MITOTIC RATES, GOBLET CELL INCREASE AND HISTO- CHEMICAL CHANGES IN MUCUS IN RAT BRONCHIAL

OXIDE EPITHELIUM DURING EXPOSURE TO SULPHUR DI-

DAVID LAMB AND LYNNE RE~D Institute of Diseases of rhe Chest, Brompton Hospital, London

PLATES XXI AND XXII

THE stigmata of human chronic bronchitis are hypertrophy of the mucous glands deep in the bronchial wall and a marked increase in the number of epithelial goblet cells, particularly in the peripheral small airways, where they are usually sparse. An increase in bronchial goblet cells beyond the normal has been produced without infection by allowing rats to inhale sulphur dioxide (Reid, 1963). In rats there is a difference in the histochemical nature of the intracellular mucus at different levels in the tracheo-bronchial tree. In the trachea the mucus is sulphated, in the proximal airways a mixture of sulphomucin and sialomucin is produced, whilst more peripherally all goblet cells produce sialomucin alone (McCarthy and Reid, 1964a).

The purpose of the experiments here reported was to discover whether epithelial destruction precedes the development of goblet cells and whether there is any increase in mitotic count; to measure gland hypertrophy, and to ascertain whether in the newly appearing goblet cells the distribution of acidic mucins is similar to the normal. A further objective was to find out whether the new goblet cells per- sisted, and whether there were further histochemical changes after the irritant was removed.

MATERIALS AND METHODS

Fortyeight rats bred from pathogen-free rats (Anticimex Stockholm) were used for this investigation. The rats were divided into three groups, one of sixteen, one of fourteen and one of eighteen. Each group was used for a different experi- ment and exposed for the periods shown in table I.

In the first two experiments the timing of exposure was so arranged that in each experiment all animals were killed at the same time, roughly 20 hr after their last exposure to sulphur dioxide. For mitotic counts three animals were used from each exposure time in the first experiment and one from each exposure time in the second experiment. The remaining animals were used for studying the uptake of radioactive sulphate. In the third experiment all were used to investigate the persistence of the changes. Three were kept as controls and the remaining 15 were exposed to SO2 for 3 wk only, after which groups of three animals were killed 1 day and 4, 7, 14 and 35 days after exposure.

The irritant, sulphur dioxide, was applied as previously described (Reid, 1963) at a concentration of about 400 p.p.m. for periods of 3 hr a day, 5 days a week.

I. PATH. BACT.-VOL. 96 (1968) 97 G

98 DAVID LAMB AND LYNNE REID

, I

1 , 4

2 3 2 1 3 : 3

15 3 3 O ( O I

I - -__--

The concentration was determined by withdrawing samples from the chamber through fine polythene cannulae distributed throughout the chamber and kept permanently in position. The air withdrawn from the chamber was absorbed into H202 solution and titrated against 0 . 0 2 ~ borax solution; Concentrations varied between 380 and 450 p.p.m.

Between exposure periods the exposed and control animals were kept in adjacent cages under identical conditions of feeding, supply of drinking water, cage cleaning and ventilation. All animals were weighed daily. The animals were killed by intraperitoneal injection of 1-2 ml. Nembutal solution (Pentobarbitone Sodium, B.P., 60 mg. per ml., Abbott Laboratories Ltd). For mitotic counts

TABLE 1

Plan of experiments

I I--- -

16

3 1 14

18 I

animals were given an injection of 0.1 mg. colchicine per 100 g. body weight 4 hr before they were killed; animals for histochemical study were given, 4 hr before they were killed, an intraperitoneal injection of 5 mc of radioactive sulphate (Jsso4) in aqueous solution, carrier free and sterile, at pH 6.5-75, per kg. body weight; the 35so4 was supplied by the Radiochemical Centre, Amersham.

