Developmental Airway Cell Biology The “Normal” Young Child JONATHAN GRIGG and JOSEF RIEDLER University Department of Child Health, Leicester Royal Infirmary, Leicester, United Kingdom; and Childrens Hospital, Salzburg, Austria
There is increasing awareness in the research community that events in early childhood have a profound influence on the expression of respiratory disease in later life, but is there any value in studying airway cells from healthy children? Why not use the extensive body of information about airway cells derived from adult volunteers? Adults are easy to recruit, whereas ingenious and time-consuming ways of sampling the lower airways of children must be devised to overcome ethical problems. Even if there are developmental changes, could these be defined in animal models? There are several reasons why children must be studied. First, research published within the last 5 yr clearly shows that there are major changes in immunocompetent and inflammatory cells during normal human development. Second, there are significant species differences in the release of mediators by immunocompetent cells. Third, age-associated changes in human airway cells are a result of complex interactions between genetic and environmental factors. It is therefore impossible to model the combination of even a handful of potential variables, for example, frequency of colds, exposure to lipopolysaccharide, nutritional state, and age. The technique of bronchoalveolar lavage (BAL) before elective surgery, initially developed to overcome the ethical problems of sampling cells from normal children, is uniquely suited to studying large numbers of individuals (the mean number of children per study is currently more than 50). It is now possible to tease out the independent effects of environmental stimuli on airway cell function. Clues to the relevant factors have been provided by epidemiological research. For example, having older siblings reduces the risk of aeroallergen sensitization (1). This association, if true, must also be associated with an effect on immune function at the cellular level. The goal of pediatric airway cell research is therefore not just to generate normal values for comparison with established disease, but to focus on the critical functional mechanisms that protect the normal lung from developing disease. Although the lung of the young child may seem to be particularly vulnerable to a range of environmental insults (2), this does not necessarily reflect immaturity of airway cell function. Young children may have more respiratory infections not because of a defect in microbial killing by cells, but because their immunoglobulin repertoire is not fully developed. Similarly, early childhood may seem to be the vulnerable period for antigen sensitization, not because of a possible immaturity in the ability of alveolar macrophages (AMs) to suppress pulmonary immune cells (3), but because it is the period when the lung is first exposed to antigen. These considerations are important, because a true developmental immaturity of airway cell function may be amenable to accelerated “maturation” by exposure to an appropriate environmental factor (e.g., exposure to traditional farms) (4), or an anthroposophic lifestyle (5). Correspondence and requests for reprints should be addressed to Jonathan Grigg, M.D., University Department of Child Health, Leicester Royal Infirmary, P.O. Box 65, Leicester LE2 7LX, UK. E-mail:
[email protected] Am J Respir Crit Care Med Vol 162. pp S52–S55, 2000 Internet address: www.atsjournals.org
WHAT DO WE KNOW? Leukocyte Differentials
For most of the intrauterine development, the fetal airway is devoid of inflammatory and/or immunocompetent cells. The normal resident cell population of the human adult cannot be seen either histologically in the airway of stillborn infants (gestational age 20–40 wk) (6), or in the BAL fluid of very premature infants immediately after cesarean section (7). Thus, while BAL recovers both large numbers and a wide range of airway leukocytes from adults, BAL fluid from healthy premature infants contains some epithelial cells, but no leukocytes. The pattern of leukocytes reported for the normal adult lung varies from study to study, but in one of the larger studies using a standardized lavage technique, the median proportions for each cell type were AM 85.2%, lymphocytes 11.8%, neutrophils 1.6%, and eosinophils 0.2% (8). Thus, from just before birth to adulthood, the airway must be “seeded” with its resident leukocyte population. Does seeding occur suddenly, or during the whole period of childhood? There is good evidence, from both fiberoptic bronchoscopic