Colonic epithelium reactive monoclonal antibodies

Histochemistry (1989) 92:397-406 Histochemistry �9 Springer-Verlag 1989

Colonic epithelium reactive monoclonal antibodies Identification and immunohistochemical localization of the target epitopes

C.P.H.J. Verstijnen*, J.W. Arends**, P.T.M. Moerkerk, M. Pijls, B. Kuypers-Engelen, and F.T. Bosman Department of Pathology, State University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands

Accepted April 22, 1989

Summary. We have produced a small library of colonic mucosa and colorectal carcinoma reactive monoclonal antibodies (MoAbs) by immunizations with extracts of human colon cancer tissue and a human colon cancer cell line. Hybr idoma supernatants were tested on (normal and neoplastic) human tissues by immunoperoxidase methods to evaluate organ or tissue specificity. Initial biochemical characterization of the target antigens was performed by gelper- meation chromatography, Western blotting and competition assays.

Based upon the immunoreactivity patterns and the characteristics of the antigen four groups of MoAbs could be distinguished. The first group concerns the antibodies PAR- L A M 3, 9 and 10. These antibodies react with an 87 kDa protein moiety in high molecular weigl~t (2-5 x 106 Da) glycoproteins. In intestinal and colon mucosa these antibodies showed diffuse binding with goblet cells. In colon carcinoma decreased reactivity with these MoAbs was found.

The second group consists o f antibodies P A R L A M 8, 12 and 13. These antibodies react with large ( > 5 x 1 0 6 Da) glycoproteins, most likely with carbohydrate epitopes. By immunohistochemistry in normal colon mucosa the antibodies all show granular supranuclear reactivity with goblet cells. These antibodies show increased reactivity with colon adenomas and adenocarcinomas.

A third group is formed by PAlq:LAM 2, which also reacts with a large ( > 5 x 106 Da) glycoprotein, showing a granular distribution in goblet cells. I n colon carcinomas more extensive expression is found than in normal colonic mucosa. Finally, the fourth group consists of P A R L A M 11, which also reacts with a large ( > :5 • 1 0 6 Da) glycopro- rein, located in the brush border of colonic columnar cells.

These antibodies might be useful tools for the analysis of the expression of mucin related glycoproteins in normal, preneoplastic and neoplastic colon muLcosa.

Introduction

Several investigators have reported the generation of MoAbs against primary colon carcinoma cells, membrane preparations of colon cancer tissue or colon carcinoma cell lines (Steplewski and Koprowski 1982; Finan et al. 1982;

* Supported by grant RL 82-1 of the Netherlands Cancer Founda- tion, K.W.F. ** To whom offprint requests should be sent

Thompson et al. 1983; Morgan et al. 1984; Girardet et al. 1986; Drewinko et al. 1986). None of these appeared to react exclusively with either colon epithelium or colon carcinoma cells. One M o A b was found to detect a mucin antigen which occurred in normal as well as carcinomatous colon epithelium (Finan et al. 1982). The organ specificity of this antigen, however, was not reported. More recently, MoAbs have been obtained through immunization with normal colon crypts (Hughes et al. 1986), human colon mucin (Po- dolsky et al. 1986a, b) and colon epithelial membrane preparations (Richman and Bodmer 1987).

Using his MoAbs, Hughes et al. (/986) showed alterations of mucin antigen patterns in normal colon mucosa of colon cancer patients as well as altered mucin composition in adenoma and carcinoma tissues. Podolsky et al. (1986a, b) described a library of MoAbs which showed distinct patterns of binding to goblet cell subpopulations in normal colon mucosa which might also be of use in detecting alterations in histologically normal mucosa in patients with colon disorders. Richman and Bodmer (1987) demonstrated that MoAbs can be used to define antigen expression patterns in tumors which might assist in more accurate tumour characterization.

In the present report we describe the production of MoAbs reactive with normal and neoplastic colon epithelium, some biochemical characteristics of the target antigens and the reactivity with a variety of normal and neoplastic human tissues.

Materials and methods

Tissue and cellpreparation. Normal colon epithelium was obtained from colectomy specimens of patients with colon carcinoma. The mucosa was dissected from the bowel wall at a distance of at least 5 cm from the tumor. The epithelial cells were separated from the lamina propria according to Bull and Bookman (1977) by incubation in 0.15 M phosphate buffered saline (PBS) with ethylene diamine tetra acetate (EDTA). Dissociated epithelial cells were pel- leted by centrifugation and resuspended in PBS. 5583-S cells (Ver- stijnen et al. 1987) derived from a primary colon carcinoma were grown in suspension in Dulbecco's minimal essential medium (DMEM) + 10% fetal calf serum (FCS) and harvested by centrifugation of culture supernatant.