Preparation of lungs. The lungs and trachea were removed intact, the trachea being ligated just below the larynx before the chest was opened in order to prevent contamination with blood during removal. The lungs and trachea were fixed to pieces of card with thread, so as to maintain the normal laryngo-hilar distance. About 2 ml. of fixative were then injected into the trachea and the specimen was immersed in fixative. Heidenhain's Susa solution was used to fix lungs for mitotic counts and buffered formol-saline for lungs studied histochemically. After fixation overnight the carina and attached left lung were embedded in one block. From the upper third of the trachea 3 blocks were cut transversely, and the remainder of the trachea was embedded to be cut in longitudinal section. The blocks were embedded in paraffin and 4p sections cut. For mitotic counts and general histo- logical examination sections were lightly counterstained with eosin after Weigert's haematoxylin, as this accentuates the nuclei in mitosis. For study of the mucus, the periodic acid-Schiff (PAS) method both by itself and with alcian blue (AB/PAs) was used. Materials for both these methods were supplied by George T. Gum Ltd, the alcian blue batch number being 08247.

Mito/ic counts. In counting cells in mitosis only those in prophase or metaphase were included, later stages of mitosis being ignored to prevent confusion with pyknotic nuclei, lymphocytes migrating across epithelium and goblet cell nuclei. The number of nuclei in mitosis was expressed as a percentage of the total nuclei counted. For each bronchial region studied (see below), at least 4OOO nuclei were counted, representing for each region all nuclei from several sections.

In the growing animal the mitotic count represents the increase in size of the organ as well as the replacement of cells lost from normal wear and tear (Bertalanffy and Lau, 1962). Bertalanffy and Lau suggest that the percentage of mitoses

SULPHUR DIOXIDE BRONCHITIS IN RATS 99

representing growth can be assessed from the increase in body weight, reckoning the percentage increase in size of an organ over a given time as the same as the percentage increase in total body weight. In these experiments the weight gain of each rat over a 4-hr period was deduced from the weight gained over the previous week. This 4-hr gain was then expressed as a percentage of the weight at the beginning of the week and subtracted from the gross mitotic percentage for the 6hr period. If no weight was gained, then the gross mitotic percentage was used without adjustment.

Peripheral atrways

FIG. 1 .-Diagrammatic representation of the trachea, main bronchus and axial pathway in the left lung of the rat, showing the four regions in which histological features have been assessed.

Mitotic counts were made separately in each of the following regions (fig. 1). 1. Upper trachea. 2. Main bronchus-below carina and proximal to the first branch, and having

cartilage in its wall. 3. " Proximal airways "-lying distal to bronchi with cartilage in the wall

and proximal to region 4. 4. *' Peripheral airways "-if possible, the last 1.5 mm. of an airway seen in

ideal longitudinal section before it opens into a respiratory bronchiole; if not, cross-sections of airways less than 0.3 mm. in diameter.

In addition, in the tracheal glands, mitoses were counted separately for particular

5. Ducts-including all that part of the gland that has an obvious lumen and

6. Acini-groups of cells staining pink with AB/PAS and having canaliculi

StNCtureS.

stains dark blue with ABIPAS.

between the cells but no proper lumen.

100 DAVID LAMB A N D LYNNE REID

Trached gland size. The tracheal glands are patchy in their distribution, lying mainly between the cartilage rings. To assess the increase in their size, we measured their length and thickness, as Seen in cross-section of the trachea (see fig. 2). From each of the three blocks of the upper third of a trachea we examined sections at 7 levels 4Op apart, i.e., 21 sections in all. From each section, we determined the maximum length and the maximum thickness of a single gland (see fig. 2). The mean of the findings for the twenty-one sections as well as the maximum for each measurement taken from each section were used for comparison between control and exposed animals.

Control After SO1 exposure

FIG. 2.-Diagrammatic repksentation of the submucosal glands in the rat trachea. The dotted lines show the sites at which measurements were made. After SO2 exposure gland depth and length are both increased.

Goblet cell density. Goblet cell density is expressed by number per high-power microscopic field, i.e., per 0.3 mm. of wall. Goblet cells were counted in consecutive fields, and as far as possible, each value given is the mean of the findings of twenty fields.

Hisrochemistry of goblet cells. Sialomucins were localised by staining adjacent sections, one of which had been pre-treated at 37°C for 18 hr with sialidase (Spicer and Warren, 1960), with AB/PAS (Mowry, 1956). The sialidase was receptor- destroying enzyme (RDE) from Vibrio cholerae (Burroughs Wellcome and Co., London). If the cells contain sialic acid susceptible to sialidase, the blue staining of goblet cells with AB/PAS stain, indicative of acid glycoprotein, is lost after sialidase pre-treatment, the dark blue giving way to shades of red or red-purple. Acid glycoprotein resistant to the enzyme may contain, as acidic groupings, sialic acid not susceptible to sialidase, or sulphate radicles.