and nonbronchoscopic (suction catheter) BAL, that the profile of leukocytes in the lower airway of children does not change significantly after 3 yr of age. Heaney and colleagues (9) found no change in the bronchoalveolar leukocyte differential between two age groups of normal children: 3–8 and 8–14 yr. Using linear regression analysis, Ratjen and coworkers (10) found no agerelated differences in the differential count of BAL fluid AM and lymphocytes in 55 children aged between 3 and 16 yr. Similarly, we found no age-associated change in the differential count and concentration of airway leukocytes in 33 normal children aged between 2 and 17 yr (11). Eosinophils are present in the airways of some of these children, but the differential count (and concentration) is low. For example, the range of eosinophils was found to be between 0 and 3.6% (median, 0.4%) (10). Mast cells and basophils in the BAL fluid have been looked for in some, but not all, studies of normal children. Where reported, the median percentage of basophils and mast cells in BAL fluid is less than 0.2% (9, 10, 12). Is there any evidence of leukocyte “seeding” of the human airway in the first few days or years of life? Histologically, newborn human infants surviving for more than 48 h do have macrophages in the alveoli, suggesting that there is a rapid expansion of the AM population in the perinatal period. Indeed, the 2-d-old Macaca nemestrina monkey has 33 times the number of AMs in BAL fluid compared with the term fetus, with an additional 4-fold increase in AM by 3–4 wk of age (13). Using nonbronchoscopic BAL we have found both significantly higher differential count and concentrations (adjusted for dilution) of AMs in 30 children less than 24 mo of age compared with two older groups (2–5 and 6–17 yr) (11). When viewed as a continuous process, our data indicate that the differential count of AMs remains high (⬎ 97%) over the first months of life, then falls gradually until 2 yr of age, and thereafter remains static (Figure 1). Associated with this fall is a concomitant rise in the BAL fluid lymphocyte differential count, and to a lesser extent, absolute lymphocyte concentration. Our fiberoptic bron-
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choscopic study of young children (12), found an increase in the BAL fluid lymphocyte differential count, but not a decrease in AM differential count during the first 24 mo of life. However, the number of infants/children aged between 3 mo and 2 yr was small. Similarly, no significant change was found in the BAL fluid AM differential count in 8 children under 3 yr of age (9). Overall, these data suggest that developmental changes in the lower airway cell profile are limited to the first two years of life, but more young children need to be studied to fully define the changes in leukocyte subpopulations. Possible mechanisms that could drive developmental changes in the lower airway inflammatory cell profile may be inferred from animal models. For example, in the newborn monkey, the expansion of the AM population mirrors the increase in alveolar surfactant levels (13). In vitro, surfactant-associated protein A is chemotactic for monocytes (14). Could the initial seeding of the lung be a result of a genetically determined increase in alveolar surfactant secretion? This hypothesis would be plausible if the expansion of AMs were a result of recruitment of precursors from the interstitium, rather than a result of local proliferation. However, to date the origin of AMs in young children remains unclear. In contrast, there is evidence that viral infections may play a role in the age-associated increase in airway lymphocyte numbers. Some asymptomatic adults have high proportions of airway lymphocytes (⬎ 30%), and it has been speculated that this is because of an unrecognized cold prior to BAL (15). Direct evidence of an effect of trivial colds comes from a group of normal children lavaged after the resolution of coryzal symptoms (i.e., no symptoms for at least 24 h and no active viral infection). These children have significantly higher proportions of lymphocytes in their BAL fluid when compared with age-matched controls with no coryzal symptoms for 8 wk (16). Thus, developmental change in airway cells may not be a smooth
Figure 1. Age-associated changes in the percentage of (A) alveolar macrophages and (B) lymphocytes in the bronchoalveolar lavage fluid obtained from healthy children during elective surgery. ***p ⬍ 0.001 versus children aged ⬍ 24 mo. (Reproduced with permission from Grigg, J., J. Riedler, C. F. Robertson, W. Boyle, and S. Uren. Eur. Respir. J. 1999;14:1198–1205 [11].)
