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Mast Cells and Paneth Cells in Ulcerative Colitis
Vol. 51, No.5, Part 2 Printed in U.S.A.
GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co.
MAST CELLS AND PANETH CELLS IN ULCERATIVE COLITIS SHELDON
C.
SoMMERs,
M.D.
Department of Pathology, Columbia University College of Physicians and Surgeons, Francis Delafield Hospital, New York, New York
In the last 5 years . it has become clear that the tissue mast cells and eosinophils are particularly important in nonspecific ulcerative colitis. A primary biological factor that EngeF believed to characterize individuals who are susceptible to develop ulcerative colitis evidently involves the basophil granulocytes, eosinophils, and mast cells in the connective tissue of the large intestine. Forty years ago, Buie 2 recognized four sigmoidoscopic sequential stages in the activation of ulcerative colitis: (1) diffuse hyperemia, (2) mucosal edema with hyperemia and friability, (3) miliary mucosal abscesses , and (4). ulcers formed from the ruptured abscesses. Crypt abscesses have been regarded until recently as the earliest lesions characteristic of ulcerative colitis. 3-5 Newer evidence supports Manson-Bahr's6 belief that ulcerative colitis begins in the submucosa and affects the mucosa secondarily. A review of mast cell structure and functions will be helpful in understanding Buie's stages 1 and 2 in the development of ulcerative colitis. Tissue mast cells normally contain granules heavily stained with polychrome dyes, such as the ·wright and Giemsa methods. Mast cells are quite inconspicuous with hematoxylin and eosin stains. The cytoplasmic granules are periodic acidSchiff (PAS) positive, sudan black B positive after periodic acid and alcian blue-safranin positive at pH 1.0. 7-9 Toluidine-blue metachromasia characterized the mast cell granules, as well as intravital tetrazolium binding to acid groups. 9 - 10 DiAddress requests for reprints to: Dr. Sheldon C. Sommers, Department of Pathology, Columbia University College of Physicians and Surgeons, Francis Delafield Hospital, New York, New York 10032.
amine methods stain sulfated mucosaccharides that occur in a variety of cells with coarse cytoplasmic granules, including the polymorphonuclear leukocytes, immature eosinophils, mast cells, Paneth cells, and enterochromaffin cells; all except the last possess strongly basic proteins histochemically. 11 Toluidine-blue and diamine staining reflect a concentration of heparin in the mast cell granules, which appear by electron microscopy to develop in the hyaloplasm_12 The heparin-containing granule is a polysaccharide-protein ionic complex that incorporates considerable sulfate. 13 · 14 Mast cells are rich in histamine, 13 p.g per 106 cells, which is apparently bound to the granules electrostatically. 14 • 15 Human mast cells synthesize histamine but not 5hydroxyJryptamine ....(5JIT) (serotonin) ,1 6 alHiO'U:'gh ~ -mouse mastocytoma cells apparently do so. 17 Chymase proteolytic enzymes also are localized in the granules.14· 18 Dopamine, glucuronidase, leucine aminopeptidase, ascorbic acid, and a variety of other biologically active substances occur in mast cells. 15 • 17 - 19 Histamine, serotonin, heparin, and chymase are considered the most important in ulcerative colitis. Mast cells were named by Ehrlich 9 because they appeared well fed. They preferentially absorb and store for variable periods, by electrostatic binding or otherwise, various substances such as metal cations, basic dyes/ 0 iodine, and, in particular, 5-HT, both in human and animal tissues, to the level of 1 p.g per 106 cells. 15 · 2 1 - 24 The local concentration of 5-HT determines the extent of its uptake by mast cells. 22 The process is evident with 5-HT labeled either by 14 C or 3H. 23 The pres841
