A Review of In Vitro Modelling Approaches to the Identification and Modulation of Squamous Metaplasia in the Human Tracheobronchial Epithelium

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein–cadaverine (FC) into bis(γ-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.

Intraspinal Bronchogenic Cyst: Ultrastructural Study of Abnormal Cilia

Intraspinal cyst formed of tissue proper to the tracheobronchial tract has been referred to as bronchogenic cyst. Six cell types of the cyst epithelium have been identified, i.e., ciliated cells, non-ciliated cells, goblet cells, basal cells, Kulchitsky's cells and undifferentiated cells. This report described the detailed morphological alterations of cilia of the lining epithelium of three cases of intraspinal bronchogenic cyst and compared the findings with the ciliary abnormalities of the tracheobronchial epithelium.Ultrastructural abnormalities of cilia of the intraspinal bronchogenic cyst could be classified into five categories:(A)Cilia showing addition, deletion or disorganization of axonemal minotubules.(B)Swollen cilia containing a single axoneme in an excess amount of matrix: (a) normal 9 + 2 axoneme,(b) stretched axoneme bending toward one side, (c) abnormal axonemal pattern.(C)Compound cilia with multiple axonemal microtubules ensheathed by a common ciliary membrane: (a) two or more normal 9 + 2 axonemes, (b) two or more normal 9 + 2 axonemes sharing doublets, (c) giant compound cilia with numerous normal and abnormal axoneme in random orientation, (d) compound cilia containing cytoplasmic organelles, (e) compound cilia with excessive matrix.

Epithelial stem cell-mediated development of the human respiratory mucosa in SCID mice

We have developed an in vivo assay for progenitor cells of the human tracheobronchial epithelium relying on the transplantation of human prenatal respiratory tissues into severe combined immunodeficiency mice. Engrafted embryonic or fetal open tracheobronchial rudiments are rapidly closed at each end by a neoformed membrane that we named the operculum. After 2–4 weeks, differentiated human respiratory epithelium covers both the native airway matrix and the new operculum. Human epithelial cells dissociated from either emerging embryonic lung primordia or mature xenografts were seeded in host human airway grafts, of which native epithelium had been eliminated by several cycles of freezing and thawing. All grafts seeded with donor epithelial cells and implanted back into SCID mice recovered a surface mucociliary epithelium expressing expected markers and secreting mucus. Spontaneous epithelium regrowth was never observed in control unseeded, denuded grafts. In some experiments, donor epithelial cells and host denuded airway were sex-mismatched and the donor origin of newly formed epithelial structures was confirmed by sex chromosome detection. After two rounds of seeding and reimplantation, a normal epithelium was observed to line the 3rd generation operculum. These observations substantiate a functional assay for human candidate airway epithelium stem cells.

Respiratory mucins: identification of core proteins and glycoforms

At least eight mucin apoproteins are expressed by the tracheobronchial epithelium, but it is not known which, if any, of these are major constituents of the respiratory secretions responsible for the formation of the mucus gel. To address this we have isolated mucins from normal, asthmatic and chronic bronchitic secretions. The asthmatic mucin reduced subunits were fractionated into four populations (I–IV) by anion-exchange HPLC. Amino acid and monosaccharide compositional analysis, as well as Mr and size measurements, indicate that two of these populations (I and II) are glycoforms of the same or related apoprotein(s) and that the other populations contain two different apoproteins. A panel of antibodies and antisera recognizing the variable number tandem repeat (VNTR) of specific mucin apoproteins did not, as predicted, react with the glycosylated molecules, but after deglycosylation the majority of these probes (with the exception of those to MUC2, which were negative) reacted at a low level with each of the subunit populations. In contrast, an antiserum against a non-VNTR sequence of MUC5AC identified one of the populations (III) as the MUC5AC mucin. The MUC5AC reduced subunit had an Mr of 2.2×106 and an RG (radius of gyration) of 57 nm. The genetic identities of the major mucin (populations I and II) and a minor component (population IV) were not established. The MUC5AC mucin was also identified as a major component in the pooled normal secretions from 20 individuals, whereas in a chronic bronchitic sample it was only a minor constituent. Furthermore, in all these different respiratory secretions the MUC5AC mucin appears as a similar biochemical entity, as assessed by Mono Q chromatography and agarose electrophoresis, suggesting that it may have a well-defined pattern of glycosylation in the respiratory tract.