In Vitro Models for Airways Mucin Secretion

Pulmonary Pharmacology & Therapeutics (1997) 10, 145–155 PULMONARY PHARMACOLOGY & THERAPEUTICS

Review In Vitro Models for Airways Mucin Secretion C. William Davis∗†‡, Lubna H. Abdullah† †Department of Physiology, ‡Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599, USA intact tracheal or bronchial explants, but those relevant to cellular regulation may best be answered using a cell culture model specific to superficial epithelium or submucosal glands.

INTRODUCTION Because of multiple sources of mucin and the morphological complexities inherent in the lung and the histology of the epithelium lining the airway lumen, in vitro models have become important tools in studies of the mechanisms and regulation of airway mucin secretion. As shown for the cartilaginous airways in Figure 1, goblet cells in the superficial epithelium and the mucous cells in the submucosal glands represent cellular stores of mucin. In the lungs of healthy humans and most other larger mammals, goblet cells comprise approximately 5 to 25% of the columnar cells in the superficial epithelium in both the cartilaginous and the larger noncartilaginous airways. In human noncartilagnous bronchioles less than approximately 2mm, goblet cells are rare but may be increased in number by a variety of experimentallyinduced inflammatory conditions.1–8 It is notable that goblet cells represent the sole source of stored mucin in the noncartilaginous airways as a result of the strict association between submucosal glands and the cartilaginous plates. Thus, in the obstructive diseases, at the sites of low, laminar airflow and mucus plugging in the smaller airways, the goblet cell represents the primary source of stored mucin. Although it is not known whether goblet and mucous cells are different cell types, it is quite likely that the agonists which stimulate mucin secretion from the superficial epithelium and submucosal glands are different (Table 1), a phenomenon proposed as early as the 1930s by Florey.9 As a consequence of potential differences in cell type and in regulation, the airway model used in a particular experimental situation should be carefully matched to the question asked. Questions relevant to the tissue level, for instance, might be answered satisfactorily using explants of

MUCIN DETECTION ASSAYS Studies of mucin-secretion and its regulation depend critically upon good assays for mucin for their success. Mucin is a very high molecular weight glycoconjugate which exists as long, linear polymers through extensive disulfide bonding. By weight, mucins are generally 80 to 90% carbohydrate (primarily O-linked) and are heterodisperse in terms of size, due variously to differences in protein (there are presently seven known secreted mucin proteins), in polymer length, and in glycosylation.10–18 As a consequence, assays specific to an individual species of mucin, i.e., a specific mucin protein bearing a specific pattern of glycosylation, are essentially nonexistent. At best, antibodies against synthetic peptides derived from specific mucin genes are just beginning to appear,19 and should these reagents allow identification of specific mucin proteins in the near future, they will represent a clear improvement over existing assay systems. The mucin assays in present use have evolved over the past several years in two general approaches. First, radioactive tracers (3H- or 14C-labelled N-acetyl glucosamine or glucose, or 35S-Na2SO42−; 20) have been extensively to label high molecular weight glycoconjugates (HMWG). In subsequent steps, proteoglycan-degrading enzymes and Sepharose or BioGel column chromatography, or polyacrylamide gel electrophoresis, are used to isolate the mucins prior to quantification (e.g., see21–24). Alternatively, the mucins in collected secretions are assessed directly in binding assays using mucin antibodies or lectins (ELISA or ELLA; e.g., see25–32). Since lectins and most mucin antibodies interact with carbohydrate epitopes on mucin, ELLA and ELISA assays are generally

‡Author to whom correspondence should be addressed at: CF/ Pulmonary Res. & Treat. Center, 6009 Thurston-Bowles—CB 7248, University of North Carolina, Chapel Hill, NC 27599, USA. Email: cwdavis @med.unc.edu 1094–5539/97/030145+11 $25.00/0/pu970088

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Fig. 1 Cellular sources of mucin in cartilaginous airways. Top: A section of canine trachea stained with AB/PAS, shows goblet cells in the superficial epithelium and mucous cells in submucosal glands. The luminal aspect of the tissue is indicated. Bottom: Following removal of the superficial epithelium by an overnight digestion in the cold with protease XIV, the submucosal glands (shown selectively) appear intact and unaffected. (Both: 15X objective).

considered to be only semi-specific for mucin. Because both proteoglycans and mucins are secreted in the airways33–35 and because these compounds share many biochemical and biophysical properties,11,13 it is essential to demonstrate selectivity for mucins over other secreted HMWG using rigorous biochemical techniques for each experimental model in which a semi-specific mucin assay is used.