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ence of 5-HT in mast cells is best demonstrated by fluorescence microscopy. 22 · 24 In summary, the mast cell is the chief connective tissue storehouse of histamine and heparin, a depot for 5-HT, and a source of proteolytic enzymes. Discharge of the mast cell contents is selective, so that histamine is preferentially secreted and heparin retained, unless the stimuli are severe. Thus in human mastocytomas, symptoms from histamine release are common and from heparin, rare. 10· _25 · 26 Free histamine is readily released and spreads fast, so that the fixed connective tissues become activated, according to Riley. 27 Later heparin is slowly released, followed by the phagocytosis and digestion of metachromatic material by activated cells which utilize it to synthesize ground substance. ! Many different stimuli produce mast cell secretion, including autolysis, distilled :water, mechanical friction, synthetic : amines like compound 48/80, dextran, polyvinyl-pyrrolidone (PVP), antigens, ovomucoid, bacterial toxins, snake venom, tissue extracts, alkaloids, antihistaminics, surface active agents, turpentine, ultraviolet light, etcY· 28 -30 Various metal ions, sulfhydryl or amine reagents, and oxidative inhibitors prevent degranulation. In anaphylaxis the guinea pig mast cells were significantly damaged and released histamine and slow reacting substance, except for the intestinal mast cells. 31 Soluble antigen-antibody complexes were ineffective. In rats histamine release required the activation of a chymase to initiate the inflammatory response in antigen-antibody reactions. 32 -34 Immunological injury in rodents, with mast cell degranulation, was followed by increased connective tissue viscosity and a "gelatinous state." 35 Delayed type allergic reactions in guinea pigs also depended on the liberation of biologically active substances from the mast cells.30 Mast cell granules discharged by nonspecific stimuli have an increased avidity for metals, like lead, and a secondary attraction for calcium and phosphate that initiates intense calcification, the calcergy phenomenon of Selye. 37
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Endocrine factors alter the mast cell population and activities. Cortisone increased both the number of mast cells and their degranulation in experimental granulomas of rats and human or rat gastric biopsies. 38 -40 Estrogen increased the total mast cells, their granule discharge and regranulation in human oral biopsies. Estrogen thus prevented the edema that ordinarily followed surgical injury and increased the connective tissue acid mucopolysaccharides and vessel wall metachromasia.41 Lindholm 42 · 43 described the embryogenesis and cell density of mast cells in the human and animal gastrointestinal tract. In childhood the mast cells increased to an average of 2000 per mm 3. In adults still greater numbers were present in the esophagus and rectum. Hamsters had more mast cells than rats, and the smallest numbers occurred in the large intestine. Kraft and Kirsner, 44 and Norris, Zamchek, and Gottlieb 45 investigated mast cells in the human gastrointestinal tract. Cell counts were highest in the mucosa of the appendix, stomach, and duodenum, and in the submucosa of the large intestine, jejunum, and ileum. By electron microscopy the human gastric mucosa contained two types of mast cells, spindle shaped and rounded. The second type was more common ; the first type was chromaffin positive and disappeared after corticoid administration.46 Note again the local absorption of 5-HT (serotonin) by mast cells. The relevance of the foregoing information to ulcerative colitis was first pointed out by McGovern and Archer. 47 Based on an analysis of 32 surgical and 20 autopsy specimens, they concluded that the fundamental reactions were typical of histamine-release phenomena, with a depletion of granules from the mast cells found in abundance in the human colonic submucosa and muscular coats. They considered that psychosomatic influences would liberate more acetylcholine in the colon and provoke an increased mast cell degranulation. The pathological consequences were congestion and edema-most prominent in the submucosa-muscle spasm, and hypertrophy.