The assessment of these changes was made for the same anatomical regions as for the mitotic counts. Because of the findings a more detailed analysis of the proximal airways was made.

Airtortzdiography and sulphate uptake, Sulphomucins were localised by auto- radiography after injection of radioactive sulphate into the animals. For auto- radiography Kodak AR 10 stripping film was mounted over PAS-stained 4p sections and exposed at 4°C in the dark for 4 wk. Development was carried out at 20'C for 5 min. with Kodak D-19b developer, and fixation for 15 min. in FIX-SOL (Johnson) diluted 1 in 5. After one hour's washing in tap water, sections were dried and mounted in Canada balsam. The silver grain density was measured on the autoradiograph by comparison with standard density charts ranging from 10 to 5 0 0 grains per unit area.

SULPHUR DIOXIDE BRONCHITIS IN RATS 101

RESULTS Histological changes in airways

Normal airways. The rat trachea (fig. 3) and main bronchi are lined by pseudostratified ciliated columnar epithelium, which becomes progressively thinner peripherally until, in the distal two-thirds of the airways, the epithelium consists of a single layer of cells, at first low columnar, more peripherally cuboidal. Goblet cells are present in the trachea and more proximal airways, whereas in the distal one-third or so of an airway of the normal rat no goblet cells are found. In the wall of the trachea there are mixed sero-mucous glands, opening by ducts on to its surface and lying mainly between the rings of cartilage. These glands are larger and more numerous in the proximal part of the trachea, and are not found in the main bronchi or beyond.

Airways after exposure. After 2 days’ exposure to sulphur dioxide cilia are lost throughout the trachea and main bronchi and patchily in proximal airways. The normal epithelium in these regions is replaced by one or two layers of small rather flat cells with an irregular but mainly horizontal alignment. The presence of pyknotic nuclei and the separation of cells one from another suggest cell death. The most peripheral airways show no histological evidence of damage.

After 4 days’ exposure these epithelial changes are even more severe in proximal airways, there being fewer cells. This is the time when mitoses are very obvious (fig. 4). The peripheral airways are still normal in appearance.

By 3 wk the epithelium in the more affected regions appears rather thicker than normal, having several layers of cells. Areas of flat regenerating epithelium are still present. Cilia, shorter than are normally seen, are irregularly distributed in main bronchi. There is marked variation in appearance, airways showing patches of flat regenerating epithelium giving rise to side branches showing no damage. By 6 wk cilia have returned to parts of the trachea, but they are still stunted and the epithelium is still thicker than normal (fig. 5). At no time is the epithelium completely denuded nor is there evidence in the submucosa of severe inflammation. After 4 days’ exposure, however, an occasional polymorphonuclear leucocyte can be seen migrating through the epithelium. The submucous glands of the trachea show no evidence of damage, the ciliated cells of even the main ducts appearing normal throughout.

In the animals exposed to sulphur dioxide the number of mitoses is significantly increased in the trachea, main bronchi, proximal and distal airways, the counts for the distal airways being lower than for the other 3 regions (fig. 8). By 4 days a large number of mitoses is present in the trachea, main bronchi and proximal airways; the difference in mean values between these sites is not statistically significant. In peripheral airways the number of mitoses

Mitotic counts.

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- 7 0.8 c - U =I c c 0 W 0.4 e, c u W a Y

seen is significantly less than in more proximal airways and is not significantly increased over levels in control animals.

By 3 wk the mitotic counts in trachea, main bronchi and proximal airways have fallen from the high values seen after 4 days’ exposure,

-

Gland ducts

Gland acini

-. --. ---------____ ,-----L ---- -

Distal airways *--- --------_______ ? 1 2 3 4 5 6

Non-exposed animals

Length of SO1 exposure (wk)

FIG. 8.-The mtiotic count (4-hr period) after SO? exposure for periods up to 6 wk. The mitoses are represented as a percentage of the total nuclei counted. In the nonexposed animals the counts are low at all levels in the bronchial tree. At 1 wk after acute damage, there is a great increase in mitotic counts in the trachea and large airways; by 3 wk even here the number has fallen. In the distal airways the increase is scarcely significant.

