ACTA HISTOCHEM. CYTOCHEM. Vol. 3, No. 3, 1970

IMMUNOHISTOCHEMICAL INVESTIGATION OF AMINOPEPTIDASE

TOSHIOSUZUKI, KUNIOTAKANO AND KENJIROYASUDA

Departmentof Anatomy,School of Medicine,Keio University,Shinjuku, Tokyo

Receivedfor PublicationJune 2, 1970

The localization of aminopeptidase was examined on the porcine intestine by use of fluorescent antibody method. Aminopeptidase was present diffusely in the striated border of mucous epithelial cell of and , supra-nuclear region of epithelial cell, intestinal glandular cells and in . The condensed materials in the crypts showed specific fluorescence. The was not always observed in every cell mentioned above, and the distribution pattern of antigen varied according to the stage of . The enzyme was concentrated in the goblet cells of the but not in those of the . Brun- ner's gland of did not contain any aminopeptidase. Though amino- peptidase in kidney and pancreas had immunological common factor with that in the intestine, specific fluorescence was not observed on the tissue sections. For comparative purposes, histochemical staining was carried out by means of Burstone and Folk's method. As a result, the goblet cell of the small intestine did not show any cytochemical reaction, though the specific fluores- cence which showed the site of the enzyme protein was concentrated in it. As far as the striated border of both mucous epithelial cell and intestinal glandular cell is concerned, the results of the cytochemical reaction coincided with those obtained by immunohistochemical study. The reason for the discordance with regard to the distribution of cytochemical reaction and the immuno- fluorescence in the goblet cell remained unknown.

There have been very few bistochemical investigation on the localization of exo-and endopeptidases. Burstone and Folk (1956) have demonstrated the location of aminopeptidase on the tissue by using L-leucyl-ƒÀ-naphthylamide and DL-alanyl- naphthylamide as the substrate. From the same purpose, Nachlas et al. (1957, 1960) used L-leucyl-4-methoxy-ƒÀ-naphthylamide as a substrate. Takamatsu (1963) has reported a histochemical method for the demonstration of proteinase, using silver protein as a substrate. More recently, the immunohistochemical study on the localization of trypsinogen and chymotrypsinogen in the acinar cell of porcine

pancreas by means of the fluorescenct antibody method, has been made by Yasuda and Coons (1965). Further, by the same technique, was demonstrated in the bovine (Yasuda, Suzuki and Takano, 1966). Though, a number of attempts for the demonstration of peptidase or proteinase have been tried by many investigators from the early years of the histochemistry, and though there are many excellent techniques for the demonstration of enzymes

130 IMMUNOHISTOCHEMICAL STUDY OF AMINOPEPTIDASE 131

other than protein catabolising enzymes, the histochemistry of protein and protein catabolising enzyme is not yet well developed. This study dealt with the localization of aminopeptidase in the porcine intest- inal mucosa by means of fluorescent antibody technique. In addition, the results were compared with those obtained by the histochemical method developed by Burstone and Folk (1956) for the demonstration of aminopeptidase.

MATERIALS AND METHODS

The antigen used in this study was crystalline porcine aminopeptidase of a commercial source (Sigma Co.), prepared from the intestinal mucosa. According to the data sheet from the company, 1 unit will liberate 1 ƒÊ mol ƒÀ-naphthylamine

per min from L-leucyl-ƒÀ-naphthylamide at pH 7.0 at 37•Ž. 40mg of the antigen dissolved in the phosphate buffered saline was mixed with an equal volume of Freund's complete adjuvant and injected intramuscularly into rabbits. A week after the first injection, 10mg of antigen dissolved in the same solution as mentioned above was mixed with an equal volume of incomplete adjuvant and injected in the same manner. This procedure was repeated four times in order to obtain as high titer antiserum as possible. Five weeks after the first injection, each rabbit recieved an intravenous injection of 10mg of the antigen dissolved in the same buffer as the final booster injection. Six days after the final injection, the animals were bled by heart puncture. The serum was separated and stored in the presence of merthiolate at the concentration of 1: 10,000 at -20•Ž until use. The antiserum contained 70mg/ml of protein and had a titer of 1: 163,840 by the passive hemagglutination test of Stavitsky (1954), using 10mg of antigen. This anti-aminopeptidase serum was conjugated with FITC and purified by the chromatography on Sephadex G-25 (Yasuda et al., 1963, 1964, 1965) and DEAE cellulose columns according to the technique of McDevitt et al. (1963). The molar F/P ratio (McDevitt et al., 1963) of the conjugated antibody solution was 0.92. Precipitin reactions were carried out on the Ouchterlony plate using 0.8 per cent Noble agar (Difco Co.). Consequently, two precipitin bands were encountered between antigen and antibody (Figl 1). The frozen sections of porcine intestine were cut at 4ƒÊ thick in a cryostat at

