The Panethcell: a Source of Intestinal Lysozyme'

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

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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). Ten minutes after the administra- hours. It will be seen from the table that a significant tion of pilocarpine fluorescent material, granular rise in lysozyme secretion was observed in all five in appearance, was found on top of the villi (fig 3). mice studied, the mean peak rate being 3-9 ± 1-9 SD. In their electron microscopical study of Paneth cell secretion in mice, Trier et al (1967) found that secre- PERFUSION STUDIES tion in Paneth cell granules was stimulated by the The results of a typical experiment are presented in injection of 4 mg of pilocarpine intraperitoneally. figure 4. The initial fluid contained high levels of Few mice survived the intravenous injection of such amylase, which decreased progressively over a a dose for more than 30 minutes, and although a 'washing-out' period of approximately 2-5 hours to a strong increase in lysozyme concentration was stable low basal value. In contrast, the lysozyme found, these results were discarded. level in the perfusion fluidwas low from the beginning of the experiment and remained low during the basal Discussion period. The intravenous injection of 1 mg of pilo- carpine provoked a sharp rise in the lysozyme level, Our results confirm and extend previous observa- whereas the concentration of amylase was un- tions concerning the presence of lysozyme in the affected. Subsequently the lysozyme output de- Paneth cell. They also demonstrate that the lysozyme creased but remained at higher than basal values present in the succus entericus is, at least in part, until the animal was sacrificed. derived from the Paneth cell, which secretes this Initially the animals were not starved before the enzyme into the crypt lumen. experiment. Later, when it became apparent that the Immunofluorescence has been used to demonstrate emptying of the intestine before the start of the lysozyme activity in human bone marrow (Briggs, perfusion was often difficult, the animals were Perille, and Finch, 1966), blood cells (Asamer, starved for 24 hours, although they were given water. Schmalz, and Braunsteiner, 1969), the parotid gland http://gut.bmj.com/ On analysis of the data it was found that the basal (Kraus and Mestecky, 1971), distinct epithelial cells values for lysozyme (mean lysozyme concentration of the oral cavity (Raeste, 1972), and in mouse and in the perfusate before pilocarpine stimulation) were rat macrophages (Glynn, 1964). Evidence for the lower in starved animals (x = 0-16 ,ug/ml; SD = presence of lysozyme in the Paneth cell was until 0-086; n = 12) than in unstarved (x = 0-31 ,ug/ml; recently based mainly upon histochemical methods SD = 0-19; n = 10). The difference between these using substrate films of Micrococcus lysodeikticus two values is significant at the 5% level. The data (Geyer, 1973) or chitin (Ghoos and Vantrappen, on September 30, 2021 by guest. Protected copyright. obtained in five mice before and after stimulation by 1971). In cell fractionation studies (Deckx et al, 1 mg of pilocarpine are summarized in table I. The 1967) particle-bound lysozyme activity has been peak rate for lysozyme was computed from the found in a fraction containing Paneth cell granules highest value for 45-minute collections, described as from mouse small intestine. Recently Erlandsen et the basal value. Because of the 'wash-out' phenom- al (1974) used the unlabelled antibody technique to localize lysozyme in Paneth cells at the ultra- structural level. Their findings are in complete agree- ment with those presented in this paper. Erlandsen Experiment Lysozyme Amylase No et al (1974) found specific staining for lysozyme over Basal Peak Initial Level Wash-out secretion granules in the apical cytoplasm, within (j.g/ml) (vg/ml) (SU/ml) (hours) the region of the Golgi apparatus, and within some I 1 0-27 5-1 6-9 21 lysosomes. They suggested that in substrate film 2 0-12 3-7 6-5 21 studies, lysozyme from a lysosomal origin may have 3 0-21 6-5 7-5 21 4 0-06 1-8 5-7 3 contributed to a considerable extent to the localiza- 5 0-07 2-6 6-1 2i tion of enzyme. Although the same argument could be used against our method, the staining pattern Table Summary of lysozyme and amylase after pilocarpine administration, combined with the determinations in the perfusate of the small intestine results of the perfusion studies, offive mice before and after stimulation by I mg of suggest that staining pilocarpine is due to lysozyme in secretory granules. The absence of fluorescence in the crypt region shortly after 'Unfasted mouse secretory stimulation, when the lysosomal apparatus Gut: first published as 10.1136/gut.16.7.553 on 1 July 1975. Downloaded from The Paneth cell: A source ofintestinal lysozyme 557 is presumably still intact, indicates that immuno- Already the phagocytosis of an intestinal parasite, fluorescence is not sensitive enough to detect lyso- Hexamita muris (Erlandsen and Chase, 1972a), and zyme within the lysosomes. Therefore, the results of spiral microorganisms (Erlandsen and Chase, obtained with the extremely sensitive unlabelled 1972b) by the Paneth cell has been demonstrated. antibody technique and our observations taken Although the bactericidal properties of lysozyme together suggest that in the Paneth cell most of the itself are limited, they are enhanced in the presence lysozyme is located within the granule itself. of other substances such as immunoglobulins and Trier et al (1967) demonstrated that the number of complement (Bladen, Hageage, Harr, and Pollack, granules remains constant during pilocarpine 1973; Glynn, 1968). It would be interesting to find stimulation and concluded that pilocarpine results out whether the Paneth cell granules themselves do not only in a stimulation of the secretion but also in not contain agents which act synergystically with an increased synthesis of the granules. Our observa- lysozyme. tions indicate that pilocarpine stimulation leads to a complete disappearance of fluorescent granules from This work has been supported by grant no. 20:59 the Paneth cells and to the appearance of fluorescent from the Fonds voor Wetenschappelijk Genees- material in the crypt lumen. These two observations kundig Onderzoek (Brussels, Belgium). The authors are not necessarily contradictory because our histo- gratefully acknowledge the skilful technical assist- chemical data relate to the first 10 minutes of ance of Mrs S. Spee. stimulation whereas those of Trier et al (1967) bear on a much later period (three and six hours after the References administration of pilocarpine). On the other hand, level which was still found in our Asamer, H., Schmalzl, F., and Braunsteiner, H. (1969). Der immuno- the high lysozyme zytologische lysozymnachweis in menschlichen Blutzellen. perfusion experiments, for a period of up to three Acta haemat. (Basel), 41, 49-54. hours after stimulation, may be due to an increased Bladen, H., Hageage, G., Harr, T., and Pollock, F. (1973). Lysis of certain organisms by the synergistic action of complement and rate of synthesis. However, it may also reflect the lysozyme. J. dent. Res., 52, 371-376. time taken for the secreted material to reach the Briggs, R. S., Perille, P. E., and Finch, S. C. (1966). Lysozyme in bone marrow and peripheral blood cells. J. Histochem. Cytochem., intestinal lumen and the time needed to solubilize 14, 167-170.