INTACT AIRWAYS Airway explants Many studies have successfully utilized explants of trachea or bronchus for studies of mucin secretion by

intact airways; indeed, explants represent the original in vitro model and have been in use for over 60 years. In its contemporary use, the explant model generally employs trachea or cartilaginous bronchus, and HMWG are radiolabeled prior to the experiments while the tissue is maintained in organ culture. Results derived from studies of airway explants include the observations that, b-adrenergic agonists,36 gastrin-releasing peptide and bombesin,37 and phorbol ester38 all stimulate mucin release from feline trachea. It is also possible to mount tissues as flat sheets in Ussing chambers and to measure the mucins released due to secretagogue challenge or electric field stimulation,39,40 or to simultaneously determine the ion transport properties of the airway epithelium.41 While explants have provided useful information

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Table 1 Agonists regulating airway mucin secretion. Submucosal glands (after Shimura & Takishima53) Cholinergic stimulation muscarinic (M1 & M3)—very potent secretagogues Adrenergic stimulation a1—secretagogue (serous>>mucous?) —stimulates gland contraction a2—inhibits b-adrenergic stimulation b?—secretagogue Peptidergic stimulation SP—secretagogue —stimulates gland contraction VIP—secretagogue —augments muscarinic responses Histaminergic stimulation H2—secretagogue Purinergic stimulation P2U—secretagogue Prostaglandin stimulation PGA2, PGD2, PGF2a—secretagogues PGE2—inhibitory Leukotriene stimulation LTC4, LTD4, 15-HETE—stimulatory in intact tissues, but no effect on isolated glands PAF stimulation stimulatory in isolated glands, but only in presence of platelets Endothelin stimulation secretagogue

Superficial epithelium (after Davis45) No effect (SPOC1 cells) No effect (explants of superficial epithelium) ND∗ No effect (SPOC1 cells) ND ND No effect (SPOC1 cells) Secretagogue (many preparations) ND ND ND

Stimulatory in primary cultures, but mediated by prostaglandins and/or leukotrienes; no effect in SPOC1 cells ND

∗Not determined.

regarding the regulation of mucin secretion in the airways, it is important to bear in mind the limitations of their use. A common notion, which arises from the extensive submucosal gland system of the cartilaginous airways, is that mucous cells supply the greater volume of mucin from intact tissues. Consequently, it is generally assumed in studies on airway explants that the mucins measured emanate primarily from mucous cells. This presumption, however, may be incorrect on two counts. First, recent morphometric studies in the cartilaginous airways of monkeys have shown the goblet cells in the superficial epithelium to harbor a greater volume of secretory material than mucous cells, despite their lower cell number.42–45 The reason for this greater volume may, in fact, be appreciated from the AB/PAS-stained section of canine trachea shown in Figure 1 by noting individual goblet cells to be far more robust than mucous cells. Second, it appears likely that mechanisms exist which couple the secretion of goblet cells to that of submucosal glands in the intact tissue. Good examples of this possibility are studies of vagus nerve stimulation on mucin release which show both mucous cells and goblet cells to respond despite the lack on extensive innervation of the superficial epithelium.46–48 Similarly, goblet cells respond, in vivo, to capsaicin,49 despite a poor innervation of the superficial epithelium by the NANC nervous system. Thus, the release of mucins

from intact airways following submucosal gland stimulation may very well include, or be dominated by material released from the superficial epithelium. Interestingly, the crosstalk between submucosal glands and the epithelium may be bi-directional: studies with isolated glands (below) have indicated that the epithelium elaborates a factor which inhibits mucouscell mucin production.50,51 As a consequence of this potential crosstalk in tissue-level studies, conclusions as to mechanism of action for mucin secretion in the airways requires the use of experimental preparations in which submucosal glands and/or superficial epithelium are studied in isolation.