All further preparations were done on ice. Colon epithelial cells or 5583-S cells were homogenized and particulate matter was removed by centrifugation. The supernatant, containing the mucin glycoprotein, was used for enzyme immunoassay and gelfiltration chromatography after determination of protein content by measur-

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ing the absorbance at 280 nm. For perchloric acid (PCA) extrac- tion, 1 vol of supernatant was added to I vol of 1 M PCA and stirred for 20 rain. The precipitated material was centrifuged (20000g, 15 min) and the supernatant was extensively dialyzed against distilled water. The precipitate was centrifuged and the supernatant was used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Generation of MoAbs. As immunogens for the production of MoAbs we used colon carcinoma tissue and a colon carcinoma cell line (5583-S). Primary tumor tissue fragments were collected, washed in PBS and pulverized in a Spex tissue homogenizer under liquid nitrogen, Balb/c mice were immunized intraperitoneally with 100 gg of tumor homogenate in 1 ml PBS and boostered twice at monthly intervals. This resulted in the MoAbs PARLAM 2 and 3. Alternatively Balb/c mice were immunized with 5583-S cells. A similar immunization schedule was followed as for the first, using two immunizations with 200 gl of packed cells for initial injection and 100 gl of packed cells for the booster injection. This approach resulted in the MoAbs PARLAM 8-13. Four days after the final booster injection spleen cells were fused with SP 2/0 mye- loma cells and antibody producing hybridoma cells were recloned untill monoclonal by limiting dilution. Initial supernatant screening was performed by ELISA using cytosol of colon carcinoma tissue and 5583-S cells as antigen preparation. ELISA was performed after attaching the desired amount of antigen to the plates by overnight evaporation at 37 ~ C in a dry incubator. MoAb containing tissue culture supernatant or aseites was diluted in PBS-Tween- BSA and incubated 1 h (37 ~ C). After washing, the plates were incubated with rabbit anti-mouse immunoglobulin-peroxidase conjugate (Dakopatts). After final washing, peroxidase activity was visualized with orthophenylene diamine as chromogen. The reac- tion was stopped after 15 min with 2 N H2SO4 and the absorbance was measured at 492 nm. Antibody producing clones were further selected on the basis of tissue immunoreactivity patterns obtained through immunoperoxidase staining of frozen and paraffin sections. MoAbs Ig subclass was determined by an immuno spot test (Beyer 1984) using Ig subclass specific goat anti mouse immuno- globulins (Nordic, Tilburg, The Netherlands).

Tissue panel. Fresh normal colon and colon cancer tissue was obtained from colectomy specimens of patients with colon carcinoma and used for immunohistological studies on unfixed cryostat sections. Reactivity of the MoAbs was determined on a panel of normal and neoplastic human tissues, embedded in paraffin, which were retrieved from our files including 9 specimens of normal colon, 10 of colon adenomas and 13 colon carcinomas as listed in Table 2. A variety of other normal and neoplastic tissues was tested as listed in Tables 3 and 4. Paraffin embedded tissues of fetal and newborn colon, small intestine and stomach were obtained from Prof. J. Huber (Pediatric Pathology, State University Utrecht, The Netherlands). Specimens were available of mid-gestation (week 12, 16.5, 19, 22, 24, 25, 26 (2x) , 27 (3x ) und end-gestation (week 29, 30, 33 and 36) and the early postnatal period (2weeks, 10 weeks, 12 weeks and 16 months).

Species specificity was investigated on paraffin embedded specimens of rat (Wag/ry) and mouse (Balb/c and NMRI) colon and small intestine.

Immunocytoehemistry. Details of the immunoperoxidase technique have been reported previously (Arends et al. 1983). Briefly, paraffin sections were subjected to 0.1% trypsin in 0.1% CaC12 in distilled water (pH 7.8) for 25 min prior to immunostaining. Appropriately diluted MoAbs and rabbit anti-mouse immunoglobulin peroxidase conjugate were applied 30 min at room temperature. Peroxidase activity was visualized with 0.01% H202 and 0.05% diamino-ben- zidine in Tris-HC1 buffer (pH 7.6). Negative controls included re- placement of specific antibodies with SP 2/0 supernatant or ascites. The tissue sections were viewed independently by two of us (JW.A., C.V.). In most instances observations were confirmatory. Whenev- er discrepancies occurred, the slides were jointly reviewed and a single score was assigned by mutual agreement.

Antigen identification. SDS-PAGE was performed according to the method of Laemmli (1970) on a 5% gel. After electrophoresis the separated proteins were transferred onto nitrocellulose and immu- nostained according to Towbin et al. (1979). Cytosol of normal colon mncosa and 5583-S cells in PBS containing 2 m M phenyl- methanesulphonylfluoride (PMSF) was eluted on a sepharose 4B column (2.6 x 85 cm, Pharmacia) using (PBS) as eluent (11.5 ml/h). Protein content of each ehient fraction (2.5 ml) was determined by OD 280 measurement and the reactivity of each fraction with the various MoAbs was tested by ELISA.

