Gut: first published as 10.1136/gut.16.7.553 on 1 July 1975. Downloaded from Gut, 1975, 16, 553-558 The Paneth cell: A source of intestinal lysozyme' T. PEETERS AND G. VANTRAPPEN From the Departement Medische Navorsing, Laboratorium voor gastrointestinale fysiopathologie, Katholieke Universiteit te Leuven, Belgium SUMMARY An antiserum prepared against lysozyme isolated from mucosal scrapings of mouse small intestine was used to stain sections of mouse small intestine with the indirect fluorescent anti- body technique. Mucosal fluorescence was confined to the base of the crypts of Lieberkiihn, where Paneth cells are located. After the intravenous administration of 4 mg of pilocarpine fluorescence was no longer found in the Paneth cell but in the crypt lumen. Perfusion studies confirmed these findings. The basal lysozyme output of 0-1 to 0 4 ,ug/ml was raised to peak rates of 1 8 to 6-5 ,g/ml after the intravenous administration of 1 mg of pilocarpine. Our results demonstrate that the lysozyme of the succus entericus is, at least in part, derived from the Paneth cell, and is probably present in the Paneth cell granules. Its secretion is stimulated by pilocarpine. Our model could be very useful for studying the function of the Paneth cell, which probably forms part of an intestinal defence system. The characteristic features of the Paneth cell, which antibody technique (Erlandsen, Parsons, and is located at the base of the crypts of Lieberkuhn in Taylor, 1974). man and some other animal species, is the presence In this paper the presence of lysozyme in the of large granules in the apical part. Paneth himself Paneth cell of mice, its secretion into the crypt lumen http://gut.bmj.com/ recognized their secretory function as early as 1888 and its ability to serve as a marker of Paneth cell (Paneth, 1888). However, the contents of these function are demonstrated by the indirect fluores- granules and their function are still largely un- cent antibody technique and by perfusion studies. known. Speculative reasoning led Creamer (1967) to Materials and Methods suggest that Paneth cells regulate crypt cell turn- over, and experimental evidence for the secretion of FLUORESCENCE MICROSCOPY free amino acids by the Paneth cell has been pre- Lysozyme was isolated from mucosal scrapings of on September 30, 2021 by guest. Protected copyright. sented (Gent and Creamer, 1972). According to mouse small intestine by combined ammonium Riecken and Pearse (1966), the Paneth cell granules sulphate precipitation, ion exchange, and molecular fulfil the enzymatic criteria of lysosomes; they sug- sieve chromatography. The purity of the isolated gested that Paneth cells are intestinal glands par- product was established by cellulose acetate electro- ticipating in the breakdown of intestinal contents. phoresis, polyacrylamide electrophoresis, isoelectric However, Lewin (1969) was unable to find pro- focusing and gel filtration. The amino acid composi- teolytic enzymes in the Paneth cell. tion resembled that of hen's egg white lysozyme al- Speece (1964), who used an indirect histochemical though basic residues were more abundant. (Details method, was the first to suggest that lysozyme is of the isolation procedure and the characteristics of present in the Paneth cells of mice. This finding has this lysozyme will be published elsewhere.) been confirmed by cell fractionation studies (Deckx, Rabbits were immunized by the injection of 1 mg Vantrappen, and Parein, 1967), histochemical studies of pure lysozyme dissolved in 1 ml of NaCl 09 Y% using substrate films of chitin (Ghoos and and emulsified with 1 ml of complete Freund's Vantrappen, 1971), and recently by the unlabelled adjuvant (Difco Laboratories, Detroit, Mich, USA). The antigen was injected in the hind footpads and into several subcutaneous sites. This procedure was repeated one week later and the rabbits were bled 'Presented in part at the autumn meeting of the British Society for one week after the second injection. The mono- Gastroenterology, Birmingham, 1974 specificity of the antisera was tested by immuno- Received for publication 21 April 1975. diffusion and immunoelectrophoresis in agarose. 553 Gut: first published as 10.1136/gut.16.7.553 on 1 July 1975. Downloaded from 554 T. Peeters and G. Vantrappen The goat antirabbit immunoglobulin antiserum and Papapavlou, 1971). The method of Caraway labelled with fluorescein isothyocyanate was (1959) was used to determine amylase activity. The obtained from Nordic Pharmaceuticals and Diag- enzyme activites are expressed in ,ug/ml hen egg white nostics, Berchem (Belgium). lysozyme equivalents for lysozyme and as Somogyi Mice were killed with a sharp blow on the head, units per ml perfusate (SU/ml) for amylase. the small intestine was quickly removed and washed with ice-cold 09% NaCl. Small sections were snap Results frozen and cut in a cryostat at 4 ,ug. The tissue slices were fixed in cold ethanol for 20 minutes and FLUORESCENCE MICROSCOPY air-dried. After rehydratation they were