SUBMUCOSAL GLANDS Isolated submucosal glands Isolated submucosal gland preparations have been developed in which the intact glands are isolated individually and studied intact, or isolated en masse and studied as dispersed cells. The intact preparation, developed by Shimura and colleagues52 has been the more useful and has yielded a large body of physiological information (for review, see53). Table 1 summarizes some of this information, and shows that intact submucosal gland mucin secretion is elicited

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with a wide range of secretagogues. Note that the intact gland preparation may be used to distinguish mucin secretion, per se, from the ejection of mucin by glandular contraction.52 While the dispersed cell preparation has seen a more limited use, it has been used to establish the time-course of mucin release from mucous cells during cholinergic stimulation.28 The remarkable findings from this study, were (i) the rapid onset of the mucous cell response to 30 second agonist exposures, and (ii) the relatively brief period of time (<5 minutes) mucin secretion occurred during continuous agonist exposure before apparent receptor desensitization terminated the response.

Primary cell cultures and cell lines Primary cultures of submucosal gland secretory cells have been available for several years, from which cell lines are readily derived by passaging.26,33,54,55 In studies with cells grown on human placental collagen-coated supports, the cultures possess cells exhibiting a mixed serous-mucous cell phenotype;55 however, after several passages, only serous cells appear to survive and these serous cell lines secrete proteoglycans, not mucins.33 The cells resulting from submucosal gland culture may be sensitive to the type of connective tissue matrix used as a substratum, since when grown on type IV collagen (Vitrogen) the cultures possess cells with a mucous-cell phenotype.56 Unfortunately, the secretion of mucins in these cultures has not been studied. Serous cells have been found to respond to adrenergic, cholinergic, and purinergic agonists, several inflammatory mediators, and to elastase and other mast cell proteases.57–60 Recently, a SV-40-transformed serous cell line has been produced from human trachea, which secretes SLPI in response to challenge with carbachol, isoproterenol, and ATP.61

SUPERFICIAL EPITHELIUM More effort has been applied to developing culture techniques for the superficial epithelium than for submucosal glands, with the result that more experimental models have been developed and a correspondingly larger pool of data has been derived from their use.

Epithelial explants Our laboratory found that cm-sized sheets of superficial epithelium could be removed intact from large airways62 and human turbinates63 and transferred

to a nitrocellulose substratum to which the cells reattach. Figure 2 shows two examples of mucin secretion from these epithelial explant cultures following stimulation by luminal ATP, using video microscopy and lectin binding assays as detection systems. Note that the patterns of purinergic-stimulated exocytosis and mucin release (Fig. 2A and C) are complementary: (i) a burst of exocytotic activity occurs immediately following agonist exposure, and mucin release rises rapidly to a peak, and (ii) the vigorous, early exocytotic response is followed by a slower, plateau rate that eventually stops, and mucin release correspondingly declines. The decline in mucin release is apparently due to desensitization of the response, since reapplication of ATP following a brief respite results in a second mucin secretory response. Note also the similarities between the dose-effect curves for ATP that result from the two assays, each giving a K0.5 in the low lM range. Epithelial explants are an attractive model because the goblet cells studied presumably reflect a phenotype that developed under the influence of a host. Working with explants, however, is very labor-intensive: the dose-effect curves depicted in Figure 2B and D, for instance, were generated over periods of approximately 5–6 and 3 months, respectively. Consequently, primary culture systems or cell lines may represent more efficient models with which to conduct basic research on the mucin secretory process in the superficial epithelium.

Primary cell cultures Airway epithelial cells were initially cultured in FBScontaining medium in 1980,64 and in serum-free, defined media in 1985.65 The latter effort has given rise to the five systems presently in use and for which mucin assay procedures have been developed, as listed in Table 2. It is commonly assumed, if implicitly, that the cells which develop in primary cultures of airway epithelial cells reflect the superficial epithelium from which the cells originated. As demonstrated in Figure 1, digestion of airways tissue with protease XIV (pronase), indeed, rather selectively removes the cells of the superficial epithelium leaving the remainder of the airway tissue, including the submucosal glands, intact. It should be noted, however, that when grown in tracheal xenografts in either syngeneic or immunecompromised hosts these proteolytically-derived cells develop into epithelia containing gland-like invaginations (S. H. Randell, personal communication). Hence, the progenitor cells in the superficial epithelium which give rise to submucosal glands are most likely present in primary cultures, and one cannot rule out the possibility that at least some of the mucin-secreting

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Fig. 2 Effects of ATP on canine tracheal goblet cells. Panels A and C depict the results of individual experiments in which mucin secretion was monitored as individual exocytotic events by video microscopy (A) or by SBA-ELLA (C). In A, experiments on two separate explants show the effects of either 1 or 10 l luminal ATP. In C, ATP (100l) was applied to the luminal solution perfusing an explant during the periods indicated by the bars. Panels B and D depict the dose-effect relationships derived from video microscopy (B) or by SBA-ELLA (D). In B, the total number of degranulations observed over a 10 minute period at each dose is shown, whereas, in D the integrated, suprabasal responses during 40 minute exposures are plotted.