To investigate whether MoAbs were directed against identical or closely related epitopes competition experiments were performed by ELISA using 5583-S cytosol as antigen. To this end each MoAb was labeled with horseradish peroxidase (Sigma, grade IV) according to the method of Wilson and Nakane (1978). Competition was performed using a 50% saturating concentration of peroxidase conjugated MoAbs and a 50-fold excess of homologous or hetero- logous unlabeled competition antibody (1 h, 37 ~ C).

In a series of experiments protease sensitivity, thermal stability, sensitivity to periodate oxidation and exoglycosidase digestion, and competition with lectins of disaceharides was investigated in order to obtain an initial characterization of the target epitope. In all experiments immunoreactivity in ELISA was used as detection sys- tem.

Pronase sensitivity was tested by pronase digestion of 5583-S cytosol Pronase coupled to sepharose beads (Pierce, 6 units/ml in PBS) was incubated with cytosol at 37 ~ C under continuous stirring for 5 h. After incubation the beads were removed by centrifugation. As a positive control, cytosol incubated under identical conditions but without pronase was applied.

Periodate oxidation of the terminal sugar residues was performed according to Woodward et al. (1985). 5583-S cytosol was coated on ELISA plates, which were incubated overnight with 50 m M NaIO4 in 0.1 M sodium acetate buffer (pH 5.4). As a control 5583-S cytosol was incubated under identical conditions but without periodate.

For the MoAbs showing reduced binding after periodate oxidation, the nature of the involved sugar residue was further investigated by digestion of coated antigens with a variety of exoglycosidases and by competition of antibody binding with lectins and monosaccharide solutions. Of all tested exoglycosidases only treat- ment with galactosidase (Behring) 1.2 mU/well in 0.01 M sodium acetate buffer (pH 5, 64 h, 37 ~ C) appeared to influence MoAb immunoreactivity. As lectins Peanut agglutinin (PNL), Soybean agglutinin (SBA), Concanavalin A, Pisum sativum agglutinin, Ulex europeaus agghitinin, Dolichos bifloris agglutinin. Pokeweed mito- gen aggtutinin, (all from Sigma) were applied. The wells were incubated with 2 gg of lectin in 50 gl of peroxidase conjugated MoAb in PBS with 0.05% Tween 20 and 0.1% bovine serum albumin (1 h, 37 ~ C). After washing the conjugate binding was determined as described earlier.

Competition experiments with sugar residues were performed by simultaneous incubation of peroxidase conjugated MoAb and monosaccharides in a concentration range from 0.0003% to 1% in PBS-Tween. The following sugars were tested: neuraminic acid, galactose, N-acetylgalactosamine, fucose, mannose and glucose. Antibody binding was determined as described earlier.

Results

Reactivity in normal colonic mucosa

Init ial ly, the react iv i ty o f the 8 co lon m u c o s a react ive M o A b s was tested on f rozen sections as well as on paraf f in sections. N o signif icant differences were observed be tween the s taining pat terns on f rozen and paraf f in e m b e d d e d tissue. Subsequen t studies were therefore l imi ted to paraf f in e m b e d d e d mater ia l . Tab le 1 shows the react ivi ty pa t t e rn o f our M o A b s with n o r m a l co lon mucosa . F o u r dis t inct s ta ining pa t te rns could be discerned in n o r m a l co lon mu-

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cosa as i l lustrated in Fig. 1: diffuse cytoplasmic reactivity with all goblet cells ( P A R L A M 3, 9 and 10), focal supranuclear reactivity, especially with the crypt base goblet cells ( P A R L A M 8, 12 and 13), focal coarse granular staining with goblet cells ( P A R L A M 2), and a predominant ly apical immunoreact ivi ty with columnar cells ( P A R L A M 11). P A R L A M 12 and 13 also occasionally showed diffuse cytoplasmic staining with goblet cells limited to the top of the crypts. Wi th the exception of P A R L A M 8, all monoclonal ant ibodies reacted with the major i ty of normal colon tissue specimens (Table 1).

MoAb reactivity in various species

In compar ison with human intestinal mucosa, mouse and rat small and large intestinal mucosa reacted only weakly with P A R L A M 11 and 13. P A R L A M 11 reacted focally with goblet cells and the apical side and brushborder of columnar cells. P A R L A M 13 showed supranuclear reactivity especially in superficial goblet cells in colon and on villi

Table 1. Staining pattern of PARLAM MoAbs on normal colon epithelium

Immunoreactivity pattern No of specimens reactive with PARLAM

3 9/10 8 12 13 2 11

Columnar cells

Brushborder overall . . . . . . 5 Brushborder focal . . . . 2

Goblet cells

Mucus overall 8 7 - - - 1 - Mucus focal 1 2 - 1 2 - 1 Mucus superficial crypt - - - 5 2 - - Supra nuclear granules overall - - - 2 3 Supra nuclear granules focal - - t 2 Supra nuclear granules - - 3 2 2

sporadic Coarse granules overall . . . . . 2 Coarse granules focal . . . . 3 Coarse granules sporadic - - - 3 1

Total no staining 9 9 3 8a 9" 9 9

in the small intestine (data not shown). Other M o A b s did not react with mouse or rat tissues.