process, especially if driven by environmental stimuli. If, for example, colds profoundly influence the lower airway milieu, permanent changes in the cell profile may be discontinuous and rapid, and superimposed on wide day-to-day fluctuations. The intrasubject variation in the airway leukocyte profile is unknown. However, support for the hypothesis that the environment has a role in pulmonary maturation is provided by studies of germ-free lambs. When compared with healthy, normally reared animals, healthy germ-free lambs have significantly fewer neutrophils in their BAL fluid (8 versus 1%) (17), suggesting that a permanent neutrophil response (at least in this model) results from inhalation of microbes. Immune Receptors
Developmental changes in the proportion and number of airway leukocyte subpopulations do not prove that immune cell function alters with development. One step closer to assessing function is the measurement of immune receptor expression. T lymphocytes are key cells in pulmonary immune functioning, and can be divided into two subclasses: helper cells, which when stimulated by antigen-presenting cells proliferate and release cytokines that stimulate the antibody response, and cytoxic T cells that kill cells expressing particular antigens. In general, the immune receptor CD4 defines a predominantly helper subset, whereas CD8⫹ cells are predominately cytotoxic (18). Immunocytochemical techniques have rarely been applied to BAL cells from normal children. However, two studies of receptors on BAL lymphocytes show that the ratio of CD4⫹ to CD8⫹ T lymphocytes is 0.6 for children aged between 3 mo and 10 yr (12), and 0.7 for children aged between 3 and 16 yr (19). Both values are significantly lower than the ratio of 2.7 reported for normal young adults (8). Although there is a significant correlation of the BAL CD4/CD8 ratio with age, the proportion of B cells, T cells, and natural killer cells in BAL fluid of healthy children seems to be within the normal range for adults (19). Assessment of the absolute number of lymphocyte subpopulations in BAL fluid suggests that the lower CD4/CD8 ratio in children (versus adults) is because of a higher absolute number of CD8⫹ cells (19). Thus the CD4/CD8 ratio increases during a period in which there is no change in the BAL lymphocyte differential count (i.e., 3–16 yr), but it remains unclear if this change is most profound in early childhood. During adulthood, the BAL CD4/CD8 ratio continues to increase with age, reaching 7.6 at age 64 to 83 yr (20). However, unlike the CD8-driven change with age in children, the process in aging adults is driven by an increase in the absolute numbers of CD4⫹ cells. The role of airway lymphocytes in the immunity of the normal adult lung is unclear. BAL lymphocytes from healthy adults are difficult to clone, and may be in a state of partial anergy, or even committed to cell death (21). How developmental changes in lymphocyte subsets affect the ability of children to react to antigen and infection will be difficult to assess. Unlike airway lymphocytes, there is a clear link between the immunophenotype of AMs and functional effect. Although AMs have some of the receptors for presenting antigen to T cells, they lack the critical B7 costimulatory molecule necessary for inducing lymphocyte proliferation (22). They are therefore ineffective antigen-presenting cells. In contrast, through a combination of soluble and contact-mediated mechanisms, AMs are effective suppressors of T cell proliferation stimulated by other (more important) antigen-presenting cells (3, 23). Elimination of AMs in vivo in an animal model therefore increases IgE responses to inhaled antigen (24). The suppressor ability of AMs can be defined by expression of RFD1 and RFD7, with RFD1⫹ 7⫹ as the “suppressor” phenotype.
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There are no published data on these receptors in AMs in normal children. We have, however, compared the expression of HLA-DR and intercellular adhesion molecule 1 (both immunomodulatory receptors) on AMs from children 0–24 mo, and 25 mo to 17 yr of age, and found no significant difference between the two age groups (11). The bronchial epithelium may have an important role in modulating inflammatory response to environmental stimuli. The ability of bronchial epithelial cells to release proinflammatory mediators such as cytokines and immunomodulatory mediators, such as nitric oxide, could change during normal development, but this has not yet been studied.
important antigen-presenting cells in the lung. Recovering dendritic cells from the interstitium of normal children is impossible, but in the adult, small numbers (⬍ 0.4%) are found in the BAL fluid. It may therefore be possible to model the complex interaction between a stimulus (e.g., antigen or pollutant), AMs, dendritic cells and peripheral T lymphocytes in vitro. 6. What is the function of airway lymphocytes? If these can be cloned, the profile of cytokine release could be compared with the systemic profile.
