The Journal of Pathology

Vol. 116 No. 1

HI STO CHEM I CAL I DENT1 FI CAT1 ON 0 F GLY COP ROTEINS I N P I G BRONCHIAL EPITHELIUM: (a) NORMAL A N D (b) HYPERTROPHIED FROM ENZOOTIC PNEUMONIA

ROSEMARY JONES, A. BASKERVILLE* AND LYNNE REID Department of Experimental Pathology, Cardiothoracic Institute,

Brompton Hospital, London. S. W.3

PLATES I AND I1

BRONCHIAL gland hypertrophy and extension of goblet cells into small airways, with a change in the histochemical nature of the glycoproteins produced by these various cell types, are characteristic of the pathological changes seen in human chronic bronchitis (Reid, 1960; de Haller and Reid, 1965; Lamb and Reid, 1969a). Such changes have been produced experimentally in a variety of animals by exposure to sulphur dioxide (Lamb and Reid, 1968b; Mawdesley- Thomas, Healey and Barry, 1971 ; Baker et al., 1973) to nitrous oxide (Freeman and Haydon, 1964) and to tobacco smoke (Leuchtenberger, Leuchtenberger and Doolin, 1958; Lamb and Reid, 1969b; Jones, Bolduc and Reid, 1973), and by the administration of isoprenaline and pilocarpine (Sturgess and Reid, 1973). Each of these techniques provides an animal " model " of the human disease, in which gland and goblet cell changes can be studied experimentally.

The role of infection as an initiator of chronic bronchitis in man is uncertain and a direct causal relationship between infection and bronchial gland hyper- trophy has never been established either in man or in experimental animals. However, in the course of studies on naturally occurring and experimentally induced enzootic pneumonia of pigs, a common disease caused by mycoplasms (Goodwin, Pomeroy and Whittlestone, 1965; Gois, Valicek and Sovadina, 1968), it was found that the bronchial gland to wall ratio (Reid, 1960) was increased (Baskerville, 1972). The pig thus offers not only an additional model for experiments on chronic bronchitis (it has moreover a lung volume similar to that of man and is suitable for certain tests of pulmonary function) but, more importantly, it is the first naturally occurring lung disease in animals in which

Received 8 May 1974; accepted 10 June 1974. The Veterinary Research Laboratories, Stormont, Belfast.

* Present address: Microbiological Research Establishment, Porton Down, Salisbury, Wiltshire. 1. PATH.-VOL. 116 (1975) 1 A

2 ROSEMARY JONES, A. BASKERVILLE AND LYNNE REID

the gland changes of chronic bronchitis are recognised as being related to infection. Since hypertrophy of the submucosal gland occurs in airways with no evidence of invasive infection, this raises the question of the cause of the gland hypertrophy.

This study describes the histochemistry of the normal bronchial submucosal gland of the pig and the changes which occur in the hypertrophied gland of pigs with enzootic pneumonia. Other epithelial changes occurring in pigs with enzootic pneumonia are described, including the increase in height of the bronchial epithelium, the distribution of goblet cells and their glycoprotein content.

MATERIAL AND METHODS

This study is based on the same material as that on which Baskerville (1972) reported bronchial gland hypertrophy. From the tissue available from his original group of 56 pigs, tissue including lobar bronchi from the anterior lobes was selected at random from four control and eight experimental animals"-wk-old large white pigs, male and female. The animals were reared conventionally at the Veterinary Research Laboratory of the Ministry of Agriculture for Northern Ireland, Belfast, in a minimal disease herd in which pneumonia had never been detected. All pigs included in the experiment were housed in special indoor accommodation in an isolation block, were fed a standard ration of pellets containing no antibiotic, and were allowed water ad libitum. Animals were inoculated intranasally with Mycoplasma hyorhinis from a pool of supernatant fluid obtained by centrifugation of ground infected pig's lung tissue and stored in the gas phase of nitrogen as described by Baskerville (1972). Control pigs were kept under the same environmental conditions. Three, four and five weeks after inoculation animals from both groups were killed.