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Rebuck and associates 48 · 49 contributed numerous than normal, 56 evidently disa valuable clue by showing in ulcerative charge excess histamine to initiate the excolitis patients that skin windows with aggerated flare and wheal reaction of Mirdiphtheria toxoid had increased basophil vish.57 The stimulus may be mechanical, leukocytes, a persisting neutrophil leuko- physical, chemical, or immunologicaL 58 cyte response, lakes of metachromatic ma- Certain stimuli, like endotoxin, are ineffecterial, and a delayed lymphocyte-macro- tive.59 Histamine, beyond its excessive lophage reaction. The neutrophil persistence cal production and release, might also enwould correlate with prolonged mainte- counter an altered local responsiveness, nance of an alkaline pH in the inflamed binding, or metabolism, 60 but nothing is connective tissue, according to Menkin. 50 known of this in ulcerative colitis. The sulWolf-Jurgensen et aL 51 observed basophil fated mucopolysaccharides of ground subexudation more commonly in Schick-nega- stance are increased.6 1 tive patients. They related it to a delayed Beyond the effects of histamine the hypersensitivity reaction. chronic hyperemia and edema of the coJuhlin 52 and Shelley and Parnes53 found lonic mucosa apparently reflect the disthe relative or absolute blood basophil charge of serotonin downward from the leukocyte counts to be increased in active argentaffin mucosal cells and its temporary ulcerative colitis but normal during remis- storage in adjacent mast cells, as alluded to sions. Basophil leukocyte counts are . re- previously.22 • 46 The persistence and severduced after corticosteroids. McAuley and ity of the edema are probably attributable Sommers 54 and Hiatt and Katz 55 reported to the greater potency of 5-HT.6 2, Ga The increased mast cells in the lamina propria, resulting vasodilation is largely venular. 64 submucosa, and muscularis of surgical Blotchy hypervascularity is demonstrated specimens of ulcerative colitis. In biopsies by microangiography. 65 The biological of active ulcerative colitis Bercovitz and tragedy in ulcerative colitis is that abnorSommers 56 found mast cells in the lamina mally large numbers of mast cells are propria increased to 8.5 times normal and available to store and to release serotonin 1.7 times the number present in ordinary close to where the bulk of it is formed. 66 As concerns Buie's stage 3, or crypt abinflammation. Submucosal mast cells were increased 2- or 3-fold. Sequential biop- scess formation, EngeJl pointed out that sies in 3 patients demonstrated mast -cells ulcerative colitis never occurred spontanein the lamina propria to decrease in re- ously in animals. Mecholyl led to experimissions from 8.7 to 3.7%, from 1.8 to mental colonic vasodilation, vascular stasis, 0.4%, and from 3.8 to 1.9% of the total and hemorrhagic necrosis with ulceraconnective tissue cells. In a subsequent tions in some dogs, but not a true ulceraexacerbation the last case had a rise to tive colitis. 67 Crypt lesions or crypt ab15.0%. Submucosal mast cells changed scesses have now been observed in at least three experimental situations: (1) irradisimilarly. The dysfunctional sigmoidal changes ated rats, particularly when adrenalectomay now be visualized in ulcerative colitis mized,68 (2) irradiated mice treated with in Buie's stage 1 of hyperemia and stage 2 homologous lymphocytes, 69 (3) neonatally of edema. Mirvish57 observed that light thymectomized hamsters. 70 All three situpressure on the mucosa in remissions of ul- ations have involved inadequate regeneracerative colitis produced a characteristic tion of the crypt epithelial cells and either abnormal response involving (1) immedi- insufficient or abnormal trephocytic lymate development of a red edematous patch phocytes. Rebuck's 49 observation of the at least 2 mm high, (2) formation in 10 sec inhibited lymphocyte-macrophage reto 6 min of a soft, sharply demarcated sponse in skin windows may be pertinent. wheal, and (3) obscuring of the superficial In biopsies of ulcerative colitis epithelial network of arterioles . and, sometimes, nuclear fragmentation seems to be the punctate hemorrhages.· The submucosal earliest evident pathological change.5, 5G In mast cells, which are about 3 times more biopsies crypt abscesses occur less com-