covered A typical result obtained when sections of mouse successively with a drop of the antilysozyme anti- small intestine were stained immunochemically for serum and, after being washed with Coons buffer lysozyme is shown in figure 1. Fluorescence is found (pH 7 2), with the fluorescent antirabbit immuno- at the bottom of the crypts of Lieberkuhn, where the globulin. After a final washing they were mounted Paneth cells are located. In the other cells of the in aquamount (Gurr, Ltd, London, UK). Incuba- mucous membrane the nuclei are seen as dark spots tion was carried out in a humid chamber, at room surrounded by faint cytoplasmic fluorescence. In temperature, for 30 minutes. The antisera were spun the connective tissue there is also some fluorescence at 4000 g for 15 min and their optimal dilutions which is probably due to the presence of leucocytes. determined from a checkerboard titration. For both Fluorescence was absent when normal rabbit antisera a 1:8 dilution provided the most satis- serum was used instead of the specific antilysozyme factory results. antiserum or when the specific antiserum was pre- The preparations were observed in a Wild micro- incubated with the pure antigen. The intravenous scope (Heerbrugg, Switzerland) equipped with an ultraviolet light source (Osram HBO 200), a high aperture, dark field condenser (N.A.: 1P4, Wild), excitation filters BG 23, GG 23 (Leitz) and FITC (Wild), and a secondary filter FITC/313742 (Wild). Photographs were taken on Ektachrome EH-135 http://gut.bmj.com/ film with the aid of a photographic chamber with automatic exposure (Photomat Mka 5, Wild). PERFUSION TECHNIQUE Female mice of the NMRI-strain, weighing ap- proximately 20 g, were anaesthetized with urethane (0-1 mg/g body weight, injected intraperitoneally). on September 30, 2021 by guest. Protected copyright. A laparatomy was performed and the jejunum was incised 4 cm and 24 cm distal to the pyloric sphincter. Soft plastic tubes were ligated in place, and the inlet tube was connected to a roller pump. The 20 cm jejunal segment was perfused with physiological saline at a rate of 4 0 ml/hr. The outlet tube was connected to a fraction collector. Fractions of 1 ml were collected, and the sampling rate was recorded. In all experiments the output rate deviated little from the input rate, so that one fraction cor- responded to a perfusion of 15 minutes. Pilocarpine was dissolved in physiological saline and injected in one of the caudal veins. After each experiment the position of the inlet tube relative to the common pancreatic bile duct was checked stereoscopically. In all experiments contamination of the perfusion Fig 1 Frozen section. ofmouse small intestine, stained with rabbit antilysozyme antiserum and FITC-conjugated with bile or pancreas fluid could be excluded. goat antirabbit gamma globulins. The structure of the villi and the muscle layers are easily seen as a faint ENZYME ASSAYS backgroundfluorescence. The FITC-fluorescence is Lysozyme was assayed turbidimetrically using hen largely confined to the base of the crypts, where the egg white lysozyme as a standard (Gorin, Wang, Paneth cells are located ( x 1000). Gut: first published as 10.1136/gut.16.7.553 on 1 July 1975. Downloaded from The Paneth cell: A source of intestinal lysozyme 555 r [1 4.0o 10.0 0~~~~~~~~~~~~~~~~~~~~~I I. Fi2.0 F5.0 Fig TIE(hus 24 6~~~~~~~~~~~~6 1.0 2 6 Fig 4 TIME (hours) Fig 2 http://gut.bmj.com/ Fig 2 Frozen section of mouse small intestine, prepared five minutes after the intravenous administration of4 mg pilocarpine, and stained with rabbit antilysozyme and FITC-conjugated goat antirabbit gamma globulins. The lumen is at the lower right corner. One villus occupies the centre and the spaces between this villus and the two on September 30, 2021 by guest. Protected copyright. adjacent ones are filled with a stream offluorescent material, granular in appearance ( x 2000). Fig 3 Frozen section of mouse small intestine, 10 minutes afterpilocarpine administration, stained as described. The strongly fluorescent material is located at the tops of the villi ( x 2000). Fig 4 Effect ofpilocarpine upon the release of lysozyme (open bars) and amylase (closed bars) into the perfused small intestine of mouse. Perfusion was done with physiological saline at a constant input rate of4-0 ml/hr. Fractions represent 1 ml collections, ie, time intervals ofapproximately 15 minutes. After 10 fractions (arrow) 10 mg pilocarpine was injected intravenously. Fig 3 Gut: first published as 10.1136/gut.16.7.553 on 1 July 1975. Downloaded from 556 T. Peeters and G. Vantrappen injection of 4 mg of pilocarpine five minutes before enon observed with amylase both these values are the dissection of the mice resulted in the disappear- mmeaningless for this enzyme. In the first fraction the ance of all fluorescence from the crypts, while a amylase concentration was always 5-6 SU/ml and stream of fluorescent material appeared between the the amylase became undetectable after two to three villi (fig 2).