Non-exposed animals

Length of SO1 exposure (wk)

FIG. 9.-The mitotic count (as in fig. 8) for the gland ducts and acini. The increase is slight but significant and persists.

but they are still six times the control levels, and still significantly higher than the counts in distal airways, in which the figure is the same as at 4 days. After 6 weeks’ exposure the mitotic counts in trachea and main bronchi are the same as after 3 wk: in proximal airways, however, the counts have fallen slightly. At 6 wk the count

SULPHUR DIOXIDE BRONCHITIS IN RATS 103

in distal airways has fallen below the 3-wk value and to a level below that of the controls. All figures for distal airways are so near zero that analysis of their significance is unsatisfactory.

In the ducts and acini of the tracheal submucosal glands, despite lack of histological evidence of damage, the mitotic counts are in- ’ G L 150;

*.’---# Distal airways

Non-exposed animals

Length of SO1 exposure (wk)

FIG. 10.-Goblet cell density expressed as the number of goblet cells per 5 high-power microscopic fields. In large airways there is a fall during the 1st wk associated with the acute damage and then a steady rise, seen also in distal airways.

Proximal rnm. along proximal airway

FIG. 11 .-Increase in goblet cells along proximal airways.

creased (fig. 9). After 4 days’ exposure, the values are significantly higher than in control animals and these levels are maintained at 3 and 6 wk.

Goblet cell counts. 1rl the fist days of exposure to sulphur dioxide there is a reduction in the number of goblet cells in the trachea and main bronchi, particularly in the trachea (see fig. 10). After 3 wk the

104 DAVID LAMB A N D LYNNE REID

0 1 3 6

number of goblet cells in the regions where they are normally present has increased and, in addition, they have appeared in the distal airways where they are not normally found. By 6 wk they have increased still more in the trachea and proximal and distal airways but not in the main bronchi (figs. 6 and 7). By 3 wk there is a marked increase in size of the individual goblet cells.

A more detailed representation of the increase in goblet cells along the proximal airways is shown in fig. 11, which gives the total for each successive 1.5 mm. length. This shows that the marked increase in goblet cells is found evenly throughout this proximal region.

TABLE I1

Increase in traclieal gland size afer exposure to sulphur dioxide

: 11.8 19.8 1 1-1 1.8 13 22 1.3 1.75 15.6 24.5 1.4 2.0 21 32 1 1.7 2.2

Tracheal gland i Length of j

exposure (wk) length (mm.) thickness (mm.)

I

I I Mean i Maximum i Mean ’

Seven levels on 3 rings of trachea measured for each case.

Increase in tracheal gland size. The mean and maximum values for the length and thickness of the tracheal glands are shown in table I1 for each exposure period. The gland size increases proportionately to the time of exposure to sulphur dioxide. The mean values give statistically rather better correlations than the maximum values. After 3 weeks’ exposure to SO2 the mean increase in both length and thickness is significant (P <0.01).

Histochemical properties of intracellular inucus In the normal rat there is a characteristic distribution of goblet

cells with different types of intracellular mucus (McCarthy and Reid, 1964~) . In the trachea goblet cells containing acid glycoprotein are resistant to sialidase; in bronchi and upper proximal airways they are partly susceptible and in more distal parts of proximal airways they are completely sensitive to sialidase. These findings were confirmed by the results in the present investigation (see results for the non- exposed animals in fig. 12).

After only 4 days’ exposure to sulphur dioxide there is marked increase in the proportion of goblet cells producing acid glycoprotein.

LAMB AND REID PLATE XXI

SULPHUR DIOXIDE BRONCHITIS IN RATS

FIG. 3.-Tracheal epithelium from control rat. Susa, Weigert’s haematoxy l in - eosin (WHE). x 330.

FIG. 4.-Bronchial epi- thelium (main bron- chus) from rat after 4 days‘ exposure to sulphur dioxide. Two layers of flattened epi- thelium with no cilia or goblet cells and several mitoses. Susa, WHE. ~ 3 3 0 .