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monly and may be regarded as secondary phenomena. 71 In stage 4 the mucosal ulcerations develop. M ast cells in the mucosa are now 8 t imes more numerous than normal and twice as numerous as the average found in ordi na ry acute or chronic inflammation.56 In the submucosa a pproximately half the mast cells are completely degranul ated, and the total population is approxim ately twic e normal and 1.5 times the average in acute or chronic infl amma ti on.56 Comp let e mast cell degra nul ation occurs a bout bYice as commonly in ulcerative colit is as in ordinary infl ammation , ''"it h a consequent release of 5-HT, heparin, and chymase. 56 · 72 · 73 It should be recalled again that the mast cell secretory pro cess is selective, hist amine first and serotonin second . H eparin and proteolytic enzymes are discha rged only after drastic stimuli.'2· 73 Since heparin interferes with plasma coagulation, and fibrin blo ck age of the venules and lymphatics is an important mechanism th at localizes infla mmation, a more widespread reaction results. Three or more proteolytic enzy mes a re released in act ive ulcerative colitis : (1) a mast cell a lkaline protease or chym ase wit h optimal activity at pH 8.9 or higher, and possibly also a tryptase,73 (2) a protease with mucinoly ti c properties, active at acid pH 6.1 ,74 and (3) a cy tolyt ic or acantholy ti c factor most active at neut ral pH of 7.2 to 7.8, described by Stought on. 75· 76 The wide pH range of the proteolytic enzymes released would fur t her exacerbate t he inflammation and increase t he associated t issue necrosis. Eosinophils are t he most notably increased mucosa l connective t issue cells in biopsies of ac tive ulcerative colit is, 30 times norm al a nd 6 times t he number present in ordinary inflammation. 56 They also occur in large numbers in cy tological prepa rations taken from the sigmoid mucosal surface77 and in the abnormal inflammatory sequence in skin windows of ulcerative colitis patients.49 The excessive local exudation of eosinophils is explained by t heir at t ra ction to tissue n ecrosis. A st atistically significant increase of the blood eosinophils also occurs during rel apses of
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ulcerative colitis.78 It is claimed that eosinophils pha gocytize antigen-antibody complexes and t hat their granules are a source of profibrinolysin.' 9 • 80 Explanations of t he relationship of eosinophils to ulcerative colitis await a better underst anding of their functions. Their a bundance suggest s less than adequat e corticoid levels.' 8 l'l'!ucosal plasma cells in biopsies of active ulcerative colit is are increased up to 15 t imes norma l and 4 times the number found in ordinary infla mmation. 56 This may reflect an antibody reaction to incidental bacteri al infection or t he form at ion of aut oant ibodies t o mucus. 80· 81 Ulcerative colit is t hus involves an abnormal inflammatory process attribut able t o a n excess of histamine-releasing mast cells t hat may also absorb 5-HT from the adj acent rich source of serotonin. The sub sequent release of 5-HT would considerably exacerb at e colonic hy peremia and edema . Complete mast cell granule discharge adds the furth er deleterious effects of heparin and a wide spect rum of proteolytic enzymes. The colonic contents do not seem to init iat e ulcerative colitis, which has occurred for example in an a rtifi cial vagina m ade 17 year s before from the sigmoid colon. 82 Ulcerative colitis no longer appears t o be a primary disease of t he ground substance or connective ti ssue fibers, as had been thought .83 · s4 P anet h cells are ordinarily r are in the colon but are increased t o 200 times normal in ul cerative colitis, proba bly through metaplasia of t he regenerating epithelium.85-87 However , very few occurred in sigmoid or rectal biopsies.85 One study of ulcerative colit is also reported the a r gent affin serotonin-containing mucosal cells to be increased,85 and anot her described them as decreased, with a cellular serotonin concentration only one-fifth normal. 87 P a neth cell metaplasia appeared correlated wit h the duration and degree of colitis. Other inflammatory intestinal diseases during regeneration show increased P an eth cells in the mucosal crypts.ss A lack of knowledge of the P aneth cell hampers interpreting its relationship t o ulcerative colitis. These cells are normally