Reactivity in extracolonic epithelia

The pat tern of reactivity of our M o A b s on various human tissues is listed in Table 2. In small intestinal mucosa most M o A b s showed diffuse cytoplasmic staining of goblet cells. However, P A R L A M 11 did not show any reactivity in the small intestine. Bile duct and gall b ladder epithelium showed variable reactivity with the M o A b s in the apical cytoplasm of mucin producing cells. In the s tomach mainly a diffuse cytoplasmic reactivity with gastric foveolae was found; gastric glands did not show any reactivity. Goble t cells of respira tory epithelia in the nasal cavity of bronchus reacted with most of the MoAbs . Less reactivity was found with acinar cells in the prostate. The endocervical mucosa showed reactivity with P A R L A M 9, 10, 11, 12 and 13. The endometr ium showed occasional immunoreact ivi ty with P A R L A M 3, 9, 10 and 13 especially in the secretory phase. Other tissues reacted to a variable extent with P A R L A M 2, 9, 10 and 13. Pancreas, kidney, adrenal gland, ur inary bladder, epidermis, esophagus, thyroid, muscle, lymphnode, spleen and testis were entirely negative. I t is clear therefore that our panel of M o A b s reacts predominant ly with mucin producing cells. Exceptions were P A R L A M 9 and 10, which react with Hofbauer cells in the placenta, known to contain high molecular weight glycoproteins and PAR- L A M 2 and 13 which reacted with surface epithelial cells of ectocervix and esophagus. The M o A b s P A R L A M 2 and 12 occasionally bound to endothelium.

Fetal tissues

The results o f testing on a panel of fetal tissues are shown in Table 3. P A R L A M 9, 10 and 11 showed reactivity with fetal colon mucosa of various stages of development. Reac- tivity of P A R L A M 2, 3, 12 and 13 for colon tissues was confined to mid to end gestation. P A R L A M 2, 9, 10 and 13 consistently reacted with goblet cells of fetal small intestinal mucosa. P A R L A M 9, 10 and 13 also showed reactivity with the antral foveolae in the stomach. The reactivity patterns corresponded with those in adul t tissues (data not shown).

- = negative sporadic = 0- 5% of cells positive focal = 5- 80% of cells positive overall = 80.100% of cells positive

a In some specimens PARLAM 12 and 13 show more than one staining pattern

Colorectal adenoma and adenocarcinoma reactivity

The reactivity of our M o A b s with neoplast ic colonic lesions is listed in Table 4. In compar ison with normal colon PAR- L A M 3, 9 and 10 showed decreasing immunoreact ivi ty in adenomas and even more so in carcinomas (Fig. 2). In con-

Table 2. Immunoreactivity of the various monoclonal antibodies in colon mucosa, colon adenomas and colon carcinomas

Percentage of specimens reactive with PARLAM

Nr. of 3 9/10 8 12 13 2 11 specimens

Normal colon 9 100 100 33 (33) 77 (44) 66 (44) 66 88

Adenoma 10 70 90 60 (60) 80 (80) t00 (90) 90 70

Carcinoma 13 46 46 61 (0) 46 (0) 69 (0) 77 77

Between brackets the percentage of specimens with supranuclear staining

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t ras t , r eac t iv i ty w i t h P A R L A M 8, 12 a n d 13 i nc r ea sed in a d e n o m a s a n d c a r c i n o m a s (Fig. 3). A l so the p a t t e r n o f im- m u n o r e a c t i v i t y d i f fe red f r o m t h a t in n o r m a l c o l o n m u c o s a . T h e l imi ted s u p r a n u c l e a r a n d c y t o p l a s m i c reac t iv i ty in n o r - m a l c o l o n (Fig. 1 B) c h a n g e d to i n t ense s u p r a n u c l e a r s t a in - ing in a d e n o m a s (Fig. 3A) . In c a r c i n o m a s m o s t l y ap ica l c y t o p l a s m i c a n d m e m b r a n e s t a in ing was o b s e r v e d (Fig. 3 B). T h e n u m b e r o f t i ssues wi th i m m u n o r e a c t i v e cells as well as the ex t en t o f i m m u n o r e a c t i v i t y in a g iven neo- p l a s m t e n d e d to d i m i n i s h in c o m p a r i s o n w i th a d e n o m a s . T h e p r o p o r t i o n o f spec imens reac t ive for P A R L A M 2 a n d 11 was s imi la r in n o r m a l m u c o s a , a d e n o m a s a n d ca rc ino - mas . In a d e n o m a s a n d c a r c i n o m a s P A R L A M 2 s t a in ing was focal c y t o p l a s m i c as well as apical . P A R L A M 11 pre- d o m i n a n t l y s h o w e d apica l m e m b r a n o u s react iv i ty . All tes ted co l lo id c a r c i n o m a s reac ted w i th all M o A b s .