FIG. 5.-Bronchial epi- thelium (main bron- chus) from rat after 6 weeks’ exposure to sulphur dioxide. Tall epithelium with goblet cells and a cell in mitosis; a layer of mucus is apparent on the surface. Susa, WHE. x330.

LAMB AND REID PLATE XXII

SULPHUR DIOXIDE BRONCHITIS IN RATS

FIG. 6.-Epithelium from proximal airway of control animal. Only an occasional goblet cell (black). Buffered formol-saline, periodic acid-Schiff. x 475.

FIG. 7.-Bronchial epithelium from rat exposed to sulphur dioxide for 6 wk. Numerous goblet cells (black), each larger than in the normal. Buffered formol-salinc, PAS. x 475.

SULPHUR DIOXIDE BRONCHITIS IN RATS 105

There is also a marked change in the susceptibility of the acid glyco- protein to sialidase (see fig. 13). In regions where the goblet cells are normally sensitive to sialidase, as in proximal airways, a marked increase in resistance can be detected. In fig. 13 a more detailed

'0

L _ _ _ L ---- I

,' Proximal airways

Distal airways

I

c 2 Non-exposed Length of SO1 exposure (wk) animals

FIG. 12.-Effect of SO2 exposure on neuraminidase (RDE) susceptibility of rat goblet cells. The ordinate shows the percentage of cells containing acid mucopolysaccharlde (blue with -/PAS stain) resistant to sialidase. At all sites this increases. In distal airways the cells on their first appearance are sensitive.

7 5 7 - - . ... .. . - -

o Control /

/ /

All 3 and 6 weeks' exposure - - 0 ~- - - - .. -- 0 2 4 6 8 10

Proximal mm. along proximal airway

FIG. 13.-Increase in resistance to neuraminidase after SO2 exposure. In control animals most of the goblet cells over the 10 mm. of the proximal airways studied are sus- ceptible; as exposure increases the number susceptible falls so that by 3 wk almost all cells are resistant to the enzyme.

representation of the effect of sialidase on goblet cells is given. After 4 days the reduction in sensitivity is most marked proximally, but by 3 wk all goblet cells in this region are resistant to sialidase.

The findings in the distal airways are of particular interest. In this region there are normally no goblet cells, but as they appear all

106 DAVID LAMB AND LYNNE REID

those containing acid glycoprotein are sensitive to sialidase. Three weeks after, all such cells are resistant to sialidase (see fig. 12).

Uptake of radioactive sulphate In the autoradiograph of the rat lung the grain density over goblet

cells in the trachea and proximal airways is low-10-20 grains per unit

I

t 1 2 3 4 .5 6 Non-exposed

animals Length of SO, exposure (wk)

FIG. 14.-Uptake of radioactive sulphate by goblet cells. The grain density representing uptake of sulphate increases with increasing exposure.

TABLE 111

Number of goblet cells and resistance of their mucins to neuraminidase up to 35 days after exposure to sulphur dioxide has ceased

1 Number or percentage in proximal airways in I

Measurement ~ control ~ exposed animals when interval between exposure and death was non-

! exposed 1 animals 1 1 day 4 days 1 7 days 1 14 days ~ 35 days

I 1 4-8 ? ’ 14.5 ‘ 12.8 12.6 I 10.3 i I

Number of goblet cells I per high-power field

affected by neuram- inidase

Percentage of goblet cells ~ 3 5 1 6 I 1 1

I I I I

area-but after sulphur dioxide exposure for only 2 days the grain density in the trachea is increased nearly 3 times and in proximal airways more than 4 times (see fig. 14). Further increase in radioactive sulphate uptake occurs with increased length of exposure; by 6 wk the uptake by tracheal goblet cells is 10 times that at the control level. In the normal animals the mucous cells of the tracheal glands have

SULPHUR DIOXIDE BRONCHITIS IN RATS 107

well marked radioactive sulphate uptake and this also increases si&- cantly on exposure, until after 6 weeks’ exposure the level is 3 times that in the control animals. This suggests increased activity of individual cells.

Persistence of histological changes In the third group of animals killed at intervals after removal

from sulphur dioxide the proximal airways still show an increase in goblet cell number, which falls only slightly after removal from the irritant (table 111). Over the proximal airways 65 per cent. of cells are susceptible to sialidase in the controls; this falls to 10 per cent. after 3 weeks’ exposure and does not revert to normal over 35 days.