At necropsy the larynx, trachea, lungs and heart were removed en bloc and the great vessels of the heart were ligated. The lungs were then fixed by infusion of 10 per cent. buffered neutral formal saline via a cannula tied in the trachea. After fixation for 1 h. in this way blocks were excised from intrapulmonary portions of all lobar bronchi of both lungs and further fixed in the same fixative. Sections from paraffin-wax embedded tissue were cut at 4 pm.

To identify the type of glycoprotein in the submucosal gland and goblet cells of the surface epithelium each of three consecutive tissue sections was stained in one of the following ways: (i) ABpH 2-6-PAS, (ii) sialidase ABpH 2.6-PAS, (iii) ABpH 1.0-PAS. These techniques and their assessment (see below) have been described in detail (Jones and Reid, 1973a and b).

Assessment of staining and type of glycoprotein After each method of staining four colours can be identified within cells-blue, blue-red,

red-blue and red. By a comparison of the gland area staining blue or blue-red after each technique, the area containing acid glycoprotein and each of its types is calculated. Briefly, after ABpH 2.6-PAS the blue and blue-red region of the gland contains acid glycoprotein: the red region contains neutral glycoprotein only and the red-blue virtually all neutral glycoprotein. In the red-blue area the trace of blueness shows the presence of a few acid radicles, but since the amount is minimal it may be taken that most of a cell staining in this way contains neutral groups. Pretreatment of the section with sialidase removes the enzyme- sensitive sialomucin leaving the enzyme-resistant sialomucin and sulphomucin which stain blue or blue-red. As acid groups fail to stain with AB the glycoprotein stains only with PAS.

Quantitation The proportion of the bronchial gland staining in a given way is calculated by point-

counting (Lamb and Reid, 1972). In the present study only the mucous cells are quantified: the morphology of the serous cells does not allow the application of the point-counting

GL YCOPRO TEINS IN BRONCHIAL EPITHELIUM 3

technique since the cells are small and the acini too sparse within the gland region. The staining response of the serous cells is assessed qualitatively.

Since the method of point-counting requires that gland acini fill any one microscopic field, each field including 25 points superimposed from an eyepiece graticule, the magnifica- tion used in this study was x 8 eyepiece with x 100 objective (field size 0.18 mm). Since the glands were so small in two of the control animals (1 and 2), two bronchi of similar size were used from each to provide the 200 points used for quantitation. In any one bronchial section from a pig with enzootic pneumonia the gland was plentiful.

Measurement of epithelial height

The height of the epithelium, from the basement membrane to the base of the cilia, was measured in nine bronchi of control pigs and in 12 bronchi of pigs with enzootic pneumonia. The mean of ten measurements was taken as the value for the epithelial height of each bronchus, for each of the airway sizes included.

RESULTS Bronchial subrnucosal gland

The submucosal glands are significantly increased in size in the infected animals (P = <0.001). The mean gland to wall ratio is 0.53 (f0.0258) for the infected, 0.27 (&O-OOSl) for the controls. A comparison of the staining of the bronchial gland of control pigs and pigs with enzootic pneumonia suggests that this disease causes a histochemical shift in the proportion of the different glycoproteins found intracellularly. Qualitative analysis suggests an increase in the volume of the gland containing sulphomucin relative to sialomucin when the bronchial gland has undergone hypertrophy with enzootic pneumonia (see

Mucous cells. The results of quantitative analysis of the mucous cells of the gland regions of three control and three experimentally infected animals are shown in tables I and I1 respectively: the percentage volume of the mucous cells staining blue, blue-red, red-blue or red, assessed by the method of point- counting, is given for tissue sections stained in one of each of three ways.

In control animals, virtually none of the gland stains blue with ABpH 2-6-PAS, most stains blue-red or red-blue and a small amount stains red. After sialidase, some of the gland stains blue, that staining blue-red is less in amount, while that staining red-blue is substantially increased. At pH 1.0 all blue staining is lost, blue-red staining is greatly reduced and most of the gland stains red-blue or red. There is no increase in AB staining of the gland region at this pH (see below).

Table I11 (based on values given in tables I and 11) give the proportion of each glycoprotein present in the mucous cells for each animal group. In the normal bronchial gland of the pig roughly the same number of mucous cells contain neutral as contain acid polysaccharide. The acid glycoprotcin includes sialidase-sensitive sialomucin, sialidase-resistant sialomucin and sul- phomucin.

