Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from Gut, 1979, 20, 420-431

Progress report The Paneth cell

It was Schwalbel in 1872 who first described the morphological appearances ofthe pyramidal cells at the bases ofthe small intestinal crypts of Lieberkiihn. Some 16 years later, Paneth2 reinvestigated the cells by light microscopy and left with them his name. The alternative name for the Paneth cell is 'enterocytus cum granulis acidophilis' which in part describes their location, morphology, and histochemistry. Human Paneth cells are usually found at the bases of crypts of Lieberkiihn in the , , and . In normal adults they occur only sparsely in the proximal colon and , but Bloch3 has shown that Paneth cells are common in the of neonates. Paneth cells are also found in mammals and birds, with the notable exception of cats and dogs and their distribution is typically human. They are also present in many amphibians and reptiles where they occur throughout the intestinal tract4'5. The Figure shows a schematic diagram of a Paneth cell depicting its truncated pyramidal shape and a short brush border (microvilli) at the apical There is a basal irregularly shaped part projecting into the crypt lumen6'7. http://gut.bmj.com/ nucleus with a well-developed nucleolus. The characteristic features of the cells are the apically located large cytoplasmic secretory granules8'9. An elaborate, highly developed, dilated, rough endoplasmic reticulum that is intimately involved inthe elaboration of the secretory material virtually fills the cytoplasm not occupied by the organelles. This endoplasmic reticulum is most extensive in the paranuclear basal portion of the cell, with extensive

Golgi apparatus of small membrane bound vesicles in the supranuclear on September 28, 2021 by guest. Protected copyright. cytoplasm'0. As early as 1888 Paneth2 had recognised the likely secretory function of these cells. Their ultrastructural organisation is very similar to that of other cell types such as pancreatic acinar cells which elaborate abundant protein- rich secretions". The secretions are granular and described as varying from 1-3 ,um, and being either homogeneous or double-structured with a dense core and clear halo'2,13,"4,15 There is some evidence that granule morphology may be determined in part by fixation technique16'17. The granules take up numerous dyes and consist of a carbohydrate-protein core and an acid-mucopolysaccharide capsule'8"19. There may be species dif- ferences, for Sheahan and Jervis20 could demonstrate acid mucosubstances only in rodent Paneth cells. They also contain lysosomal enzymes2122 23 and some heavy metals, especially zinC6'24 as well as mercury, lead, and coblat25. Lewin 26 has detailed their staining reactions, methylene blue, Lendrum's phloxine-tartarazine, and Masson's trichrome being the most effective. Like the zymogen rich cells of the pancreas, however, they quickly degranulate during autolysis and are destroyed by acetic acid fixation. The literature 420 Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from The Paneth cell 421