-18•Ž . The other blocks were fixed with several kinds of fixatives at 0•Ž, de- hydrated with ethanol and embedded in paraffin. The fixatives employed in this study were twenty-two kinds. Among these fixatives, 1 per cent glacial acetic acid in 99 per cent ethanol was the most satisfactory for the preservation of the antigen. The hematoxylin-eosin staining, PAS staining and toluidine blue staining were carried out to observe the histological detail of the porcine intestine. The histo- chemical method to demonstrate aminopeptidase (Burstone and Folk, 1956) was also applied to the tissue to compare with the immunohistochemical results.

RESULTS In general, the histological pattern of porcine small intestine is rather similar to that of cat and dog than that of man. Goblet cells are abundant, while the Paneth cells are very scanty or almost lacking. Furthermore, the most characteristic feature 132 SUZUKI, TAKANO AND YASUDA IMMUNOHISTOCHEMICAL STUDY OF AMINOPEPTIDASE 133 of pig small intestine is that the crypt of Lieberkuhn is much longer than that of man and other higher vertebrates. Duodenum: The epithelial cells covering the free surface of the villi are simple columnar. The wall of the crypt of Lieberkuhn is lined with a low columnar epithelial cells. The striated border is noticed at the free surface of the epithelial cells of the and of cells covering the crypt of Lieberkiihn. The few goblet cells are irregularly scattered among the mucous epithelial cells and intestinal glandular cells. The observation by fluorescence microscope revealed the concentration of antigen in the goblet cells. But, the specific fluorescence is not encountered in both mucous epithelial cells and the intestinal glandular cells. The striated border of the mucous epithelial cells and intestinal glandular cells is occasionally bright. Es- pecially, the fluorescence recognized on the striated border of the cells lining the crypts of Lieberkuhn is intensely bright. The fluorescenct materials are not seen in lamina , smooth muscle fibers, and in blood cells.

Jejunum and ileum: The histological features differ much from those of the duodenum except for the presence of Brunner's gland. The intestinal crypt is much deeper than that of the duodenum. Goblet cells are much more abundant than in duodenum. The abundance of goblet cells resembles to the histological pattern of human colon. Bright green fluorescence is observed in the striated border and in the cytoplasm of the supra-nuclear region of each goblet cells and mucous epithelial cell (Figs. 2,3, and 4). The cells which line along the crypt are filled with fluorescent material. The apical portion of cytoplasm of Paneth cell-like cells situated at the bottom of the intestinal crypt is also fluorescent (Fig. 2). The masses of secretory products which are projecting into the crypt and lumen of the show brilliant flu- orescence (Fig. 3). The nucleus of every cell shows the negative image. Large intestine: Goblet cell, mucous epithelial cell, intestinal glandular cell and striated border of these cells are quite free of antigen.

Fig. 1. Precipitin reaction on the agar plate (Ouchterlony technique). Large center cup contained anti-aminopeptidase serum, surrounding cups are filled with different dilutions of the antigen, con-

taining 20mg/ml, 10mg/ml, 5mg/ml and 2.5mg/ml respectively clockwise from the upper left cup. Two precipitin lines are found between antigen and antiserum. Fig. 2. The cross and longitudinal section of the intestinal gland of the porcine ileum. In the intestinal crypt, diffuse brilliant specific fluorescence is observed in the goblet cells. And

intense fluorescence is also found in cytoplasm of the cells lining along the crypt. On the upside of this figure, the cross section of secretory portion of the gland of Lieberkiihn is observed. Besides the findings that specific fluorescence is found in goblet cells, Paneth cell-like cell (P) is stained with labeled anti-aminopeptidase serum. The lumen of the intestinal gland is partly filled with the material which shows the bright specific fluorescence. •~250. Fig. 3. The crypt of porcine intestinal gland is shown in this picture. The secretory product

excreted from goblet cells shows the bright specific fluorescence, which is going to spread over the striated border. •~400. Fig. 4. The crypt of porcine intestinal gland. Specific fluorescence is observed in goblet cells and along the striated border of crypt cells. •~400. 134 SUZUKI, TAKANO AND YASUDA