D i s t r i b u t i o n o f i m m u n o r e a c t i v i t y in o t h e r c a r c i n o m a s

I m m u n o r e a c t i v i t y o f o u r M o A b s a p p e a r e d n o t to be re- s t r ic ted to c o l o n n e o p l a s m s ( T a b l e 5). M o s t M o A b s also r eac t ed w i th c a r c i n o m a s o f the s t o m a c h a n d gall b l adde r . All M o A b s s h o w e d reac t iv i ty w i th a s igne t r ing cell c a rc ino - m a o f the s t o m a c h . G o b l e t cells in a d e n o c a r c i n o m a s o f the l ung also d e m o n s t r a t e d c o n s i s t e n t i m m u n o r e a c t i v i t y . M u c i n o u s c y s t a d e n o m a s a n d c y s t a d e n o c a r c i n o m a s o f the o v a r y a n d e n d o m e t r i u m c a r c i n o m a s also s h o w e d focal im- m u n o r e a c t i v i t y w i t h m o s t o f the M o A b s . O t h e r c a r c i n o m a s were nega t ive ( T a b l e 5).

Immunoblotting and gel filtration chromatography

G e l f i l t r a t i o n c h r o m a t o g r a p h y was p e r f o r m e d w i t h ex t rac t s o f 5583-S cells a n d n o r m a l c o l o n m u c o s a . W i t h these p r e p a - r a t i o n s a l m o s t iden t i ca l resu l t s were o b t a i n e d excep t for P A R L A M 8, 12 a n d 13, w h i c h were n o t p r e s e n t in de tec t - ab le a m o u n t s in n o r m a l h u m a n c o l o n ex t rac t s u n d e r o u r e x p e r i m e n t a l cond i t i ons .

G e l f i l t r a t i o n e lu t i on p a t t e r n s o f P A R L A M t a r g e t an t i - gens a re s u m m a r i z e d in T a b l e 6. M o s t a n t i g e n s e lu ted in the v o i d v o l u m e ( P A R L A M 2, 3, 11, 12 a n d 13) o r in the f r ac t i ons i m m e d i a t e l y t h e r e a f t e r ( P A R L A M 8 a n d 10) cor- r e s p o n d i n g to a mol .wt , exceed ing 5000 k D a a n d all be fo re the m a j o r p r o t e i n peak . T h e a n t i g e n s o f P A R L A M 9 a n d 10 e lu ted in the a s c e n d i n g s lope o f the p r o t e i n peak , cor re - s p o n d i n g w i t h a mol .wt , o f 2 - 5 0 0 0 k D a .

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Table 3. Immunoreactivity of P A R L A M monoclonal antibodies with normal human tissues

Tissue type N o of specimens reactive with P A R L A M

N 3 9/10 8 12 13 2 11

Small intestine 3 3 3 2 3 3 Gall bladder 9 2 8 2 8 4 7 Stomach 7 0 6 2 0 4 4 1

Bronchus 4 2 4 2 - 4 4 4 Nasal mucosa 4 1 2 1 1 4 4 3 Prostate 3 1 1 3 3 3 3 - Endocervix 3 0 3 0 0 3 3 3 Endometrium 8 1 1 - 1 5 Serous salivary gland i 1 - - 1 1 1 Breast 4 - 1 ] - - 2 - Acinar pancreas 3 - 2 - 2 2 - Oesophagus mucous 2 2 - - 1 - -

gland Skin sweat gland 4 nt 4 - 2 nt Ectocervix keratinizing 2 . . . . 2 2 -

cell borders Oesophagus keratinizing 3 . . . . 3 3 -

cell borders Placenta Hofbauer cells 2 2 -

nt-~ not tested

Table 4, Immunoreactivity of the MoAbs with fetal and newborn tissues

P A R L A M Organ Mid Late Newborn gestation gestation

3 Colon - + + Small intestine - - + Stomach - - -

9/10 Colon + + + Small intestine + + + Stomach + + +

12

Fig. 1 A-J. Immunoreactivity of P A R L A M MoAbs on normal colon mucosa (immunoperoxidase, counterstained with hematoxy- 13 lin). A Immunoreactivity for P A R L A M 113. Note diffuse cytoplasmic reactivity of all goblet cells ( x 100). B Immunoreactivity for P A R L A M 3. The pattern of staining closely corresponds to that of P A R L A M 10 ( x 100). C Detail of B. Note slight inhomogeneity 2 of staining of the mucin goblets ( x 400). D Immunoreactivity for P A R L A M 8. Note granular supra nuclear staining of a proport ion of the goblet cells (arrows; x 100). E Immunoreactivity for PAR- LAM 12. The pat tern of staining corresponds closely to that of 11 PARLAM 8 ( x 100). F Detail of E. The immunoreactivity is dis- tinctly granular ( • 400). G Immunoreactivity for P A R L A M 11. Note apical and apicolateral staining of columnar cells (x 100). H Detail of G ( x 600). I Immunoreactivity of P A R L A M 2. The pattern (granular supranuclear staining of some goblet cells) resem- bles that of P A R L A M 8 (x 100). J Detail of I ( x 400)