brush border secretory granules

golgi apparatus -

basement membrane endoplasmic reticulum

Figure The Paneth cell http://gut.bmj.com/ contains numerous references to Paneth cells and their alteration in disease based upon the examination of tissue fixed in acid media. These are invalid and histological examination should therefore always be preceded by for- malin or mercuric fixation27. The apical part of the cells, show glucose 6- phosphatase, carbonic anhydrase, and strong monoamine oxidase activity28. (muramidase) was first demonstrated in Paneth cells by Speece29 and later by others21'22'30'31'32'33, and more recently immunocytochemical on September 28, 2021 by guest. Protected copyright. staining has disclosed the presence of immunoglobins IgA and IgG22t34035,36. Development and life cycle Irradiation studies37 and autoradiographic investigations13 indicate that Paneth cells are derived from crypt base columnar cells. This is contrary to an earlier view that they were extraepithelial in origin38 and possibly derived from the neural crest39. Paneth cells do not synthesise DNA, or divide, but have a turnover time of about three weeks'3. Keren et aL.40, however, claim to have observed mitoses of the Paneth cells in the isolated perfused ileal loop in the rabbit. Georgieva and Gerov41 showed that porcine Paneth cells were not evident until the time of birth. In the mouse the Paneth cells with small granules first appear at about 1 week of age and larger adult granules are acquired at about 4 weeks old6. Subbuswamy42 demonstrated that, during infancy, three immature forms of Paneth cell and a form intermediate between a Paneth cell and a exist. The presence of an intermediate cell"'43'44456, though Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from 422 M. J. Sandow and R. Whitehead disputed4, may indicate a common origin with the goblet cell. Indeed, trans- formation one to the other has been postulated42'46. Recently, the demon- stration of intestinal cells containing both mucin and lysozyme reinforces the concept of intermediary cell types47. The total number of Paneth cells ofthe human gut increases to its maximum in early adult life, and after 70 years of age shows a significant decrease. The secretory function in old age is also reduced48. Unlike the other cell types of the crypt, Paneth cells do not migrate up the villous and thus are not lost at the surface13. It is claimed that they undergo degeneration and phagocytosis by mononuclear cells in the crypt base17. It is plain that the precise embryological origin of Paneth cells, their capacity to divide, and their turnover time needs further study. Function That Paneth cells promote intraluminal of ingested food stuffs has been a popular theory for some time12'49 50 and is included in some texts5l1 although published accounts of the evidence for this are often contradictory and indirect. A prolonged milk diet, for example, was reported to double the number of Paneth cells in the mouse intestine52. Balass3 noted increased secretion by Paneth cells after refeeding after fasting and Ahonen and Penttilii54 noted an increased granule count of the Paneth cells after fasting for two days with a significant reduction in the number of granules upon refeeding. Kilkowska and Zegarska55 found evidence of 3-galactosidase and