Table 2 Primary cell culture systems derived from superficial epithelium. Tissue

Composition of columnar cells in mature cultures∗

References

Hamster trachea Guinea pig trachea Rat trachea Cat trachea Human trachea and bronchus

Mucin-secreting cells only Ciliated, mucin-secreting Ciliated, mucin-secreting Mucin-secreting cells only Ciliated, mucin-secreting

11,65,66,92–94 30,95 96,97 29 76,77,98,99

∗At the time the cells are generally used for experimentation.

cells which develop therein are mucous, and not goblet, cells. This potential problem notwithstanding, primary cultures of airway cells have been extremely important in developing our present understanding of goblet cell function. In primary cultures of hamster tracheal epithelial cells, which have been studied for the longest period of time and by several laboratories, a progression in cell composition is observed with time in culture, from undifferentiated to secretory to a mixed ciliated and secretory cell population; this progression reflects the development of the epithelium in the host.66,67 The

development the airway epithelium in the mammalian lung, however, is complicated and the inter-relationships between unidentified progenitor cells and ciliated, goblet, and basal cells are not clear.68–72 Despite the uncertainties in developmental relationships, there is good agreement that goblet cells arise from an intermediate secretory cell.72–74 In the cultures derived from guinea pigs, rats, and humans and used for studies of mucin secretion, the columnar cells are comprised of both ciliated and goblet cells, suggesting that cultures are ‘mature’. In contrast, in cultures derived from hamster and cat airways, ciliated cells

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are absent when studied for mucin secretion.75 Because these cultures are used before acquiring a mixed ciliated and secretory cell phenotype, it is not clear whether the mucin-secreting cells therein represent mature goblet cells, or an intermediate secretory cell type. It is also unknown how intermediate secretory cells and goblet cells compare in terms of mucin secretory activity and its regulation. An important recent advance in epithelial cell culture has been the use by two laboratories of passaged primary cultures∗ of human airway epithelial cells.76,77 These culture systems have the obvious advantage of allowing the expansion in cell number and freezing of precious human cells, and consequently they are very likely to prove important tools in our developing knowledge of airway cell biology. One drawback of these cultures, however, is the change in phenotype which may occur with continued passaging. Gray et al77 found, for instance, that the ability of the cultures to develop a transepithelial electrical potential and to respond to purinergic stimulation with mucin secretion declined beyond passage 2. Because of these possible cell culture artifacts, the successful use of these passaged primary cell cultures in physiologic and pharmacologic investigations will depend critically upon good time-based control studies. Primary cultures of airway epithelial cells have proven useful in recent years in understanding the agonist regulation of goblet cell mucin secretion. In fact, most of the data on the regulation of goblet cell mucin secretion summarized in Table 1 were derived from experiments which employed these cultures.45 As may be appreciated from the table, goblet cells respond to few of the secretagogues which elicit mucin secretion from submucosal glands. Hence, an important advance which has occurred over the past five years in understanding the regulation of goblet cell secretion was the recognition of nucleotide triphosphates (ATP and UTP) as potent mucin secretagogues: mucin secretion from goblet cells in primary epithelial cell cultures derived from the airways of hamsters,78 rats,32 and humans77 is stimulated by these agonists (see Fig. 3). Primary cultures have also been used to study the effects of lipid mediators on goblet cell mucin secretion, with most of the work focusing on platelet activating factor (PAF). In cultures derived from both guinea pig and feline airways, PAF has been shown to elicit mucin secretion in a dose-dependent manner.29,79 Interestingly, in both cases PAF-induced mucin release was blocked not only by PAF receptor antagonists, but also by NDGA, an cyclooxygenase and lipoxygenase inhibitor suggesting that the effects of PAF to elicit mucin secretion are indirect. In guinea ∗ Passaged primary cell cultures are frequently termed, ‘secondary cell cultures’.

pig epithelial cell cultures, HETE production was induced by PAF, and mixtures of exogenous 5-, 12-, and 15-HETEs stimulated mucin secretion—but not by the individual HETEs.79 Most recently, the effects of PAF, as well as reactive oxygen species, was found to be blocked by L-NMA, the inhibitor of nitric oxide synthase suggesting that NO may be an active mediator in the train of events which leads to mucin secretion.80,81 The precise role of NO in this process, including the cell type(s) in which it functions, however, remains to be established.