Colon Small intestine + and - - Stomach + and - -

Colon + Small intestine - Stomach

Colon + Small intestine + Stomach +

+ a n d - - -

+ and - --

+ and - - + + + +

Colon + and - - - Small intestine + + + Stomach - - -

Colon + + + Small intestine - - - Stomach - - -

+ =present in 75-100% of the specimens tested + and - = present in about 25-75% of the cases tested - = present in less than 25% of the specimens tested

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Fig. 2A and B. Immunoreactivity pattern of PARLAM 3 with an adenoma (A) and a carcinoma (B) respectively. Note supranuclear staining of mucin vacuoles in a varying proportion of tumor cells. Significant heterogeneity is apparent in the carcinoma (B). Immunoperoxidase, counterstained with hematoxylin ( x 100)

Fig. 3A and B. Reactivity of PARLAM 8 with an adenoma (A) and a carcinoma (13) respectively. Note fine granular supranuclear immunoreactivity in adenoma cells (A) and predominance of apical membrane associated reactivity in the carcinoma (B). Immunoperoxidase counterstained with hematoxylin (A x 100; B x 250)

Antigens of P A R L A M 3, 9 and 10 appeared to be heat sensitive and therefore boiling was omitted. Under these conditions target antigens in a western blot appeared as a single protein band of 87 kDa (Fig. 4). Of all other MoAbs the target antigens did not enter the gel, consistent with a mol.wt, of over 5000 kDa.

Competition experiments

Binding of conjugated P A R L A M 9 could be blocked completely by unconjugated P A R L A M 10. In contrast PAR-

LAM 9 did not block binding of conjugated P A R L A M 10. The same phenomenon was observed with P A R L A M 12 and 8. P A R L A M 8 blocked binding of conjugated PAR- LAM 12 but P A R L A M 12 did not block binding of PAR- LAM 8. In other combinations monoclonal antibodies did not or only weakly inhibit each others binding. Therefore, P A R L A M 9 and 10 and P A R L A M 8 and 12 were either directed to the same epitope but with different affinity, or the epitopes o f the MoAbs partially overlapped. Other P A R L A M antigens were unique.

TabLe 5. Immunoreactivity of various neoplastic human tissues

Tumor type No of specimens reactive with PARLAM

Nr. of 3 9/10 8 12 13 2 11 specimens

Stomach carcinoma 6 2 2 3 0 4 5 5

Intestinal metaplasia 3 1 3 1 1 2 2 0 stomach

Gall bladder carcinoma 2 1 1 0 1 1 1 1

Lung adenocarcinoma 4 nt 1 3 2 4 nt 2

Endometrium 9 0 3 1 0 7 9 3 carcinoma

Mucinous cystade- 4 2 2 1 0 2 3 0 noma ovary

Serous cystadenoma 1 0 0 0 0 0 0 1 ovary

Fibrocystic breast 6 0 2 2 0 2 2 0 disease

Ductal breast 4 0 1 1 0 2 0 0 carcinoma

Lobular breast 2 0 0 2 0 2 0 1 carcinoma

Salivary gland 4 0 0 3 0 2 0 0 carcinoma

Lund squamous cell 4 0 0 0 0 3 2 0 carcinoma

nt = not tested

Table 6. Elution profiles of MoAbs on 5583-S and normal human colon extracts

Molecular weight category a

>5.106 Da <5.106 Da <700 kDa (void volume)

5583-S extract

2 + - 3 + - 8 + - 9/10 - +

Ii + -

12 + - 13 + -

Normal human colon extract

2 + - 3 _+ - 8 - + 9/10 - +

11 + - 12 - - 13 - -

m

m

m

m

m

m

m

m

Determined according to the elution position of the molecular weight markers Blue Dextran (2 x 106 Da), thyroglobulin (700 kDa) and ovalbumin (45 kD)

Antigen identification

Table 7 summar izes the effect o f var ious t rea tments on antigen reactivity. The react iv i ty o f the target epi topes o f P A R - L A M 2 and 3 was reduced by heating, pe r ioda te oxyda t ion