a highly viscous product. It should be noted in this Caraway, W. T. (1959). A stable starch substrate for the determina- http://gut.bmj.com/ that the for tion ofamylase in serum and other body fluids. Amer. J. clin. respect, 'wash-out' period amylase was Path., 32, 97-99. already two to three hours. In earlier studies (Klein, Cordier, R. (1923). Contribution a l'ktude de la cellule de Ciacco- 1906; Miram, 1912; Cordier, 1923) a 'secretory Masson et de la cellule de Paneth. C. R. Soc. Biol., 88, 1227- 1230. cycle', consisting in the accumulation of granules Creamer, B. (1967). Paneth cell function. Lancet, 1, 314-316. and sectretion after feeding, has been described. Deckx, R. J., Vantrappen, G. R., and Parein, M. M. (1967). Localiza- tion of lysozyme activity in a Paneth cell granule fraction. Trier et al (1967) found no evidence for such a cycle, Biochim. Biophys. Acta (Amst.), 139, 204-207. although our observations of higher basal lysozyme Erlandsen, S. L., and Chase, D. G. (1972a). Paneth cell function: levels in mice support the hypothesis of phagocytosis and intracellular digestion of intestinal micro- on September 30, 2021 by guest. Protected copyright. non-fasting organism. I. Hexamita muris. J. ultrastruct. Res., 41, 296-318. some stimulation by feeding. Our model provides Erlandsen, S. L., and Chase, D. G. (1972b). Paneth cell function: the means to investigate this and other factors or phagocytosis and intracellular digestion of intestinal micro- organisms. II. Spiral microorganism. J. ultrastruct. Res., 41, agents responsible for Paneth cell secretion in a 319-333. relatively simple way. Erlandsen, S. L., Parsons, J. A., and Taylor, T. D. (1974). Ultra- structural immunocytochemical localization of lysozyme in the The action of pilocarpine is of course not limited Paneth cells of man. J. Histochem. Cytochem., 22, 401-413. to the Paneth cell. In this study attention was paid to Gent, A. E. and Creamer, B. (1972). Paneth cell secretion. Digestion, a possible contamination of the perfusate with 7, 1-12. Geyer, G. (1973). Lysozyme in Paneth cell secretions. Acta histochem. material originating from the pancreas. Such con- (Jena), 45, 126-132. tamination could be excluded on two grounds: Ghoos, Y., and Vantrappen, G. (1971). The cytochemical localiza- tion of lysozyme in Paneth cell granules. Histochem. J., 3, 175- the position of the inlet tube and the absence of a 178. rise in amylase concentration after stimulation by Glynn, A. A. (1968). Lysozyme: antigen enzyme and antibacterial agent. Sci. Basis Med. Ann. Rev., 31-52. pilocarpine. Gorin, G., Wang, S. F., and Papapavlou, L. (1971). Assay oflysozyme It seems unlikely that lysozyme should be the by its lytic action on Micrococcus lysodeikticus cells. Analyt. only secretory product of the Paneth cell. The Biochem., 39, 113-127. Klein, S. (1906). On the nature of the granule cells of Paneth in the investigations of Gent and Creamer (1972) provide intestinal glands of mammals. Amer. J. Anat., 5, 315-330. evidence for the secretion of free amino acids. This Kraus, F. W., and Mestecky, J. (1971). Immunohistochemical localiza- tion of amylase, lysozyme, and immunoglobulins in the human point should be reinvestigated in the light of our parotid gland. Arch. oral. Biol., 16, 781-789. present knowledge. In view of the bactericidal Lewin, K. (1969). Histochemical observations on Paneth cells. J. Anat. (Lond.), 105, 171-176. properties of lysozyme, the Paneth cell probably Miram, K. (1912). Zur Frage uber die Bedentung der Panethschen forms part of a defence system in the small intestine. Zellen [des Dunndorm-epithels]. Arch. mikr. Anat.,79, 105-113. Gut: first published as 10.1136/gut.16.7.553 on 1 July 1975. Downloaded from 558 T. Peeters and G. Vantrappen

