especially the older children and adults. However, the nature of the tissue is such that we were unable to have an equal age distribution. We also acknowledge that the three age groups are made up of subjects from different areas, and that these are not longitudinal data. Despite the above limitations, we consider the large number of subjects, whose deaths were not respiratory related, and the wide age range included provides a strong basis for establishing the time of appearance and pattern of thickening that occurs to the bronchial RBM, and its positive relationship with epithelial height, during normal development. These data indicate that future paediatric and adult biopsy studies assessing RBM thickness should include age-matched controls.