Leukocyte Function
1. Strachan, D. P., L. S. Harkins, and J. Golding. 1997. Sibship size and selfreported inhalant allergy among adult women. ALSPAC Study Team. Clin. Exp. Allergy 27:151–155. 2. Holt, P. G. 1995. Postnatal maturation of immune competence during infancy and childhood. Pediatr. Allergy Immunol. 6:59–70. 3. Holt, P. G., J. Oliver, N. Bilyk, C. McMenamin, P. G. McMenamin, G. Kraal, and T. Thepen. 1993. Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J. Exp. Med. 177:397–407. 4. Braun-Fahrlander, C., M. Gassner, L. Grize, U. Neu, F. H. Sennhauser, H. S. Varonier, J. C. Vuille, and B. Wuthrich. 1999. Prevalence of hay fever and allergic sensitization in farmer’s children and their peers living in the same rural community. SCARPOL team. Swiss Study on Childhood Allergy and Respiratory Symptoms with Respect to Air Pollution. Clin. Exp. Allergy 29:28–34. 5. Alm, J. S., J. Swartz, G. Lilja, A. Scheynius, and G. Pershagen. 1999. Atopy in children of families with an anthroposophic lifestyle. Lancet 353:1485–1488. 6. Alenghat, E., and J. R. Esterly. 1984. Alveolar macrophages in perinatal infants. Pediatrics 74:221–223. 7. Grigg, J., S. Arnon, A. Chase, and M. Silverman. 1993. Inflammatory cells in the lungs of premature infants on the first day of life: perinatal risk factors and origin of cells. Arch. Dis. Child. Fetal Neonatal Ed. 69:40–43. 8. Anonymous. 1990. Bronchoalveolar lavage constituents in healthy individuals, idiopathic pulmonary fibrosis, and selected comparison groups: The BAL Cooperative Group Steering Committee. Am. Rev. Respir. Dis. 141:S169–S202. 9. Heaney, L. G., E. C. Stevenson, G. Turner, I. S. Cadden, R. Taylor, M. D. Shields, and M. Ennis. 1996. Investigating paediatric airways by nonbronchoscopic lavage: normal cellular data. Clin. Exp. Allergy 26: 799–806. 10. Ratjen, F., M. Bredendiek, M. Brendel, J. Meltzer, and U. Costabel. 1994. Differential cytology of bronchoalveolar lavage fluid in normal children. Eur. Respir. J. 7:1865–1870. 11. Grigg, J., J. Riedler, C. F. Robertson, W. Boyle, and S. Uren. 1999. Alveolar macrophage immaturity in infants and young children. Eur. Respir. J. 14:1198–1205. 12. Riedler, J., J. Grigg, C. Stone, G. Tauro, and C. F. Robertson. 1995. Bronchoalveolar lavage cellularity in healthy children. Am. J. Respir. Crit. Care Med. 152:163–168. 13. Jackson, J. C., S. Palmer, C. B. Wilson, T. A. Standaert, W. E. Truog, J. H. Murphy, and W. A. Hodson. 1988. Postnatal changes in lung phospholipids and alveolar macrophages in term newborn monkeys. Respir. Physiol. 73:289–300. 14. Wright, J. R., and D. C. Youmans. 1993. Pulmonary surfactant protein A stimulates chemotaxis of alveolar macrophage. Am. J. Physiol. 264: L338–L344. 15. Laviolette, M. 1985. Lymphocytes fluctuation in bronchoalveolar lavage fluid in normal volunteers. Thorax 40:651–656. 16. Grigg, J., J. Riedler, and C. F. Robertson. 1999. Bronchoalveolar lavage fluid cellularity and soluble intercellular adhesion molecule-1 in children with colds. Pediatr. Pulmonol. 28:109–116. 17. Weiss, R. A., A. D. Chanana, and D. D. Joel. 1986. Postnatal maturation of pulmonary antimicrobial defense mechanisms in conventional and germ-free lambs. Pediatr. Res. 20:496–504. 18. Leahy, D. J. 1995. A structural view of CD4 and CD8. FASEB J. 9:17–25. 19. Ratjen, F., M. Bredendiek, L. Zheng, M. Brendel, and U. Costabel. 1995. Lymphocytes subsets in bronchoalveolar lavage fluid of children without bronchopulmonary disease. Am. J. Respir. Crit. Care Med. 152: 174–178. 20. Meyer, K. C., and P. Soergel. 1999. Variation of bronchoalveolar lym-
The small number of leukocytes that can be recovered from children makes functional studies difficult, and most of the evidence for developmental immaturity of leukocyte function comes from animal models (25–27). As discussed above, AMs are important suppressors of immune responses, but they also have the vital role of removing and killing infectious material arriving at the air–tissue interface. Adult human AMs are more effective at killing nonfilamentous yeast, when compared with AMs from intubated term neonates (28). However, there are no data on how the ability of AMs to phagocytes and kill microorganisms changes throughout normal development. We found that the ability of AMs to reduce nitroblue tetrazolium (a surrogate marker of intracellular oxidant production) is attenuated in AMs from young children (11), but whether this is associated with defective oxidant-mediated killing of microorganisms is unknown. Bakker and colleagues (26) studied AM function in the 14-d-old rat, and found no evidence of an impairment of AM-mediated suppression of T cell proliferation. The researchers, however, do point out that while AM suppression may be mediated by nitric oxide in the rat, this mediator is not involved in immune suppression by human AMs (29). There are no data on BAL lymphocyte function in normal children.