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most numerous in the small intestine but ulcerative colitis are as yet poorly underare also common in gastric intestinal stood. Ulcerative colitis, furthermore, inmetaplasia. They are characteristically as- volves several processes that parallel allersociated with a condensed eosinophilic mu- gic or hypersensitivity diseases, but how cus,80 which is one histological characteris- close the relationship is remains unclear. tic of ulcerative colitis. 90 Paneth cells are Summary further thought to secrete special proteolytic enzymes that are stored in distincIn nonspecific ulcerative colitis, mucosal tive coarse protein-polysaccharide gran- hyperemia and persistent edema are atules .91-95 Their lysozyme activity ordinarily tributable to local excesses of histamine reis high and increases after irradiation. 96 leased by an increased colonic population In fine structure Paneth granules resemble of mast cells. Mast cell storage and release the prosecretory granules of serous glands, of serotonin produced in nearby argentaffin IYith transition to goblet cells. 97 The fea- mucosal cells are likely exacerbating factures described suggest that Paneth cell tors. Severe stimuli may release mast cell metaplasia is a secondary process in ulcer- granules that contain heparin and proteoative colitis. lytic enzymes, exaggerating and spreading The deranged inflammatory reactions in the associated inflammation and necrosis. ulcerative colitis provide a partial explana- Eosinophils are particularly numerous in tion of the disease. Its etiology remains the inflamed mucosa. Blood basophil and unknown. Not all ulcerative colitis pa- eosinophil leukocytes are reportedly intients have manifested an increased baso- creased when colitis is active. phil leukocyte reaction or a measurable Paneth cell metaplasia appears to be a excess of mast cells in the colon wall. The secondary phenomenon of mucosal regenervasculitis type of ulcerative colitis, which is ation in ulcerative colitis, which may have less common and clinically often fulminant, a poorly defined relationship to altered resembles periarteritis nodosa localized to lymphocytes. The abnormal inflammatory the large intestine. This type probably has process in ulcerative colitis is a newly apa quite different pathogenesis. 9 Concerning preciated characteristic of the disease. the common superficial recurrent cryptREFERENCES abscess type of ulcerative colitis, however, our knowledge has recently considerably 1. Engel, G. L. 1961. Biologic and psychologic features of the ulcerative colitis patient. increased. Gastroenterology 40: 313-322. Improved fluorescence methods are now 2. Buie, L. A. 1926. Chronic ulcerative colitis. available to identify serotonin in the coJ. A. M. A. 87: 1271-1274. lonic argentaffin cells and mast cells during 3. Warren, S., and S. C. Sommers. 1949. Patho24 98 different stages of ulcerative colitis. • genesis of ulcerative colitis. Amer. J. Path. Histochemical studies are needed to investi25 : 657-679. gate in greater detail the formation, stor4. Lumb, G. 1958. Rectal biopsy in ulcerative age, and release of histamine, 5- HT, hepacolitis. Dis. Colon Rectum 1: 37--43. rin, and proteolytic enzymes by mast cells 5. Lumb, G., and R. H. B. Protheroe. 1958. Ulin the human large intestine. The fragmencerative colitis. Gastroenterology 34: 381407. tation of crypt epithelium that leads to 6. Manson-Bahr, P. 1943. The dysenteric discrypt abscess formation and its relation to orders, p. 629. Williams & Wilkins Comaltered lymphocytes and local mitotic inhipany, Baltimore. bition by heparin 99 are worth further in7. Feagler, J . R., and J. F. A. McManus. 1964. vestigating. The conflicting endocrine data Extraction resistance of sudan stained mast indicate that corticoids and estrogen are cells after previous acid treatment. J. Ristosomehow associated with increased mast chern. Cytochem .12 : 530-532. cells or basophils and with their augmented 8. Combs, J. W., D. Lagunoff, and E. P. Benditt. degranulation. The roles of the eosinophils 1965. Differentiation and proliferation of and plasma cells remain uncertain. Paneth embryonic mast cells of the rat. J . Cell Biol. 25: 577-592. cell functions and enzymatic processes in
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DISCUSSION