In animals with enzootic pneumonia (see table 11) the staining pattern of the gland, with ABpH 2.6-PAS alone and after sialidase, is much the same as for the control animals. However, a tpH 1.0 blue staining is increased, much of the

figs. 1-3).

4 ROSEMARY JONES, A. BASKERVILLE AND LYNNE REID

blue-red staining remains, little of the gland stains red-blue and almost half of the gland stains red. Thus, in the hypertrophied gland similar types of glyco- protein are present as in the normal gland of control animals, but in different

I-

TABLE 1 Control pigs: pattern of staining of bronchial gland mucous cells, assessed by area (percentage

of total), after ( i ) ABpH 2.6-PAS, (ii) sialidase ABpH 2*6-PAS, (iii) ABpH I.O-PAS

1 2 3

1 Blue 1 Blue-red no.

0 7 7

Staining technique

ABpH 2.6-PAS

Animal no. Blue

i Red I I 50

2 ' 1 7 1 72 40 27 51

10 0 7 3 1 0 1 42

Sialidase ABpH 2.6-PAS

30 46 15

60 38 63

10 4

14

3

5 8 6

ABpH 1.O-PAS

TABLE I1 Pigs with enzootic pneumonia: as for table I

Staining technique Blue-red

64 72 80

Red-blue

32 23 20

Red

3 1 0

33 1 1 3

ABpH 2.6-PAS qg 3

Sialidase ABpH 2.6-PAS i i ; 3 1

18 30 44

45 48 47

ABpH 1.0-PAS 26 28 35

8 9

11

q s 8

3 0

57 54 55

I

proportions (see table 111). Acid glycoprotein now predominates over the neutral although not significantly so. The change in the proportion of the types of acid glycoprotein is highly significant. Sialidase-sensitive sialomucin and sulphomucin are greatly increased (P<O.OOl for both) and sialidase-resistant sialomucin is reduced (P<0-05).

Serous cells. With light microscopy the glycoprotein of the serous cells of the bronchial gland is included in discrete granules (see fig. 3). In control

GLYCOPROTEINS IN BRONCHIAL EPITHELIUM 5

animals, most of the granules of the serous cell stain red with ABpH 2.6-PAS, some stain red-blue and a few blue-red. A serous cell may contain granules staining either red or red-blue or only granules staining blue-red. Almost all serous acini contain cells in which the granules stain either with PAS alone or with both AB and PAS. Those granules staining blue-red retain their staining after sialidase treatment. At pH 1.0 virtually all granules are red but some are red-blue. Thus, most of the glycoprotein is neutral, the little acid glycoprotein present being sialidase-resistant sialomucin with some sulphomucin. The red- blue staining at pH 1.0 indicates very few sulphate radicles.

50 73 42 55 9.2

TABLE I11 Comparison of the proportion (percentage of gland area) present in bronchial gland mucous cells

in control pigs ( C ) and in pigs with enzootic pneumonia (EP)

26 , ~ 20

19 46 19 16 19 29 0.4 8.6

Animal group and number

65 76 80 13 4.4

c1 c 2 c3 Mean value SE

43 35 38 35 10 35 39 3 i 36 2.3 3.3 I 0.6

!

EP 1 EP 2 EP 3 Mean value SE

P =

~

Neutral glycopro tein

50 27 58 45 9.2

35 25 20 27 4.4

NS

i

sialidase ,

Sialomucin Sialomucin Acid I 1 glycoprotein sensitive to resistant to sialidase

I ! 5 8 6 6 0.9

I I I- I

In animals with enzootic pneumonia the serous cell granules stain blue, blue-red, red-blue or red with ABpH 2.6-PAS, there being more staining varia- tion than in the control animals. The number of serous cells in which the granules stain either blue or blue-red, or stain either red or red-blue is similar. Within a serous cell the granules may be of a single staining type or mixed, although a cell containing granules staining only red is rare and the combination of blue granules with red granules is not seen. A serous acinus usually contains cells with granules of a similar staining distribution but mixtures of cell types within an acinus are also seen. Sialidase has no effect on granules staining blue or blue-red. At pH 1.0 many more cells still contain granules staining blue-red than is the case with the control animals (fig. 3), although some cells which retain AB staining after sialidase lose their AB staining at this pH level: some cells contain only granules which only stain blue-red, other cells granules which stain blue-red or red-blue, but most contain granules staining red-blue or red either singly or in combination. Where mucous acini stain red at pH 1.0 the adjoining serous acini may contain cells staining blue-red and vice versa.