WHAT DO WE NEED TO KNOW AND HOW CAN WE ACHIEVE THIS? 1. What are the changes in the airway leukocyte profile during the first two years of life? Sampling has been difficult in very young children; however, the use of suction catheter BAL means that cells may be removed from very small infants. 2. What drives developmental changes in airway leukocytes? Large numbers of children will need to be recruited (possibly in a multicenter project) to assess the independent effects of genetic and environmental variables, that is, age, lung function, environmental tobacco smoke exposure, exposure to ozone and particulates, viral colds, diet, and hygiene. 3. What is the intrasubject variation of the BAL leukocyte profile? This can be assessed only in children undergoing repeated elective surgery. 4. How does the ability of AMs to suppress inappropriate T cell activation change with age? This has direct relevance in assessing the vulnerability of the lung to antigen sensitization. Immunocytochemistry of suppressor AMs is relatively easy to perform, especially with the new technique of laser scanning cytometry. Using laser scanning cytometry, a large amount of data can be generated from small numbers of cells (30). Only 2,000 cells are needed for laser cytometry compared with more than 15,000 for flow cytometry. 5. What are the interactions between airway and interstitial cells such as dendritic cells? Dendritic cells are the most
References
Grigg and Riedler: Developmental Airway Cell Biology
21.
22.
23.
24.
phocyte phenotypes with age in the physiologically normal human lung. Thorax 54:697–700. Garlepp, M. J., A. H. Rose, R. V. Bowman, N. Mavaddat, J. Dench, B. J. Holt, M. Baron-Hay, P. G. Holt, and B. W. Robinson. 1992. A clonal analysis of lung T cells derived by bronchoalveolar lavage of healthy individuals. Immunology 77:31–37. Chelen, C. J., Y. Fang, G. J. Freeman, H. Secrist, J. D. Marshall, P. T. Hwang, L. R. Frankel, R. H. DeKruyff, and D. T. Umetsu. 1995. Human alveolar macrophages present antigen ineffectively due to defective expression of B7 costimulatory cell surface molecules. J. Clin. Invest. 95:1415–1421. Schauble, T. L., W. H. Boom, C. K. Finegan, and E. A. Rich. 1993. Characterization of suppressor function of human alveolar macrophages for T lymphocytes responses to phytohemagglutinin: cellular selectivity, reversibility and early events in T cell activation. Am. J. Respir. Cell Mol. Biol. 8:89–97. Thepen, T., C. McMenamin, B. Girn, G. Kraal, and P. G. Holt. 1992. Regulation of IgE production in pre-sensitized animals: in vivo elimination of alveolar macrophages preferentially increases IgE responses to inhaled allergen. Clin. Exp. Allergy 22:1107–1114.
S55 25. Kurland, G., A. T. Cheung, M. E. Miller, S. A. Ayin, M. M. Cho, and E. W. Ford. 1988. The ontogeny of pulmonary defenses: alveolar macrophage function in neonatal and juvenile rhesus monkeys. Pediatr. Res. 23:293–297. 26. Bakker, J. M., E. Broug-Holub, H. Kroes, E. P. van Rees, G. Kraal, and J. F. van Iwaarden. 1998. Functional immaturity of rat alveolar macrophages during postnatal development. Immunology 94:304–309. 27. Zeligs, B. J., L. S. Nerurkar, and J. A. Bellanti. 1977. Maturation of the rabbit alveolar macrophage during animal development: III. Phagocytic and bactericidal functions. Pediatr. Res. 11:1208–1211. 28. D’Ambola, J. B., M. P. Sherman, D. P. Tashkin, and H. Gong, Jr. 1988. Human and rabbit newborn lung macrophages have reduced antiCandida activity. Pediatr. Res. 24:285–290. 29. Upham, J. W., D. H. Strickland, N. Bilyk, B. W. Robinson, and P. G. Holt. 1995. Alveolar macrophages from humans and rodents selectively inhibit T cell proliferation but permit T cell activation and cytokine secretion. Immunology 84:142–147. 30. Rew, D. A., G. Woltmann, and A. J. Wardlaw. 1999. Laser-scanning cytometry. Lancet 353:255–256.
