ACID MUCINS IN HUMAN INTESTINAL GOBLET CELLS SHELAGH M. MORRISSEY AND M. C . TYMVIOS Department of Physiology, Queen Elizabeth College, University of London, Campden Hill Road, W8 7AH
H I s T o c H E M I c A L investigations have been carried out on epithelial mucins in the digestive tract of human subjects (Spicer and Meyer, 1960; Schrager, 1964; Lev and Spicer, 1965; Lev, 1966) and the different histochemical properties of these mucins have been observed in different parts of the gastro-intestinal tract. Epithelial glycoproteins in man are usually acidic due to their sialic acid residues (Gottschalk, 1960) or sulphated (Gibbons and Mattner, 1966). Both sulphomucins and sialomucins have been studied in tracheal and bronchial mucosa (Lamb and Reid, 1972) and in the sheep’s colonic mucosa (Kent and Marsden, 1963). A qualitative and histophotometric investigation of colonic epithelial mucins has been reported (Greco et al., 1967). Parallel to these investigations, several histochemical studies on mucous glands in relation to cystic fibrosis (CF) have been carried out (Landing, 1956; Graig, 1959; Wagner et al., 1960; Belanger, 1963) but all of these have been of an essentially subjective nature. Since this disease appears to be associated with abnormalities in mucous secretion which could be due either to physical or chemical differences from normal or to the amount of secretion, it was of interest to measure goblet cell mucins quantitatively in the epithelium of the duodenum, jejunum and ileum in both CF and non-CF patients using a Vickers M85/0010 Integrating and Scanning Microdensitometer. Our objective in this investigation was to establish whether any marked quantitative differences existed in the duodenum, jejunum and ileum since in cystic fibrosis it has been established that physiological and pathological changes occur mostly in the ileum of the small intestine (meconium ileus) which usually requires ileostomy to avoid further complications. MATERIALS AND METHODS Surgical tissue was obtained from Queen Elizabeth Hospital for Children and from Withington Hospital, Manchester. Duodenal and jejunal control tissue (five cases of each) was obtained from patients who were investigated for gastro-intestinal disorders (atresia, stenosis, etc.) but in which cystic fibrosis was excluded. Cystic fibrosis duodenal and jejunal tissue was also obtained (four cases of each). These numbers are satisfactory since no clinical or pathological manifestations of the disease compel a biopsy of these areas of the small intestine. Tissue from the ileum investigation (six cases of each) was obtained from neonates (2 weeks to 12 weeks old) present with meconium ileus (cystic fibrosis) and from the same Received 10 Feb. 1978; accepted 5 ApriI 1978. 1. PATH.-VOL.
SHELAGH M. MORRISSEY AND M . C. TYMVIOS
patients at closure of ileostomy 6 mth later. In this way, any changes occurring between neonates and infants could be investigated. Control tissue for neonates and infants was also obtained (six cases of each) from children in which cystic fibrosis was excluded. The investigation for the ileum therefore comprised four groups--control neonate, CF neonate, control infant and CF infant. Tissue from patients on antibiotic treatment was excluded. All specimens obtained were fixed in formalin embedded in paraffin and sectioned at 4 pm. Sections were dewaxed and stained with alcian blue (8 GX) at pH 2.6 for acid mucins (Jones and Reid, 1973). Acid mucins were identified by staining with alcian blue at pH 2.6 and tissues stained with alcian blue pH 2.6 only, gave a blue colour. Acid mucins. The protocol followed is shown on fig. 1.