6 ROSEMARY JONES, A. BASKERVILLE AND LYNNE REID

In the serous cells, as in the mucous cells, there is a change in the proportion of the histochemical types of glycoprotein in the bronchial gland of pigs with enzootic pneumonia. Some neutral glycoprotein is present but the acid glyco- protein has increased. Sialidase-resistant sialomucin is still found but more sulphomucin has appeared.

In neither the control nor infected animals has sialidase-sensitive sialomucin been identified within the serous cells of the bronchial gland.

TABLE IV Epithelial height @m> in lobar bronchi (internal diameter range I-3 mm)

for control pigs (C) andpigs with enzootic pneumonia (EP)

Animal group and number

-

c1 c 2 c 3 Mean value SE

-

EP 1 EP 2 EP 3 Mean value SE

P = <

Airway size

3q 29 2 1 I 30 2

53 60 I :: I z

0.001 1 0.001 I 0.01

Epithelial height In table IV are given the results of measurements for the height of the

epithelium in lobar bronchi of control and infected animals. At each airway size there is a significant increase in the mean value for the height of the bronchial epithelium of the infected group as compared with the controls.

Goblet cells In the bronchi of control pigs the goblet cells are well defined and have

the shape of a chalice: the cells are regularly distributed around an airway and contain predominently acid glycoprotein (fig. 4). The acid glycoprotein is mainly sialidase-resistant sialomucin and sulphomucin. In some cells some neutral glycoprotein is found in conjunction with the acid glycoprotein, the latter occupying the apex of the cell.

In the heightened epithelium of pigs with enzootic pneumonia the goblet cells are not well defined: the cells are tall and thin, depleted in number and irregularly distributed around an airway and very little glycoprotein stains intracellularly (fig. 5). Many goblet cells contain only neutral glycoprotein

GLYCOPROTEINS IN BRONCHIAL EPITHELIUM 7

although some acid glycoprotein is found in some cells. Sialidase-resistant sialomucin and sulphomucin are the predominant types of acid glycoprotein found.

DISCUSSION The types of glycoprotein found in the airways of the normal pig are similar

to those in man (Lamb and Reid, 1969a), save that the mucous cells of the pig bronchial gland contain more neutral glycoprotein than the human. In the hypertrophied pig gland in enzootic pneumonia the mucous cells have more sulphate or sialic acid sensitive to sialidase and the serous cells more sulphate and sialic acid resistant to sialidase than is found in control animals. In the hypertrophied submucosal gland in the human chronic bronchitic there is increased production of acid glycoprotein together with reduction in sialidase sensitivity of mucous cells, although no attempt has been made to identify either sulphomucin or the type of acid glycoprotein in the serous cells (de Haller and Reid, 1965; Lamb and Reid, 1970). In the hypertrophied bronchial epi- thelium of pigs with enzootic pneumonia there were fewer goblet cells present and these seemed depleted of secretion and contained neutral rather than acid glycoprotein.

Bronchial gland hypertrophy is a well-established feature of chronic bronchitis and hypertrophy of the submucosal gland in respiratory airways an established feature of irritation produced experimentally (Lamb and Reid, 1968, 1969b; Mawdesley-Thomas, Healey and Barry, 1971; Baker et al., 1973; Jones, Bolduc and Reid, 1973). In this and in an earlier study (Baskerville, 1972) bronchial gland hypertrophy was found, for the first time, in association with infection.

The way in which mycoplasma infection has produced bronchial gland hypertrophy is not known. Baskerville (1972) showed that infection of pigs with Mycoplasma hyorhinis causes the alveolar consolidation of lobar pneumonia, but save for an occasional organism in the alveolar ducts or alveoli, mycoplasma were not found distal to bronchioli. Other studies have shown that mycoplasma colonise airways but not alveoli (Gois et al., 1971), the inference being that the alveolar region is unfavourable for their growth. The organisms are seen in the lumen of the airways, not in the tissue, and although mucus and inflam- matory cells are seen lying free in the airway lumen it is unusual to see inflammatory cell infiltration. Thus, changes in airway epithelium would not seem to arise from airway invasion by bacteria.