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Fig. 4. SDS-PAGA and Western blotting of PARLAM 3, 9 and 10 material in a normal colon mucosa extract. Immunoreactivity occurs as a distinct band of 87 kDa for all three MoAbs. Towards the left the position of the molecular weight markers is indicated. The arrow indicates the position of the stacking gel

Table 7. Influence of various antigen treatments on MoAb reactivity in ELISA a

PARLAM Heating Pronase Periodate oxydation

2 21 33 42 3 61 77 68 8 5 --1 78 9 68 46 0

10 79 35 0 11 14 4 - 10 12 0 10 15 13 - 7 15 89

a Difference between the reactivity of untreated and treated antigen, expressed as a percentage decrease of the reactivity of the untreated antigen

and p ronase digest ion, suggest ing tha t in its s t ructure car- bohydra t e as well as pro te in c o m p o n e n t s are invo lved or tha t the ep i tope is confo rma t iona l . The target epi tope o f P A R L A M 8 and 13 was only affected by pe r ioda te oxydation, indica t ing tha t it migh t be a ca rbohydra t e moie ty . The target ep i tope o f P A R L A M 9 and 10 was affected by heat ing as well as p ronase digest ion, suggest ing tha t the ep i tope resides on a p ro te in moiety . The epi topes o f P A R - L A M 11 and 12 were no t affected by any t rea tment .

Ta rge t ant igen digest ion with galac tos idase reduced b inding only o f P A R L A M 13. Othe r exoglycosidases did no t affect M o A b binding. The b inding of P A R L A M 8 was reduced by P N L and o f P A R L A M 12 and 13 by P N L and SBA. Ga lac tose reduced b inding o f P A R L A M 13 only. Other sugars did no t affect b ind ing o f any o f the M o A b s .

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Discussion

Goblet cells represent one of the major cell types of the human colon epithelium and their main product, mucin, plays an important role in normal colon function. Early studies on colonic mucin from various species suggested that colonic mucin consists of a single high molecular weight glycoprotein complex (Allen et al. 1976). More recent studies, however, have shown that colonic mucin con- tains various different glycoproteins (LaMont and Ventola 1980). Podolsky and Isselbacher (1984) purified mucin from human colon mucosa and found at least six different mucin species, each with a distinctive hexose, hexosamine and aminoacid composition. Monoclonal antibodies raised against these mucins allowed the recognition of a striking degree of heterogeneity among colon goblet cells (Podolsky et al. 1986a). In spite of increasing attention for structural and functional properties of mucin the knowledge concern- ing the molecular structure of mucin associated glycoproteins and their function in health and disease remains rather fragmentary.

Considerable attention has been paid to changes in mucin profiles in colon epithelial neoplasms and in the normal mucosa adjacent to these tumours. Abnormalities have been documented in adenomas and carcinomas as well as in the so-called "transitional" mucosa using conventional histochemical staining techniques (Reid etal. 1985; Greaves et al. 1980; Lapertosa etal. 1986) lectin probes (Boland et al. 1982, 1984; Bresalier et al. 1985) and immunocyto- chemical techniques.

In the latter, antisera have been used which were raised against high molecular weight glycoproteins such as CSA-p (Shochat et al. 1981), GOA (Rapp et al. 1979), M1 and M3 (Bara et al. 1980, 1986). Very few mucin reactive MoAbs have been described following immunization with colon cancer cells. Various MoAbs, induced against ovarian (Ma et al. 1983; Gangopadhyay et al. 1985; Mattes et al. 1985) and mammary (Hilkens et al. 1984; Stramignoni et al. 1983) carcinoma were reported to react with mucin epitopes, also present in normal colon goblet cells.

We have generated a panel of monoclonal antibodies, which identify epitopes on high mol.wt, glycoprotein antigens in colon mucosa. Most MoAbs (PARLAM 3, 9, 10, 12, 13) reacted with goblet cells in colon and small intestinal mucosa. PARLAM 8 reacted with goblet cells in some normal colon specimens but not in small intestinal mucosa. PARLAM 11, however, reacted with brushborders of columnar cells in colon mucosa but not with columnar cells in the small intestine. Variable patterns of reactivity were found for the various MoAbs with other tissues. Outside the endodermally derived epithelia only limited reactivity occurred. Notable exceptions were isolated prostatic glands (possibly corresponding to the utriculus), endocervix and endometrium, all derived from the Mfillerian duct. This pattern of expression was also reported for M1 antigens of gastric mucosa (Bara et al. 1986).

According to their staining characteristics and the available biochemical characteristics of the target antigen the MoAbs were classified into four groups. Group I consists of PARLAM 3, 9 and 10. By Sepharose 4B gelchromatography the target antigens appear to be high molecular weight (2000-5000 kDa) glycoproteins. In the Western blots these MoAbs react with a 87 kDa protein. The protein nature of the epitope is corrobated by the heat lability and

the sensitivity to pronase digestion. Homologous and heter- ologous competition experiments show that the epitopes of PARLAM 9 and 10 are closely related.