/3-glucuronidase in the Paneth cells of rats and claimed that they therefore http://gut.bmj.com/ have a role in carbohydrate metabolism. Trier et al.10, however, report that cell-free filtrates of pure duodenal secretions from experimental animals are free from lipase, protease, and peptidase activity, although they do contain amylase and enterokinase. Using labelled leucine, Trier et al.10 found that diet has no influence on the number or size of Paneth cell granules, which contradicts a role in protein digestion. The increasing number of Paneth cells towards the ileum also makes a protein digestive function unlikely. Schaaf and on September 28, 2021 by guest. Protected copyright. Wenzel56, using bats, showed that diet had no influence on the histochemistry of the Paneth cell and, in rats, Sundstrom and Helander57 were unable to demonstrate any effects from 24 hour fasting. Their zinc content9'24, as well as mercury, lead, calcium, and cobalt25'58, suggests that Paneth cells are important in the elimination of metals. Gent and Creamer59, using radioactive Zn as a marker and pilocarpine as the stimulus, found that Paneth cell secretions are rich in amino-acids. They concluded that the secretions helped to establish a concentration gradient across the intestinal mucosal cells which ensured absorption of essential amino acids present in low concentration6". In this way the cells reputedly have a function in amino acid homeostasis. Earlier Creamer6' had noted reduced Paneth cell numbers in some patients with coeliac disease and speculated that the Paneth cell secretions provided a luminal environment conducive to the rapid cell division of the . However, the deficiency of Paneth cells in some animals'0 62 and the lack of Paneth cells in other areas of the with a comparable cell turnover rate make the hypothesis unlikely. There are other theories concerning Paneth cell function for which strong Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from The Pazteth cell 423 evidence is lacking. It has been claimed that they have a role in the elaboration and control of the viscosity of intestinal mucin43,63. They are also frequently seen in the proximity of colonic and gastric neoplasms, as a metaplastic phenomenon and Black and Ogle64 suggested that Paneth cells may secrete an anti-tumour factor which exerts a control on the neoplasia. Other roles for the Paneth cell concern the absorption of monoamines6, the absorption of cholesterol19, and the digestion of chitin30'55. The Paneth cells contain the ubiquitous lysozyme and by utilising its bacteriolytic activity on Micrococcus lysodeikticus Speece29 and Geyer21 showed that they could assess the functional Paneth cell mass. Paneth cell lysozyme has since been accurately demonstrated by an immunocyto- chemical method31 32, and in man it occurs in the secretory granules, the Golgi apparatus, and in some, but not all, of the lysosomes. Whereas Paneth cells appear in the rat at about the 15th post-natal day when ordinary methods are used for their demonstration, they can be identified at days 4-6 when methods for lysozyme are used. This suggests earlier biochemical, as opposed to morphological, differentiation65 and highlights the shortcomings of simple tinctorial methods for the study of Paneth cell function. The intestinal villi are larger than normal and the Paneth cells contain abundant granules in rats reared in germ-free conditions50. This is attributed to the absence ofmicrobes, and ultrastructural studies have provided evidence that rat Paneth cells may be capable of the phagocytosis and intracellular degradation ofintestinal microbes by their extensive lysozymal apparatus66'67. Erlandsen et al.31 have thus suggested that Paneth cells contribute to the regulation of the intestinal flora by the action of lysozyme in the degradation ofthe glycopeptide cell wall ofthe and in the inactivation of . http://gut.bmj.com/ The possible role has been strengthened by the recent demon- stration, using an immunocytochemical method, that they contain immuno- globins IgA and IgG22 35'36. Such a role would explain the increasing number of Paneth cells towards the ileum. It is also possible to relate the presence of metaplastic Paneth cells in certain inflammatory states of the and large intestine to altered microbial flora and the tendency towards tissue invasion. on September 28, 2021 by guest. Protected copyright. Control of secretion There is profuse literature concerning the control of Paneth cell secretion and this probably reflects the limited knowledge of their function. As early as 191044 pilocarpine, a parasympathomimetic drug, was known to cause increased Paneth cell secretion. This has been confirmed since by several workers'0'23'54, and Barkla and Tutton68 have shown that carbochol has a similar action. The Paneth cells of the mouse take up adrenaline and noradrenaline and even more avidly their precursors L-dopa and dopamine6'69. This uptake is intensified by nialamine, a monoamine oxidase inhibitor54, and by cocaine6. Chemical sympathectomy with 6-hydroxydopamine prevents secretion of Paneth cells6 and is effective up to 12 months after treatment70. Propanolol, a ,B-adrenergic blocker, and phentolamine, an x-adrenergic blocker, however, produce no alteration of Paneth cell activity68. Serotonin administration has been shown to cause a reduction in the number of granules71. Both glucagon and insulin increase significantly the number, but not the Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from 424 M. J. Sandow and R. Whitehead size, of secretory granules of the cells, possibly by inhibition of secretion72. The effect of glucagon is similar to that which results from fasting hypo- glycaemia. Insulin and fasting increase serum glucagon, thus glucagon may be the common mediator. Vagotomy and sympathectomy cause Paneth cell granules to increase in size6. Trasylol73, a trypsin and proteinase inhibitor, produces the same effect and probably acts by delaying extrusion of the Paneth cell granules from the cytoplasm into the intestinal lumen. Thus vagotomy, sympathectomy, Trasylol, glucagon, and insulin all act to increase the granule count of the Paneth cells, probably by delaying secretion. Glucagon and insulin, however, also reduce granule synthesis, and thus do not induce increased granule size. Adrenalectomy in hamsters also increases granule size and, after an adrenal graft, the morphology of the cells returns to normal74. It seems that the adrenal cortex may have a facilitatory role in the secretion process of Paneth cells, possibly through glucocorticoid hormones. Cycloheximide is an inhibitor of protein synthesis and produces rapid vil- lous atrophy of the small intestine in treated rats. One hour after administra- tion there is a rapid release of Paneth cell granules, but, after three hours, granule synthesis has increased to replace those extruded, and the cell becomes more apparent than in the controls75. Treatment of mice with actinomycin C, an RNA synthesis inhibitor76, zinc deficiency in rats77, and whole body x-irradiation78 all produce crystalloid structures within the matrix of the Paneth cell granules. In all these situations there appears to be disturbance of granule synthesis and an impairment of secretion and yet, after x-irra- diation with 400 r, van Dongen et al.79 showed that the absolute number and localisation of the Paneth cells was unaltered, although the proliferative activity of the crypts was reduced. http://gut.bmj.com/ McDermott and Roudnew80 resected 40% of the small intestine of rats and observed an increased cell proliferation compartment in the remaining gut two months later. They recorded no significant Paneth cell changes in that time, but the fixation technique used was unsuited to their meaningful evaluation.