Heterogeneous organ:

As an organ which has aminopeptidase, the porcine kidney was selected and stained with labeled gamma-globulin solution. As a result, only autofluorescence was seen in the wall of blood vessels, but no specific fluorescence is seen in the kidney. Further, the results obtained by this method was compared with those by histo- chemical technique developed by Burstone and Folk. The intense reaction was observed in the striated border of mucous epithelial cells and intestinal glandular cells of duodenum, jejunum and ileum, showing rough granular pattern. But no reaction was observed in Brunner's gland of duodenum. Moderate enzyme activity was seen in the basal part of cytoplasm of goblet cells, while no enzyme reaction was seen in the supra-nuclear region of goblet cells. And, both the secreted materials

projected from goblet cells and the materials condensed in the crypt indicated no reaction at all. Therefore, as for the striated border, the sites of the enzyme reaction coincide with the localization of specific fluorescence. But, two results are quite contrary to each other in the goblet cells.

DISCUSSION

The presence of peptidase in the Paneth cell has been suggested in the biochemi- cal field by and LinderstrƒÓm-Lang Holter (1931, 1934). Paneth cells were dis- covered at the bottom of intestinal crypts, first by Schwalbe (1872), and a complete review was presented by Patzelt (1936). It is said that its structural organization is akin to a cell type such as the pancreatic acinar cell which secrets protein-rich materials. And it is suggested that this cell, rich in remarkable secretory granules, rough surfaced endoplasmic reticulum and well defined Golgi apparatus, is a highly differentiated cell which has a large secreting potential. Notwithstanding, with respect to the functional significance of the secretory products of the Paneth cell, there are various opinions. Van Genederen and Engel (1938) admit that the Paneth cell produces a proteolytic exoenzyme (dipeptidase), Miram (1912) suggested that the Paneth cell secrets lipase. Clara (1926) and Baecker (1934) believed that the nature of the secretory granules were related to glycoprotein. Florey, Wright and

Jennings (1962) reported that the Paneth cell contains amylase and enterokinase. Prenant (1907) and Hertzog (1937) believed that the Paneth cells were a specialized

goblet cell and produced mucous. Further, recently, Creamer (1967) has postulated that the Paneth cells are specialized nutrition units that produce the secretory substance rich in protein and carbohydrate which acts as a favourable luminal envi- ronment for crypt cells and also the epithelial cells on the villi. Riecken and Pearse

(1936) observed the presence of many kinds of enzymes in the Paneth cell by means of conventional histochemical technique. And Miller et al. (1960) noticed the

presence of zinc in the cytoplasm. Spicer et al. (1967) proved that the granules of the Paneth cell have some similar properties to lysosomes. On the contrary, no cytochemical and histochemical investigation on the goblet cell has been performed. Furthermore, the relationship between the Paneth and

goblet cells has been much obscure. Bizzozero (1889) stated the granulous cells must be the young form of goblet cells. Kull (1911, 1912) has admitted the reverse relationship between these two cells, i.e., goblet cells are formed first and become Paneth cell after several phases during the transition. Further, de Castro (1959) IMMUNOHISTOCHEMICAL STUDY OF AMINOPEPTIDASE 135 has made a histochemical investigation on the Paneth cell of the ant bear which is an suitable material for research because of the richness in Paneth cells. As a con- sequence, he has demonstrated the existence of a functional cycle between the Paneth cell and the goblet cell, admitting that the latter originates from the former. According to him, it is quite easy to identify the transitional forms. More recently, in the light and electron microscopic investigation on the Paneth and goblet cell in mouse duodenal crypts, cells which show the intermediate structure between un- differentiated cells located on the lateral wall of crypt and goblet cells, and between undifferentiated cells at the bottom of crypt and the Paneth cells were identified. Their light and electron microscopic features are described by Troughton (1969). He stated that the differentiating cells with prominent secretory granules possessed morphological features which did not permit their precise identification as either goblet or Paneth cells. These cells were arbitrarily named "intermediate cells" by Troughton (1969). As the results of his radioautographic investigation, he con- cluded that the differentiated Paneth cells do not normally proliferate, but, instead, arise by differentiation from undifferentiated crypt cells or from intermediate cells. Further, Troughton (1969) has described from a electron microscopical point of view as follows: the dense cores of goblet cell granules were generally less than 0.5ƒÊ in diameter, while much larger cores were seen in Paneth cell granule. And unlike Paneth cell granules, adjacent goblet cell granules often distorted and oc- casionally appeared to fuse with each other. The main difference of the structure among the intermediate cells and differentiated Paneth and goblet cells is that the granular endoplasmic reticulum in the latter two kind of cells is not so well developed as in the former cells. Instead, many unattached ribosomes lie free in the cyto-