Cell lines At present, a single mucin secreting cell line has been derived from superficial epithelium, the SPOC1 cell, which arose as a polyclonal line from spontaneous immortalization events which occurred in passaged primary cultures of rat tracheal epithelial cells. The cell line is diploid with minor and stable alterations of chromosomes 1, 3, and 6, and it has reduced requirements for peptide growth factors.82 Importantly, SPOC1 cells assume a goblet cell phenotype when grown in tracheal xenografts, i.e., the xenografts develop a pseudostratified epithelium comprised of a layer of basal-like cells underlying columnar cells which contain large numbers of dense-core secretory granules. The granules are positive for both carbohydrate and the rat tracheal mucin-specific mAb, RTE-11.83 When grown in culture SPOC1 cells develop a multi-layered epithelium over the period of 6–7 days post-confluence comprised, chiefly, of a layer of cuboidal, basal-like cells overlaid by cuboidal to columnar cells which stain positive with AB/PAS and immunostain with RTE-11. Small portions of the cultures will develop instead as a squamous epithelium. During this period, a transepithelial electrical potential difference and resistance also develop, which is indicative of a polarized epithelium.32,82,83 The HMWG secreted by cultures of SPOC1 cells has been shown by multiple biochemical criteria to be mucin, and the rate of mucin secretion is enhanced by purinergic agonists.32 Thus, SPOC1 cells are similar to airway goblet cells by several morphological, biochemical criteria. Importantly, they have proven to be similar physiologically, also, at least to the extent that mucin secretion is stimulated by purinergic agonists23,95 (Fig. 4). It is notable, therefore, that these cells do not respond to many agonists which elicit mucin secretion from mucous cells in submucosal glands (Table 1). Clearly, these findings need to be verified in other experimental models before we may conclude that goblet cells are not affected by these agents, but these data do support the notion that mucin secretion from the superficial epithelium and submucosal glands may be regulated differently.

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Fig. 4 Dose-effects relationships for ATPcS and UTP on mucin secretion by SPOC1 cells. The cells were grown in two 48-well plates and exposed to the indicated doses of nucleotide for 40 minutes following which the medium was assessed for mucin by SBA-ELLA. The data from the two experiments are presented as the mean values ±SE of wells studied in triplicate.

PURINERGIC REGULATION OF MUCIN SECRETION IN THE AIRWAYS The extracellular signaling activities of ATP were well-established by 1950,84 but another 35 years were

required for its actions on surfactant secretion in the lung to be discovered.85 In 1991, ATP and UTP, luminally applied, were found to stimulate Cl− secretion in cultures derived from tissues removed from both control and cystic fibrosis patients,86,87 and this

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finding precipitated the current investigations into the effects of nucleotides on mucin secretion in the airways. The effects of ATP on mucin secretion was described initially by Kim and Lee working with primary cultures of hamster trachea epithelial cells78 and by our laboratory working with epithelial explants of canine trachea.62 Subsequently, ATP has been shown to stimulate mucin secretion in human77 and rat32 in vitro systems using cells from the airway superficial epithelium (), as well as in isolated airway submucosal glands.88 That the receptor mediating these effects on mucin secretion might be the P2U purinoceptor† was first indicated by the finding with epithelial explants from human turbinates that UTP and ATP were equipotent.63 Recent evidence from laboratories working with SPOC1 cells32 and primary cultures of hamster trachea epithelial cells89 has strengthened this possibility with the findings that the P2U purinoceptor mRNA is expressed. In fact, these are the first data available for goblet cells regarding the expression of a specific receptor. † The P2U purinoceptor receptor.100,101