Lauweryns, Cokelaere and Theunynck (1972) have demonstrated the presence of innervated neuroepithelial bodies in the mucosa of the airways of a number of mammalian species including man and the newborn pig. The function of these bodies is unknown, but these authors postulated that it might be both secretory and receptor, since histochemical staining showed that these bodies produce serotonin precursors and have nerve fibre connections. They may therefore be concerned in the reflex control of mucosal secretion. No other studies appear to have been carried out on the innervation of the bronchial mucosa and submucosal glands of the pig.

8 ROSEMARY JONES, A. BASKERVILLE AND LYNNE REID

Bronchial gland hypertrophy may occur as a reflex response to a stimulus acting either directly on the overlying bronchial epithelium or in the peripheral region of the lung. It would seem likely that bronchial epithelial hypertrophy occurs in response to a local stimulus and there is evidence that the bronchial epithelium and submucosal gland are connected through nervous pathways. Mycoplasma are known to produce peroxide (Cherry, and Taylor-Robinson, 1970), which acts directly on epithelial cells and may act as an irritant to receptors within the airways. Gland hypertrophy from a stimulus received indirectly, perhaps from the alveoli, would occur in response to stimulation via a nervous reflex. That the parasympathetic nervous system causes increasing bronchial gland discharge is well established (Florey, Carlton and Wells, 1932). The role of the sympathetic nervous system is less clear. The administration of isoprenaline to rats causes tracheal gland hypertrophy (Sturgess and Reid, 1973), but in-vitro studies have suggested no increase in the rate of secretion from the mucous cells (Sturgess and Reid, 1972). Some humoral control may be involved in bronchial gland hypertrophy since it is accompanied by an increase in the number of blood vessels and vessel dilation (de Haller and Reid, 1965). Certainly the uniformity of the amount of the various types of acid glycoprotein in the airways of one individual suggests some overall control of the bronchial glands in man (Lamb and Reid, 1972). It is probable that an increase in activity, from whatever cause, leads to an increase in the size of the gland: work hypertrophy may lead to cell hypertrophy and eventually to cell hyperplasia.

The histochemical techniques used in this study have been used previously to identify glycoproteins in a variety of epithelial tissue sites (Jones and Reid, 19730 and b) and offer a relatively simple means of identifying and analysing change in the distribution of glycoproteins both within a cell and within a cell population (Jones, Bolduc and Reid, 1973). No detailed histochemical study of the glycoproteins in pig airways has been made; the only previous study used PAS staining only and reported the presence of PAS-positive material in cells of the gland and epithelium (Goco, Kress and Brantigan, 1963).

In the mucous cells of the normal submucosal gland of the pig neutral glycoprotein, sialidase-sensitive and sialidase-resistant sialomucin and sulpho- mucin were identified-the last in small amounts. In the serous cells there was no evidence of sialidase-sensitive sialomucin although each of the other types of glycoprotein was found. In addition, in the hypertrophied gland, two types of sulphomucin could be identified by their AB staining-some staining both at pH 2-6 and 1.0 and some only at pH 1.0. The latter was identified by an increase in staining intensity and in some bronchi by an increase in the area of the gland staining with AB at the lower p H level. The presence of these groups has already been described (Jones and Reid, 1973a and b) and would seem a common feature of sulphated epithelial glycoproteins, although a sulphomucin staining with AB only at pH 1.0 was not evident in the normal pig bronchial gland perhaps because so little of any sulphate was found (<7 per cent.).

Change in the proportion of the various types of glycoprotein has been

JONES, BASKERVILLE AND REID

GLYCOPROTEINS IN BRONCHIAL EPITHELIUM

PLATE I

FIG, 1.-Bronchial gland of control pig (a) ABpH 2.6-PAS: mucous acini stain blue-red or red-blue showing the presence of both acid and neutral glycoprotein, (b) ABpH 1.0-PAS: most AB staining is now lost and mucous acini stain red-blue showing that little of the acid glycoprotein is sulphated. x 750.