DISCUSSION Decrease in severity of damage peripherally

The acute damage is much more severe in the trachea than in more peripheral airways. This is unlikely to reflect any difference in the resistance of the epithelium to the sulphur dioxide, but is probably due to a reduction in the concentration of sulphur dioxide in the peripheral airways because of its high solubility. The study of Dalhamn and Strandberg (196 1) suggests that sulphur dioxide dissolves so quickly in the tissues of the trachea that its concentration has fallen significantly by the time the main carina is reached.

Recovery In view of the severe damage produced by the initial days of

exposure to the sulphur dioxide it is strange that the epithelium heals and differentiates even with continuing exposure, so that by 6 wk the epithelium is somewhat thicker than normal and the cilia reappear. It may be that the basal cells are more resistant to the sulphur dioxide than is the superficial layer of differentiated cells. During the restoration of the central epithelium there is already an excess of mucus in the bronchial lumen from the glands and from peripheral goblet cells and it may be that this “ protects ” the epithelium if only by absorbing the sulphur dioxide.

In the normal rat or human lung, there are very few goblet cells at the periphery and no evidence of mucus on the tips of the cilia. The cilia themselves beat in a fluid layer (Lucas and Douglas, 1934; Hilding, 1943), which is either transudate or secretion from cells other than mucous cells. After several weeks’ exposure a layer of mucus is often detected in the lung sections.

Mitotic counts The mitotic counts give an estimate of cell turnover, and are a

more sensitive criterion of the damage produced by SO2 exposure

108 DAVID LAMB AND LYNNE REID

than the histological appearances, since the mitotic counts in the distal airways are increased at 1 and 3 wk of exposure, although there is no histological evidence of damage. At 6 wk the fall of the mitotic count in proximal and distal airways supports the histological evidence of a decrease in the damage by sulphur dioxide.

In proximal sites the large number of mitoses seen after one week’s exposure reflects the effect of regeneration following early cell damage. By 3 wk the count stabilises to a level that is maintained at 6 wk; although the histological appearances are still abnormal there is an intact epithelium of several cell layers, suggesting that regeneration exceeds any destruction. It is not possible to say from the techniques used here whether the persistently raised mitotic count arises from a small proportion of cells that bear the brunt of the damage, die and are replaced, or from shortening of the life-span of all cells.

Tracheal mucous glands The rise in the mitotic count in the tracheal glands is not associated

with any histological evidence of cell damage. It may be that as in distal airways the damage is too slight to be detected with the light microscope, or possibly the count reflects the hypertrophy of the gland which occurs during exposure as shown by the measurements of gland size.

The mechanism that produces gland hypertrophy is not known. Florey, Carleton and Wells (1932) have shown that in the cat and dog the tracheobronchial glands secrete in response to irritation of the surface epithelium through a reflex mediated through the vagus. Irritation of the bronchial surface may lead through hypersecretion to gland hypertrophy. Studies with the salivary glands of the rat suggest that irritation of the teeth leads to salivary gland hypertrophy (Wells, 1963).

Goblet cell increase An increase in goblet cells can be produced by other irritants-

formalin (Florey et al.), nitrous oxide fumes (Freeman and Haydon, 1964)-as well as by sulphur dioxide as used here. What is not known is the relation between the severity and duration of damage and the production of new goblet cells. Goblet cells, like other epithelial cells, seem to be susceptible to the acute effects of SO2 exposure, for their numbers fall in the first days of the exposure. They reappear, and in larger numbers than normal, in all areas affected by the early fall, during continued exposure. The increase is least in the trachea where damage is most severe, either because it is not directly related to intensity of damage or because persisting exposure to the irritant does not permit of further differentiation to goblet cells; or the con- tinuing loss of cells may be counterbalanced by new production.

At the periphery of the lung where there are normally no goblet

SULPHUR DIOXIDE BRONCHITIS IN RATS 109

cells, they appear by 3 wk and continue to increase up to 6 wk, by which time they are found further out in the peripheral airways. In this region any early damage is minimal, but the increase continues even when evidence of this is no longer seen. New goblet cells seem to appear and increase in number progressively to the more peripheral regions; but not simultaneously throughout the bronchial tree.

