ABSTRACT
The airway epithelium is the first target for a wide range of inhaled molecules and particles. Mucociliary clearance is responsive for their partial, if not total, elimination as coordinated beating of cilia along the upper respiratory tract continuously drives mucus towards the nasopharynx. Ciliated cells are one of the three main cell types lining the surface of the upper airways, along with basal and secretory cells. Ciliated cell differentiation is a complex molecular process involving centriole/basal body assembly (centriologenesis) and cilia formation (ciliogenesis). Detailed ultrastructural features have been described in different species (1). However, most of the molecular events underlying the differentiation process remain to be understood. The formation of 100-200 cilia in each terminally differentiated ciliated cell requires the assembly of the same number of 155
centriole/basal bodies. Procentrioles are assembled around electron-dense cytoplasmic granules near the Golgi area. The centrioles elongate, migrate through the cytoplasm, and anchor onto the apical plasma membrane, where they trigger axonemal microtubule polymerization and cilia formation (2,3). Tubulin and cen-trin are the two most widely studied gene families involved in centriole and cilia structure and functions. The heterogeneity of a-and (3-tubulins is generated at a genetic level by several genes (4) and at a biochemical level by posttranslational modifications. Among them, polyglutamylation and polyglycylation are common, consisting of the addition of glutamyl and glycyl residues, respectively, to form polyglutamyl and polyglycyl chains at the C-terminal region of both a-and 13-tubulins (5,6). These additional peptide chains could play an important role in cytoplasmic microtubule networks as well as in centriolar and axonemal structures. Centrin proteins are associated with the distal lumen of the centrioles (7) and belong to the family of Ca2+-binding protein (8). In humans, three centrin genes have been described: HsCENl, HsCEN2 and HsCEN3. But functionally, only two distinct families of centrin proteins seem to exist, one of which has been implicated in centrosome duplication (9,10).We have developed a primary culture system of human nasal epithelial cells where the mucociliary differentiation process can be observed and quantified. We characterized markers of ciliated cell differentiation at the cellular and biochemical level by studying polyglutamylation and polyglycylation of a-and (3-tubulins and expression and cellular localization of centrin isoforms. Immunoelectron microscopy revealed that polyglutamylation is an early event in the centriole/basal body assembly process, while polyglycylation is restricted to axonemal tubulin (11). In parallel, we have shown a correlation between ultrastructural localization and function of two human centrin isoforms (12). A MODEL SYSTEM TO STUDY MUCOCILIARY DIFFERENTIATION IN VITRO
Several different methods have been developed to culture primary respiratory epithelial cells. The most frequently used technique is the creation of an air/ liquid interface, which results in mucociliary differentiation in vitro (13-15). For our studies, we used the spheroid culture model of human nasal epithelial (HNE) cells initially described by Jorissen et al. (16). In these cultures, we have quantified the mucociliary differentiation process using specific monoclonal antibodies against secretory and ciliated cell components. HNE cells were dissociated from nasal polyps or turbinates and were seeded onto a type I collagen gel where undifferentiated epithelial cells proliferated to confluence. The collagen gel was digested and cell sheets were shaken in a rotary motion for 5-8 days to induce formation of still undifferentiated epithelial spheroids (Fig. la). In these spheroids, epithelial cells polarized and differentiated. During the differentiation pro
cess, spheroids were dissociated to analyze the phenotype distribution of the cells by flow cytometry using specific antibodies. We used Ml antibodies, which recognize MUC5AC gene product and potentially other human mucins (17), to estimate the relative proportion of secretory cells and GT335 antibodies, which recognize glutamylated tubulin (a marker of centrioles, basal bodies, and ciliary axonemes), to estimate the percentage of ciliated cells. Such experiments revealed that secretory cells are detected in significant numbers early during differentiation and subsequently decrease, while ciliated cells develop at a later stage and continue to increase in number throughout the differentiation process. A representative experiment is shown in Figure lb.We also studied the effects of two Th2-response interleukins, IL-4 and IL-13, on mucociliary differentiation. These interleukins play important roles in asthma (18,19), a disease known to change the epithelium to a more secretory phenotype. In particular, IL-13 has been shown to cause mucus cell metaplasia (20). When 10 ng/mL IL-13 was added to the cultures during differentiation, the percentage of secretory cells, compared to control, increased dramatically (Fig. 2a), while the percentage of ciliated cells remained low (Fig. 2b) (21). Western blot analysis confirmed the latter result (Fig. 2b'). Interferon gamma (IFNy), a Thl cytokine, had no effect on the percentages of secretory versus ciliated cells, while IL-4 had an effect similar to IL-13 (Fig. 2c and d). CHARACTERIZATION OF CENTRIOLAR MARKERS DURING MUCOCILIARY DIFFERENTIATION Both human centrin 1/2 protein (Hscenl/2p) and Hscen3p were associated with cytoplasmic granules (Fig. 3a,b) and enriched in the distal lumen of migrating centrioles (Fig. 3a,b) and mature centriole/basal bodies (Fig. 3a',b'). Interestingly, the localization of Hscen3p was restricted to centriole/basal bodies (Fig.