Paneth cell hyperplasia has been described in isolated (Thiry-Vella) ileal on September 28, 2021 by guest. Protected copyright. loops of rabbits some four days after operation40 and this was accompanied by atrophic changes in the villi. The authors thus postulated that substances in the chyme may be necessary for normal Paneth cell activity. If the functional role of the Paneth cell is in doubt, it can be fairly said that the same is true, if not more so, for the factors which control its secretion. It certainly seems possible, however, that if function can be ascertained, knowledge of the mechanism of control of secretion, will more probably follow. No precedent will be set if this knowledge comes via the study of the Paneth cell in relation to disease. Paneth cells in disease The most notable connection between Paneth cells and disease is acro- dermatitis enteropathica. In this condition zinc deficiency and low serum zinc is accompanied by characteristic changes in the Paneth cells81. The cells often contain granules of varying sizes with irregularly formed homogeneous structures in their cytoplasm82 88. Similar lesions have been described in the intestine of zinc-deficient rats79. Lombeck et al.84, and Polanco et al.85 have Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from The Paneth cell 425 verified intestinal malabsorption of zinc in this condition and shown that dietary zinc supplements produce rapid and complete clinical remission; the ultrastructural lesions of Paneth cells however, persist85. A controversial area in Paneth cell pathology is their importance in coeliac disease. Kelley and Terry86 first noted a relative deficiency of Paneth cells in a jejunal biopsy of coeliac disease and later Pink and Creamer87 correlated a decrease or absence of Paneth cells with failure to respond to a gluten free diet. That a proportion of patients with properly diagnosed coeliac disease fail to respond to dietary modification is well known88, but Thompson89 has since shown that absence of Paneth cells makes no difference to the clinical course of the disease. There has also been considerable dispute concerning overall Paneth cell changes in coeliac disease (Table).

Table Literature on Paneth cell changes

Author Paneth cell changes Vogel9 N Thuribeck et al.9" N or ' Dissanayake" N or t Watson and Wright"93 ort Morson and Dawson"' or t Lewin"'- Pink and Creamer8' 54 patients--49 t -S5 Kelley and Terry" Ward et al.9" Otto"6 34 patients-28 t -6 Mahnovski et al.9" http://gut.bmj.com/ 43 patients-23 t -20