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In vitro models have also been useful in determining the intracellular pathways which mediate the purinergic regulation of mucin secretion in goblet cells. Using primary cultures of hamster trachea epithelial cells, in which goblet cells (or intermediate secretory cells) are the predominant cell type, Kim and colleagues have shown that ATP elicits the production of inositol phosphates, thereby suggesting activation of phospholipase C.90 More recently, as summarized in Figure 5 our laboratory has shown that SPOC1 cell mucin secretion is elicited by agents which elevate intracellular Ca2+ (ionomycin and thapsigargin) or activate protein kinase C (PMA). Additionally, we found that PKC activity was enhanced in these cells not only by PMA, but also by UTP. This latter result suggests that PKC activation may have a physiologic role in the purinergic regulation of mucin secretion.91

CONCLUSION In vitro models have played a central role in the process of understanding the regulation of mucin secretion in the airways. Initially, the various model

In Vitro Models for Airways Mucin Secretion

systems allowed the different cellular sources of mucin in the airways to be studied independently, and they are now allowing the identification of the molecular pathways which underlie the secretory process and its regulation. It will be crucial, as these model systems continue to develop, to probe in vivo function in a parallel fashion since it is always difficult to determine a priori whether a particular in vitro function possesses a homologous counterpart in the whole animal. Acknowledgements The authors thank Drs Paul Nettesheim and Scott Randell for their valuable contributions to both their work and this review. The work in the authors’ laboratory discussed herein has been supported by the Cystic Fibrosis Foundation (USA), the American Lung Association of North Carolina, and Glaxo Wellcome Corporation. References 1. Plopper C G, Mariassy A T, Wilson D W, Alley J L, Nichio S J, Nettesheim P. Comparison of nonciliated tracheal epithelial cells in six mammalian species: ultrastructure and population densities. Exp Lung Res 1983; 5: 281–294. 2. Harkema J R, Mariassy A, St George J A, et al. The airway epithelium: physiology, pathophysiology, and pharmacology. In: Farmer S G, Hay D W eds. Epithelial cells of the conducting airways. NY: Marcel Dekker, 1991; 3–39. 3. Mariassy A T. Comparative biology of the normal lung. In: Parent R A ed. Epithelial cells of trachea and bronchi. Boca Raton: CRC Press, 1992; 63–76. 4. Plopper C G, Hyde D M. Comparative biology of the normal lung. In: Parent R A ed. Epithelial cells of bronchioles. Boca Raton: CRC Press, 1992; 85–92. 5. Thurlbeck W M, Malaka D, Murphy K. Goblet cells in the peripheral airways in chronic bronchitis. Am Rev Respir Dis 1975; 112: 65–69. 6. Hogg J C, Macklem P T, Thurlbeck W M. Site and nature of airway obstruction in chronic obstructive lung disease. N Engl J Med 1968; 278: 1355–1360. 7. Kung T T, Jones H, Adams 3rd G K, Umland S P, Kreutner W, Egan R W, Chapman R W, Watnick A S. Characterization of a murine model of allergic pulmonary inflammation. Int Arch Allergy Immunol 1994; 105: 83–90. 8. Jeffery P K. Comparative morphology of the airways in asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1994; 150: (Pt 2): S6–13. 9. Florey H, Carleton H M, Wells A Q. Mucus secretion in the trachea. Brit J Exp Pathol 1932; 13: 269–284. 10. Carlstedt I, Sheehan J K, Corfield A P, Gallagher J T. Mucous glycoproteins: a gel of a problem. Essays Biochem 1985; 20: 40–76. 11. Silberberg A. Mucus glycoprotein, its biophysical and gelforming properties. Symp Soc Exp Biol 1989; 43: 43–63. 12. Jentoft N. Why are proteins O-glycosylated? Trends Biochem Sci 1990; 15: 291–294. 13. Verdugo P. Goblet cells secretion and mucogenesis. Ann Rev Physiol 1990; 52: 157–176. 14. Lamblin G, Lhermitte M, Klein A, Houdret N, Scharfman A, Ramphal R, Roussel P. The carbohydrate diversity of human respiratory mucins: a protection of the underlying mucosa? Am Rev Respir Dis 1991; 144: (Pt 2): S19–24. 15. Sheehan J K, Thornton D J, Somerville M, Carlstedt I. Mucin structure. The structure and heterogeneity of respiratory mucus glycoproteins. Am Rev Respir Dis 1991; 144: S4–9.

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Date accepted: 1 October.