FIG. 2.-Bronchial gland of pig with enzootic pneumonia (a) ABpH 2.6-PAS: more variation in the staining of cells within mucous acini-some blue, some red-blue, but most are blue-red-showing more acid than neutral glycoprotein present, (b) ABpH 1.O-PAS: many cells retain AB staining demonstrating sulphated glycoprotein. x 750.

In the animals with enzootic pneumonia there is an increase in the size of mucous acini due, at least in part, to cell hypertrophy.

JONES, BASKERV~LLE AND REID

GLYCOPROTEINS IN BRONCHIAL EPITHELIUM

PLATE IJ

FIG. 3.-Higher magnification of 2(b). x 1500. Serous cells (arrow) show positive AB staining at p H 1.0, demonstrating sulphated glyco- protein. This is not seen in the serous cells of the bronchial gland control animals.

FIG. 4.-Goblet cells in bronchial epithelium . FIG. %-Goblet cells in thickened bronchial of control pig. ABpH 2.6-PAS. Most cells epithelium of pig with enzootic pneumonia. stain blue, demonstrating acid glycoprotein. ABpH 2.6-PAS. Most cells stain red or i( 1500. red-blue, demonstrating neutral glycoprotein.

x 1500.

GLYCOPROTEINS IN BRONCHIAL EPITHELIUM 9

reported with goblet cell hypertrophy and hyperplasia in rat respiratory airways after exposure to sulphur dioxide and tobacco smoke (Reid, 1963; McCarthy and Reid, 1964; Lamb and Reid, 1968; 19693) and after isoprenaline (Sturgess and Reid, 1973). Change in the proportions of glycoproteins would seem a sensitive marker of damage to respiratory epithelium since it may also occur with cell hypertrophy but without any goblet cell increase (Jones, Bolduc and Reid, 1972, 1973).

The reason for change in the distribution of types of glycoproteins within a cell population is not clear. A cell may contain enzymes necessary for the production of a wide range of glycoproteins. From quantitative studies it has been shown that a single goblet cell may produce neutral and acid glycoprotein, the acid glycoprotein including both sialic acid and sulphate (Jones, Bolduc and Reid, 1973) and the same is true of mucous and serous cells of the human bronchial gland (Lamb and Reid, 1969c, 1970). There is evidence to suggest hormonal control of glycoproteins synthesised by certain epithelial tissues (Warren and Spicer, 1961 ; Shackleford and Klapper, 1962; Varute and Jirge, 1971) but this seems unlikely in airways.

The change in proportion of intracellular glycosyl transferases is probably an important factor in glycoprotein synthesis. Baker et al. (1973) have demon- strated an increase in glycosyltransferases from microsomal fractions prepared from respiratory tract tissue showing goblet cell hyperplasia and gland hyper- plasia, from dogs exposed to sulphur dioxide. Degand et al. (1973) have sug- gested that an increase in galactosamine leads to an increase in the length of the glycan chains and thus to a possible increase in the number of sites available for sulphation of the glycoprotein. It may be that the increase in sulphomucin seen in the cells of the hypertrophied pig bronchial gland is secondary to synthesis of galactosamine.

While a change in the type of glycoprotein may be the result of increase in biosynthesis, any change would appear to be independent of cell discharge. It seems that where there is no stimulus to increase, discharge cells produce an acid glycoprotein and that with such a stimulus production of neutral glyco- protein is favoured. For example, there is a shift to sulphated glycoprotein without an increase in discharge from goblet cells, after exposure to tobacco smoke to which an anti-flammatory agent has been added (Jones, Bolduc and Reid, 1973) or after isoprenaline (Jones and Reid, in preparation); more likely there is a retention of secretion. However, organ culture studies have shown that, unlike isoprenaline, pilocarpine increases secretion from the bronchial gland (Sturgess and Reid, 1972), and in in-vivo studies the epithelial goblet cells appear empty of secretion and an increase in goblet cell number consists of an increase in both neutral and acid glycoprotein containing cells (Sturgess and Reid, 1973). After the administration of Bisolvon a loss of goblet cells secreting acid glycoprotein, particularly sulphomucin, has been associated with an increase in cell discharge (Janatuinen and Korhonen, 1969). Certainly with infection leading to bronchial epithelial hypertrophy as reported in this study the goblet cells appear to have been stimulated to discharge with reduction in acid glycoprotein and an increase in neutral glycoprotein production.