N: normal. t : increased. decreased. There is therefore some evidence that Paneth cells may be decreased in a proportion of patients with coeliac disease, but accurate information con- cerning Paneth cell numbers in the jejunal mucosa in coeliac disease and other on September 28, 2021 by guest. Protected copyright. forms of mucosal abnormality98 is lacking. Lewin4,5 suggested that changes in the Paneth cells ofthe small intestine are either intrinsic-for example, acrodermatitis enteropathica and fibrocystic disease-or the result of direct damage to the mucosa in inflammation or as part of a general mucosal reaction to injury, when a fall in the number of Paneth cells is the usual result. The reduction in Paneth cells in Crohn's disease, ulcerative colitis with reflux ileitis, and tumours-for example, lymphosarcoma-occurs in the diseased segments of intestine only, and is ascribed to non-specific injury. Proliferation of Paneth cells, however, may occur in areas of regeneration and repair. Vogel90, however, has claimed that there is no consistent Paneth cell response in any of several small bowel enteropathies and a somewhat puzzling report by Ahonen et al.99 describes increase in the secretory granules of Paneth cells in experimentally induced amyloidosis. Paneth cell metaplasia may occur in a diversity of diseases of the stomach, colon, and . This is especially true in chronic atrophic gastritis with intestinal metaplasia, in ulcerative and neoplastic gastric disease, as well as infectious, ulcerative, and neoplastic diseases of the rectum and colon100'10. Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from 426 M. J. Sandow and R. Whitehead Paneth cell metaplasia of the colon is frequently seen in ulcerative colitis4,5"101"102"103"104, but its non-specificity in diagnostic terms needs stressing. It has, however, significance in rectal biopsy tissue, for it denotes long-standing and often mild disease and it is this group of patients who have the highest rate of malignant disease. Paneth cell metaplasia also occurs outside the gastrointestinal tract. Gordon'05, for example, has demonstrated complete intestinal metaplasia of the urinary tract epithelium in response to chronic irritation, and a similar metaplastic appearance was seen in an intestinal urinary conduit'06. The appearance of with Paneth cells in adeno- carcinoma of the gall bladder44 and in chronic cholecystitis also occurs'l07 and Patzelt'08 found Paneth cells in the pancreatic and biliary ducts in the human embryo, and Helly'09 in the adult pancreas. The metaplastic Paneth cells in ulcerative colitis and other gastroentero- pathies are histochemically and immunocytochemically identical with normal cells"1,ll0 Neoplasia completes the spectrum of Paneth cell pathology. They are often seen adjacent to mucinous carcinomas of the colon4 5'44'63'64"02. In adeno- matous polyps, however, they are observed within the lesion. They have also been demonstrated in the adenomas of familial polyposis coli42,1" and juvenile polyps"12. Cells intermediary between goblet and Paneth cells have been described"142'45 and this suggests that they have a common . It is also pertinent that in the precancerous phase of ulcerative colitis a pan- cellular dysplasia picture is described in which Paneth cells, goblet cells, and intermediate cell types are seen"13. Kawachi et al."14 and Tahara et al."15 in an noted intestinal and Paneth cell metaplasia experimentally produced http://gut.bmj.com/ precancerous change of the stomach mucosa. The proliferative ability of Paneth cells is, however, disputed and certainly pure tumours of these cells are rare. Neoplastic Paneth cells have, however, been described102"'16"'17. Scharfenberg and DeCamp"l8 reported neoplastic Paneth cells in an adenocarcinoma of Meckel's diverticulum and they were also present in the metastases. More recently, Miyajima and Takeuchi"19 de- scribed a papillary adenoma with malignant change consisting only of Paneth on September 28, 2021 by guest. Protected copyright. cells and their precursors. Conclusion The Paneth cell is the subject of considerable controversy and as yet a definitive role has not been ascribed to it. Much ofthe controversy is no doubt due to the way in which Paneth cells have been investigated. Interpretation of decreased or increased function based upon change in simple tinctorial reaction of the granules is clearly not scientific and the instability of the granules as evidenced by degranulation induced by acid fixatives adds another source of potential error. There are several instances in which attention to detail such as appropriate fixative have been overlooked and much of the discrepancy in studies concerning Paneth cell kinetics is due to poor method- ology. The staining properties of the granules-for example, with phloxine- tartarazine-formed the basis of the major part of earlier experimental work. Clearly, increased granularity of the cells can be interpreted as due to either increased synthesis or blockade of secretion and reduced granularity to increased secretion or reduced synthesis. Estimating the numbers of Paneth Gut: first published as 10.1136/gut.20.5.420 on 1 May 1979. Downloaded from The Paneth cell 427 cells solely on the basis of stainability of their granules is another obvious and significant source of error. With newer methods of investigation, which are based upon biochemical and immunochemical characteristics, real advances in our knowledge of Paneth cell function can be expected soon. There seems to be no doubt that Paneth cells secrete a proteinaceous substance into the gut lumen and that this contains immunoglobulin and lysozyme. It is clear also that their secretion is intimately related to the small intestinal environ- ment and not to that of the normal stomach and large intestine. When the intragastric and intracolonic environment changes, possibly acquiring similarities to that of the small intestine due to changes in pH and microbial flora, for example, then there is a metaplasia towards small intestinal epithe- lium including Paneth cells.

M. J. SANDOW AND R. WHITEHEAD1 Flinders Medical School Bedford Park, South Australia, 5042

'Address for correspondence and reprints: Department of Pathology, Flinders Medical School, Bedford Park, South Australia, 5042. Received for publication 2 November 1978

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