(A) Acidic Mucins
Alcian Blue pH 2.6
0.1N H+O46o0C 2 Hrs
O.1N H2S04600C 2Hrs
Akian Blue pH 2.6 Alcian Blue pH1 (Weakly Acidic) (Strongly Acidic)
(McCarthy and Reid, 1963) used for the separation and identification of acid mucins into sialo-resistantsulphomucins staining at pH 2.6 with alcian blue and sulphomucins staining with alcian bluepH 1. Groups A , B, C and D : Sections stained with alcian blue pH 2 6 were assigned as group A, total mucins. Sections stained with alcian bluepH 2.6 after sialidase digestion were assigned as group B. Sialidase digestion in the group removed the sensitive sialic acid from the polysaccharide chain and left the sialic acid which is resistant to the enzyme (sialo-resistant mucins). Group C are the weakly acidic sulphomucins stained with AB pH 2.6 after acid hydrolysis which removes all sialic acid residues. Group D are the strongly acidic sulphomucins which are stained with AB pH 1.0 (strongly acidic sulphomucins can only be demonstrated when stained with As pH 0.5-1.2) after acid hydrolysis.
Sialidase digestion. Sections were incubated in 100 cm3 of sialidase medium for 15 hr at 37°C. Sections were allowed to cool in air for 2 min. The sections were washed in distilled water and were stained with alcian blue (PH 2.6). (Sialidase-receptor destroying enzyme (RDE) from Vibrio Cholerae, Wellcome Reagents Ltd). Acid hydrolysis. Sections were dewaxed, taken down to water and were incubated in 100 cm3 of 0.1 N sulphuric acid for 2 hr at 60°C. The sections were then allowed to cool in air for 4 min. and then were carefully washed in distilled water. One-half of the batch were stained in alcian blue pH 2.6 for weakly acidic sulphomucins and the other half were stained with alcian blue pH 1.0for the strongly acidic sulphomucins. The four treatments were designated A, B, C and D as described in fig. 1.
Quantitative estimation of mucins. A Vickers M85/0010 Scanning and Integrated Microdensitometer was used on alcian blue stained sections. The microdensitometer is concerned with the precise measurement of chromophores in microscopic preparations. AS in orthodox spectrophotometry the total amount of energy absorbed will depend not only on the spectral characteristics of the chromophore but also on the total concentration of chromophore present. Absorption maxima for AB
Vickers M 8 5 Scanning 8 Integrated Microdensitometer
2 - 200
Machine one Mask size A 3 A 600 Band width 30 Spot size 1
FIG.2.-Alcian blue absorption maximum and parameters used. This figure shows the absorption for alcian blue at 600 nm, mask size, panel width and spot size.
Substances which do not naturally absorb in the visible spectrum may be stained by modifying their structure in situ to include suitable chromophores (for example by coupling alcian blue to reacting groups of the macromolecules in the section) so that they can exhibit absorption at a suitable wavelength in the visible. Fig. 2 shows the absorption maximum for alcian blue to be at 600 nm. Therefore the
SHELAGH M. MORRISSEY AND M. C. TYMVIOS
wavelength used for the reading of all slides was 600 nm. Spot size number 1 (0.25 p m in diameter) was used. Mask size A3 was selected since it covered single goblet cells. Band width was selected at 30 nm. Integrated density or extinction coefficient values were calculated according to the equation below. Integrated density or - Goblet cell reading-Background reading x 100 Standard filter reading* extinction coefficient Random count of goblet cells. The microscope slide tray on the Vickers M8.5/0010 is moved at random without looking down the eyepiece and the goblet cell closest to the spot is read, a background reading is also taken next to the goblet cell. ACID
CELLS OF HUMAN
AB pH 2 6
A: Total acid mucins stained with alcian blue pH 2.6. B: Sialomucins 3.-Duodenum. remaining after sialidase digestion. C: Sulphomucins staining with alcian blue at pH 2.6 after acid hydrolysis. D: Sulphomucins staining with alcian blue at g H 1 after acid hydrolysis.
If the spot falls at equidistance between two cells then the slide tray is moved again. Twenty goblet cell readings were taken and 20 background readings. Duplicate tests were carried out which showed that this method of counting gave reproducible results (P>0,05)(a = 20).
* Standard filter of O.D.
1.0 read after setting O.D.
on unstained (lumen) area of section.
RESULTS The results obtained for the duodenum, when plotted as a histogram are shown on fig. 3. The shaded columns represent the results obtained for cystic fibrosis duodenum and the white columns are the results obtained for the control in each respective group, A, B, C and D. Column D shows highly significant differences (P