10 ROSEMARY JONES, A. BASKERVILLE AND LYNNE REID

SUMMARY

The glycoproteins in the normal pig bronchial gland are identified by the combined Alcian Blue (AB)-periodic acid Schiff (PAS) technique, with the use of sialidase digestion and AB staining either at pH 2.6 or at pH 1.0. In enzootic pneumonia (produced experimentally by infection with Mycoplasma hyorhinis) the bronchial gland hypertrophies, mucous and serous cells both increase, in number and size; hence the total glycoprotein content of the gland increases. The distribution of glycoproteins in the hypertrophied gland differs from that in the normal. Quantitative analysis of the mucous cells shows that in the hypertrophied gland the acid glycoprotein is increased relative to the neutral. There is also a relative change in the amounts of sialidase-sensitive sialomucin and sulphomucin; both are significantly increased at the expense of the sialidase- resistant sialomucin. Qualitative analysis of the serous cells shows that in the normal gland most of the glycoprotein is neutral and that the small amount of acid glycoprotein is sialidase-resistant sialomucin. In the hypertrophied gland there is relatively more acid glycoprotein which is either sialidase-resistant sialomucin or sulphomucin; in addition, in pigs with enzootic pneumonia there is an increase in the height of the bronchial epithelium and a depletion in both goblet cell number and glycoprotein content, which latter has more neutral glycoprotein and less acid glycoprotein.

REFERENCES

BAKER, A. P., CHAKRIN, L. w., SAWYER, J. L., MUNRO, J. R., AND HILLEGRASS, L. M. 1973. Levels of glycosyltransferases in canine respiratory tissue in an experimentally induced hypersecretory state. Fed. Proc., 32, 560.

BASKERVILLE, A. 1972. Development of the early lesions in experimental enzootic pneumonia of pigs: an ultrastructural and histological study. Res. vet. Sci., 13, 570.

CHERRY, J. D., AND TAYLOR-ROBINSON, D. 1970. Growth and pathogenesis of Mycoplasma mycoids var. Capri in chickens embryo tracheal organ cultures. Infection and Immunity, 2,431.

DEGAND, P., ROUSSEL, P., LAMBLIN, G., AND HAVEZ, R. 1973. Purification et 6tude des mucines de Kystes bronchogeniques. Biochem. biophys. Acta, 320, 318.

DE HALLER, R., AND REID, L. 1965. Adult chronic bronchitis. Med. Thorac., 22, 549. FLOREY, H., CARLETON, H. M., AND WELLS, A. Q. 1932. Mucus secretion in the trachea.

Br. J. exp. Path., 13, 269. FREEMAN, G., AND HAYDON, G. B. 1964. Emphysema after low level exposure to NOz.

Arch. eno. Hith., 8, 125. Goco, R. V., KRESS, M. B., AND BRANTIGAN, 0. C. 1963. Comparison of mucus glands in

the tracheobronchial tree of man and animals. Am. N. Y. Acad. Sci., 106, 555. GOIS, M., VALICER, L., AND SOVADINA, M. 1968. Mycoplasma hyorhinis, a causative agent

of pig pneumonia. 261. vet. med., B15, 230. GOB, M., POSPISIL, Z. , CERNY, M., AND MRVA, V. 1971. Production of pneumonia after

intranasal inoculation of gnotobiotic piglets with 3 strains of Mycoplasma hyorhinis. J. comp. Path., 81, 401.

GOODWIN, R. F. N., POMEROY, A. P., AND WHITTLESTONE, P. 1965. Production of enzootic pneumonia in pigs with a mycoplasma. Vet. Rec., 77, 1247.

JANAWNEN, M., AND KORHONEN, L. K. 1969. The effect of substituted Benzylamine (Bisolvon) on mucosubstance production. Sonderdruck aus " Naunyn-Schmiedebergs Archiv fur Pharnakologie ", 265, 112.

GL YCOPRO TEINS IN BRONCHIAL EPITHELIUM 11

JONES, R., BOLDUC, P., AND REID, L. 1972. Protection of rat bronchial epithelium against tobacco smoke. Br. med. J., 2, 142.