plasm. Thus, many histological, histochemical and electron microscopic investigations have been made to find the morphological similarities and differences between Paneth cell and goblet cell. However, very little is known with regard the origin and functional relationship of both cells. As far as the Paneth cell-like cells are concerned, which are seen at the bottom of the crypts of porcine small intestine used in this study, the nature is quite unknown. The fine granules in these cells, which are stained red with hematoxylin-eosin and Mallory-azan staining, and also stained pale blue with toluidine blue staining are rather similar to mucin granules of goblet cells than those of Paneth cells. While the stainability of the granules to toluidine blue resembles that of the granule in Paneth cell. Accordingly, it can be thought that these cells correspond to the undifferentiated Paneth cell or the intermediate cell as described by Troughton (1969), or to the transitional form of the goblet cell and Paneth cell as suggested by de Castro (1959). As mentioned above, there have been several reports on the nature and the relationship of the goblet cells and the Paneth cells. But, the fact that aminopeptidase was found in the Paneth cell-like cells of porcine intestine in this study, supports the opinion which believes the pre- sence of the intermediate type between the two.

From early years, consensus of opinions has not been seen concerning the presence or absence of Paneth cells in the small intestine of the pig. Baecker (1934) and Clara (1933) confirmed the presence of Paneth cell. On the other hand, Moller

(1889), Martin (1910), Tehrer (1929), Wheeler (1964) and Creamer (1967) have denied the presence of Paneth cell. Creamer (1967) reported that Paneth cells 136 SUZUKI, TAKANO AND YASUDA were absent in pig as in dog, birds and fish. In addition, he stated that, man, rat and mice have very similar structure with short crypts, and that in the animals without Paneth cells such as cat, dog and pig, a different kind of mucous has developed. In the latter animals, the mucous epithelium is thick with very long crypts and many goblet cells are distributed scatteringly along the long crypts. The Paneth cell-like cells found at the bottom of the crypt in this study were very few, and their secretory granules were not well developed. Accordingly, the func- tional role of these cells is considered to be very small. This fact contrasts markedly with goblet cell. As described by Prenant (1907) and Hertzog (1937), if the Paneth cell is a specialized goblet cell, in the case, when differentiated Paneth cell is absent or very few and when Paneth cell is undifferentiated, goblet cell may act as the substitute for Paneth cell. If, as mentioned by de Castro (1959), a functional cycle exists between the Paneth and goblet cells, it would be suggested that the secretion and probably the production of peptidase will be carried out reciprocally by both cells. From the fact that specific fluorescence is not found in the goblet cells of large intestine, while strong specific fluorescence is seen in numerous goblet cells ofjejunum and ileum, it is suggested that the goblet cell may play a role as the protein catabolizer in porcine small intestine, but not in the large intestine. This suggestion is fairly exciting, since the goblet cell has been believed to contain only mucin which is destin- ed to be secreted on the surface of the mucous epithelium of the intestine to facilitate the passage of the intestinal content. In addition, the difference in the pattern of the distribution of the enzyme in the goblet cells, between small and large intestine, is suggestive of the difference of the function on the digestive activity in each part of the intestine. Lastly, the reason, why the histochemical reaction for aminopeptidase was negative in the goblet cell where the presence of specific fluorescence was recognized by the immunohistological method, remained unknown. The probable assumption is that the inactive form of aminopeptidase may be produced in the goblet cell and be activated by the intestinal juice as soon as it is secreted in the intestinal lumen, so that the histochemical reaction turns positive. The excreted enzyme may spread over the surface of the mucous epithelium, where the enzyme reaction is positive. Another possibility is that the discordance of the results may based on the difficulties as well as the accuracy of the techniques used.

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