Paneth, J. (1888). tJber die secernirenden Zellen des Diinndarm- Speece, A. J. (1964). Histochemical distribution of lysozyme activity Epithels. Arch. mikr. Anat., 31, 113-191. in organs of normal mice and radiation chimeras. J. Histo- Raeste, A. M. (1972). Lysozyme (muramidase) activity of leucocytes chem. Cytochem., 12, 384-391. and exfoliated epithelial cells in the oral cavity. Scand. J. Trier, J. S., Lorenzsonn, V., and Groehler, K. (1967). Pattern of dent. Res., 80, 422-427. secretion of Paneth cells of the small intestine of mice. Riecken, A. E., and Pearse, A. E. (1966). Histochemical study on the Gastroenterology, 53, 240-249. Paneth cell in the rat. Gut, 7, 86-93.

The June 1975 Issue THE JUNE 1975 ISSUE CONTAINS THE FOLLOWING PAPERS Antibodies to double-stranded (native) DNA in Assessment of simple methods of measuring intesti- active chronic hepatitis P. DAVIS AND A. E. READ nal transit times in children with gastroenteritis R. H. HIGGS, R. B. ELLIS-PEGLER, AND H. P. LAMBERT Cell-mediated immunity to hepatitis B surface anti- with persistent antigenaemia The nutritive effect of glucose on the structure and gen in blood donors self-emptying blind loops in the W. M. LEE, W. D. REED, C. G. MITCHELL, I. L. WOOLF, function of jejunal rat H. MENGE, H. WERNER, H. LORENZ-MEYER, AND I. W. DYMOCK, A. L. W. F. EDDLESTON, AND ROGER E. 0. RIECKEN WILLIAMS The influence of food intake on the development of Hepatitis-associated-antigen-positive hepatitis in a structural and functional adaptation following ileal tuberculosis unit G. R. FITZGERALD, H. GRIMES, resection in the rat H. MENGE, M. GRAFE, H. LORENZ- M. REYNOLDS, H. HITCHCOCK, AND C. F. MCCARTHY MEYER, AND E. 0. RIECKEN

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