Insofar as there is some increase in mitosis and goblet cell numbers throughout the bronchial tree it may be said that the two are related, but the goblet cell increase is not proportional to the mitotic count. At the periphery, where the goblet cell numbers continue to rise steadily through the 6 wk of exposure, the mitotic counts were only slightly raised, and to significant levels only in the first few days.

Change in intracellular mucus Along airways of the rat the type of intracellular mucus varies;

in the tracheal airways the acid mucopolysaccharide is resistant to sialidase and incorporates sulphate, whereas in the more distal airways the acid mucopolysaccharide is less and is susceptible to sialidase (McCarthy and Reid, 1964a). This has been confirmed in the present study in a different strain of rat.

During the experiments here described this distribution changed with an extension of acid mucopolysaccharide, resistant to sialidase, to the very smallest airways, those in which goblet cells newly appeared. This change in distribution of acid mucopolysaccharide could be detected in 4 days and, save for the most distal goblet cells, was complete in 3 wk.

Is this a new type of cell or is it a change in the type of mucus being produced by a cell? The fact that, at the periphery, when the cells first appear they contain a sialomucin that develops into sulpho- mucin suggests that the intracellular pattern of secretion can change. Because nearly all cells come to resemble each other it seems certain that these changes happen within the same cell.

Exposure to the irritant increases the rate of secretion of individual cells as shown by an increase in uptake of radioactive sulphate and a n increase in its discharge. The former is measured by the increase in density of sulphate uptake and is not therefore simply a question of total cell size, although cell size also increases. It might be that the switch to a sulphomucin reflects an increased rate of synthesis in the cell. Most cells, even those that predominantly produce a sialomucin, take up some sulphate, and under conditions of irritation there may be a shift from the sialo- to the sulpho-mucin. However, this argument, based as it is on the rate of secretion, does not explain the presence of sulphomucins in the trachea when all cells are of low activity.

This change in the type of mucus is seen also with other irritants such as tobacco smoke (Lamb, Passey and Reid, unpublished).

110 DAVID LAMB AND LYNNE RElD

Reversibility of these changes These changes, in particular the increase in goblet cells and the

change in the type of intracellular mucus, do not revert to normal patterns even 5 wk after exposure ceases. Although speedily produced the changes persist in the absence of the external stimulus.

Model of human chronic bronchitis The increase in size of the tracheal submucous glands and the

extension of goblet cells to peripheral airways are typical of the changes in human chronic bronchitis. In human bronchial glands there are also both sulpho- and sialo-mucins, some cells within an acinus con- taining one, others both; and in chronic bronchitis the sialidase- resistant acid mucopolysaccharide is increased (McCarthy and Reid, 19643; de Haller and Reid, 1965), including the sulphomucins (Lamb, unpublished).

Exposure to irritants causes hypertrophy of the bronchial mucous glands and extension of the goblet cells to the periphery, two key features of the lung in human chronic bronchitis (Reid, 1954, 1967); addition of the change in the type of intracellular mucus in rat lung after irritation makes sulphur dioxide bronchitis an even closer model of the human disease.

SUMMARY Rats exposed to sulphur dioxide by inhalation developed a disease

closely resembling human chronic bronchitis; an important feature of it was the relatively slight damage to the peripheral as compared with the central airways.

The mitotic count increased particularly in the first 4 days and in the central airways. In the ducts and gland cells it was also increased. Goblet cells increased at the periphery and centrally, the increase continuing even when the mitotic count had dropped back to nearly normal values. With exposure, cells producing acid glycoprotein increased and those with sulphomucin appeared in much more peri- pheral parts of the airways. New goblet cells seemed first to go through the stage of producing sialomucin; the activity of individual cells as measured by radioactive sulphate uptake increased. The gland size increased, showing that measurable hypertrophy had developed within days.

The presence of the new goblet cells and the change in the histo- chemical nature of the intracellular mucus persisted at least up to 5 wk after exposure.

The work reported in this paper was made possible by grants from the Medical Research Council and from the National Coal Board. We are grateful to Miss Jean Waldron for the charts and to Mr K. Moreman for the photomicrographs.

SULPHUR DIOXIDE BRONCHITIS IN RATS 111

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