These monoclonal antibodies react with most goblet cells in intestinal and colon mucosa and in this regard are similar to the MoAb 2, 3, 5, 11 and 12 from the Podolsky series (1986 a). The target antigens appear rather late during embryonal development: in the last trimester of gestation. Neoplastic colon epithelium shows reduced expression of these glycoproteins. A similar phenomenon was noted by Hughes (1986) and Richman and Bodmer (1987) in demon- strating that the expression of normal colon mucosal antigens in colon adenomas and carcinomas is reduced.

Group II consists of PARLAM 8, 12 and 13. By Sephar- ose 4B gelchromatography, these MoAbs appear to react with large (> 5 x 106 Da) glycoproteins. The decreased immunoreactivity of the target antigens after periodate oxydation suggests that the target epitopes reside in the carboxy- hydrate moiety, which is corroborated by the thermal stability and pronase resistance. Competition experiments with lectins and disaccharides as well as sensitivity for galactosidase suggest that galactose and N-acetylgalactosamine may be part of the epitope structure of the PARLAM 12 and 13 antigens. By immunohistochemistry these MoAbs stain the supranuclear regions of goblet cells, suggestive of a Golgi complex localization. This staining pattern corresponds with those of the galactosamine and N-acetylgalactosamine binding lectins Vicia villosa agglutinin and Grif- fonia simplicifolia agglutinin, which have been found to bind only to the Golgi complex (Kellokumpu et al. 1986). It is tempting to suggest that our group II MoAbs react with glycoproteins in different phases of posttranslational pro- cessing. The PARLAM 8, 12 and 13 antigens are expressed in colon and/or small intestine only during early develop- mental stages. Similar observations were reported for M1 and M3 antigen (Bara and Burtin 1980). PARLAM 8, 12 and 13 showed an increased reactivity in neoplastic colon mucosa and therefore may be of value to study alterations in the synthesis of mucin related glycoproteins accompany- ing neoplastic transformation, especially in the colon.

Group III consists only of PARLAM 2. The target epitope of this MoAb, resides on a large (> 5 x 10 6 Da) complex glycoprotein. We were not succesfull in our attempts to further characterize the target epitope of this MoAb. The striking granular immunoreactivity in goblet cells corresponds to that of MoAbs 4, 9 and 14 of Podolsky's series (1986a, b). It is of interest that PARLAM 2 antigen is extensively expressed in fetal small intestine but only occasionally in mid gestation in the fetal colon. The reactivity in normal tissues is not restricted to intestinal mucosa. In carcinomas fairly extensive expression, not limited to tumors in the gastrointestinal tract is found. In the colon expression is more extensive in neoplastic than in normal mucosa. In this regard further exploration of the signifi- cance of expression of this antigen in neoplasia and identification of the target epitope are warranted.

Group IV consists of PARLAM 11, which reacted with a large (> 5 x 106 Da) glycoprotein. Further identification of the target epitope was unsuccessfull. PARLAM 11 shows reactivity with the apical plasma membrane of colon columnar cells, comparable to the pattern obtained with MoAb 8 of Podolsky (1986a, b). Strikingly, columnar cells in the small intestine do not show immunoreactivity, neither in adult nor in fetal tissues. This antibody may be of interest

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in the study of differentiat ion in normal colon mucosa and in the development of the various types of intestinal metaplasia.

F o r glycoprotein reactive ant ibodies the possibil i ty that they react with epitopes related to b loodgroup antigens must be excluded (Feizi 1985). Compar ison of the staining pat terns of our monoclonal ant ibodies with the tissue localization of the A B H and Lewis antigens, suggests that they do not react with these antigens (Cordon-Cardo et al. 1986; Yuan et al. 1985), except for P A R L A M 12 which occasionally reacts with vascular endothel ium and other s t romal components . Fur thermore , Lewis A and Lewis B antigens have a lower molecular weight than we found for the PAR- L A M antigens (Blaszczyk et al. 1985). More direct tests will be necessary to completely exclude reactivity of our M o A b s with b lood group antigens.

In summary, we have generated a panel of monoclonal ant ibodies directed against human high molecular weight glycoproteins, with a dis t r ibut ion largely restricted to goblet cells in epithelia of the gastrointest inal tract. Some antibodies ( P A R L A M 3, 9, 10) show reduced expression in colon adenomas and carcinomas and might be useful for the study of goblet cell differentiation in neoplast ic colon mucosa. Other ant ibodies ( P A R L A M 8~, 12, 13) show en- hanced expression in neoplast ic colon mucosa and might be used to study al terat ions in mucin product ion during early development and the progression of neoplasia in colon epithelium. We are currently investigating the use of these M o A b s in the immunohis topathologica l assessment of neoplasia in the gastrointest inal and urogenital tract.

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Colonic epithelium reactive monoclonal antibodies