JONES, R., BOLDUC, P., AND REID, L. 1973. Goblet cell glycoprotein and tracheal gland hypertrophy in rat airways: The effect of tobacco smoke with or without the anti- inflammatory agent phenylmethyloxdiazole. Br. J. exp. Path., 54, 229.

JONES, R., AND REID, L. 1973~. The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. I. Sialomucins and sulphomucins (singly or in single combinations). Histochem. J. , 5, 9.

JONES, R., AND REID, L. 19736. The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. 11. Human bronchial submucosal gland. Histochem. J., 5, 19.

LAMB, D., AND REID, L. 1968. Mitotic rates, goblet cell increase and histochemical changes in mucus in rat bronchial epithelium during exposure to sulphur dioxide. J. Path. Bact., 96, 97.

LAMB, D., AND REID, L. 1969a. Histochemistry autoradiography, tissue culture and bio- chemical analysis in the study of bronchial epithelial glycoproteins. In : " Hypersecretion Bronchique ", Colloque International de Pathologie Thoracique, LiZZe, pp. 11-22.

LAMB, D., AND REID, L. 19696. Goblet cell increase in rat bronchial epithelium after exposure to cigarette and cigar tobacco smoke. Br. med. J., 1, 33.

LAMB, D., AND REID, L. 1969c. Histochemical types of acid glycoprotein produced by mucous cells of the tracheobronchial glands in man. J. Path., 98, 213.

LAMB, D., AND REID, L. 1970. Histochemical and autoradiographic investigation of the serous cells of the human bronchial glands. J. Path., 100, 127.

LAMB, D., AND REID, L. 1972. Quantitative distribution of various types of acid glycoprotein in mucous cells of human bronchi. Histochem. J., 4, 91.

LAUWERYNS, J. M., COKELAERE, M., AND THEUNYNCK, P. 1972. Nervo-epithelial bodies in the respiratory mucosa of various mammals. 2. ZeZZ. Forsch., 135, 569.

LEUCHTENBERGER, C., LEUCHTENBERGER, R., AND DOOLIN, P. F. 1958. A correlated histo- logical, cytological and cytochemical study of the tracheobronchial tree and the lungs of mice exposed to cigarette smoke. I. Bronchitis with atypical epithelial changes in mice exposed to cigarette smoke. Cuncer (Philad)., 11,490.

MAWDESLEY-THOMAS, L. E., HEALEY, P., AND BARRY, D. H. 1971. Experimental bronchitis in animals due to SO2 and cigarette smoke. An automated quantitative study. In Proceedings of an International Symposium on Inhaled Particles, ed. W. H. Walton, vol. 1, p. 509. London, Unwin Bros.

MCCARTHY, C., AND REID, L. 1964. Acid mucopolysaccharide in the bronchial tree in the mouse and rat (sialomucin and sulphate). Quart. J. exp. PhysioZ., 44, 81.

REID, L. 1960. Measurement of the bronchial mucous gland: a diagnostic yardstick in chronic bronchitis. Thorax, 15, 132.

REID, L. 1963. An experimental study of hypersecretion of mucus in the bronchial tree. Br. J. exp. Path., 44, 437.

SHACKLEFORD, J. M., AND KLAPPER, C. M. 1962. A sexual dimorphism of hamster sub- maxillary m u c h Anat. Rec., 142, 495.

STURGESS, J., AND REID, L. 1972. An organ culture study of the effect of drugs on the secretory activity of the human bronchial submucosal gland. CZin. Sci., 43, 533.

STURGESS, J., AND REID, L. 1973. The effect of isoprenaline and pilocarpine on (a) Bronchial mucus-secreting tissue and (b) Pancreas, salivary glands, heart, thymus, liver and spleen. Br. J. exp. Path., 54, 388.

WARREN, L., AND SPICER, S. S. 1961. Biochemical and histochemical identification of sialic acid containing mucins of rodent vagina and salivary glands. J. Histochem. Cytochem.,

VARUTE, A. T., AND JIRGE, S. K. 1971. Histochemical analysis of mucosubstances in oral 9,400.

mucosa of Cichlid fish. Histochemie, 25, 91.