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opsy in the di agnosis of ulcerative colitis. 86. P aterson, J. C., and S. H . \Vatson. 1961. P aQuart. J. Med. SO: 393-407. neth cell metaplasia in ulcerative colitis. Amer. J. Path. 38: 243-249. 72. Tocantins, L. M. 1948. Historical notes on blood platelets. Blood S : 1073-1082. 87. Verity, M . A., S. M. Mellinkoff, M. Frankland, 73. Lagunoff, D ., and E . P. Benditt. 1963. Proand M. Greipel. 1962. Serotonin content and argentaffin and Paneth cell changes in ulteolytic enzymes of mast cells. Ann. N. Y. Acad. Sci. 103: 185-198. cerative colitis. Gastroenterology 43: 24-31. 74 . Warren, S., and S. C. Sommers. 1950. Proteol- 88. Conrad, M . E., F. D . Sch'i\·artz, and A. A. Young. 1964. Infectious hepatitis: A generysis in intestinal disease. Gastroenterology alized disease . Amer. J . iVIed. 37 : 789-801. 14: 522-526. 75. Stoughton, R. B. 1952. Enzymatic cytolysis of 89. H ertzog, A. J. 1937. The Paneth cell. Amer. J . P ath. 13: 351-358. epithelium by filtrates of feces from pa- \ j 90. vVarren, S., and S. C. Sommers. 1954. Patholtients with ulcerative colitis. Science 116: ogy of regional ileitis and ulcerative colitis. 37-39. 76. Fleming, W. H., E . W. Smith, and T. R. HenJ. A. M. A. 154: 189-193. drix. 1962. Acantholytic factor and ulcera- 91. De Castro, N. M., S. ·w. D a Silva., and F. A. Saad. 1959. Preliminary observations of t he tive colitis. Bull. Hopkins Hosp. 111: 285Paneth cell of the T amand1ta tetradactyla 291. Lin. Acta Anat. (Basel) 38 : 345-352. 77. Anthonisen, P ., and P. Riis. 1961. A new diagnostic approach to mucosal inflammation in 92. Selzman, H. M., and R. A. Liebelt . 1961. A cytochemical analysis of Paneth cell secreproctocolitis. Lancet 2 : 81-82. tion in the mouse. Anat. Rec. 140 : 17-22. 78. Riisa.ger, P. M. 1959. Eosinophil leukocytes in 93. Selzman, H. M., and R. A. Liebelt. 1962. The ulcerative colitis. Lancet 12 : 1008-1009. 79. Litt, M. 1964. The acute inflammatory rePaneth cell granules of the mouse intestine. sponse: Eosinophils and antigen-antibody Anat. Rec. 142: 278. reactions. Ann. N. Y. Acad. Sci. 116: 964- 94. Selzman, H . M., and R. A. Liebelt. 1962 . Paneth cell granule of mo use intestine. J. 985. 80. Koffler, D ., S. Minkowitz, W. Rothman, and Cell Bioi. 15: 136-139. J. Garlock. 1962 . Immunocytochemical stud- 95. Taylor, J . J., and R. C. Flaa.. 1964. Histochemical analysis of P aneth cell granules in ies in ulcerative colitis and regional ileitis. Amer. J. Path. 41: 733-745. rats. Arch . Path. 77 : 278- 285. 81. Harrison, W. J. 1965. Autoant ibodies against 96. Speece, A. J. 1964. Histochemical distribution of lysozyme activity in organs of normal intestinal and gastric mucous cells in ulcermice and radiation chimeras. J. Histochem. ative colitis. Lancet 1: 1346-1350. Cytochem. 12: 384-391. 82. Metz, A. 1962. Ein Fall von Colitis ulcerosa in einer Vagina art ificialis. Gastroenterologia 97. Geyer, G., and W. Linss. 1965. Histochemie und Feinstruktur der Prosekret-granula in (Basel) 98: 113-116. den Panethschen Zellen der Ma.us. N atur83. Levine, M. D., J . B. Kirsner, and A. P. Klotz. wissenschaften 52 : 14-15. 1951. New concept of pathogenesis of ulcerative colitis. Science 114 : 552-553. 98. Barter, R., and A. G. E. P earse. 1953. D etection of 5-hydroxytryptamine in mammalian 84. Streicher, M . H., H. R. Catchpole, and C. L . enterochromaffin cells. Nature (London) Pi rani. 1956. Chronic ulcerative colitis: Histochemical studies. Illinois Med. J . 110: 172: 810-811. 99. Paff, G. H., H. T. Sugiura, C. A. Bacher, and 172-176. 85. Watson, A. J ., and A. D . Roy. 1960. P aneth J. S. Roth. 1952. The probable mechanism of heparin inhibition of mitosis. Anat. Rec. cells in t he large intestine in ulcerative colit is. J. Path. Bact. 80 : 309-316. 114: 499-505.
DISCUSSION OF PAPER PRESENTED BY DR. SOMMERS DR. D AVID LAGUNOFF (Seattle): There are fashions in research much as in dress, the mast "cellologist" in the company of students of inflammation finds himself somewhat in the position of a high school
girl with her skirt around her ankles. The recent lack of interest in mast cells stems from a disillusion in any role for histamine in inflammatory responses. I, therefore, would like to emphasize one
1\' ovem ber 1966
DISCUSSION
of the points D r. Sommers made, namely, that there are other substances in mast cells that qualify as potential chemical media tors \Yhich can play a role in inflammation. Chief among these are chymase and tryptase. As far as human mast cells go, the evidence is reasonable that a trypsin-like enzyme does occur in mast cells in addi tion to a chymotrypsin-like enzyme. In rodents, t here is only a chymotrypsin-like enzyme. Both these enzymes are unique in tissues in that t hey have an alkaline pH optimum, unlike t he cathepsins t hat are common constituents of cells. In fact, the alkaline pH optimum proteolytic enzymes shO\m histochemically occur only in mast cells. There are two interesting features of th e~e enzymes. First, they come packaged in an active form. They do not have to be activated like chymotrypsinogen or trypsinogen which are found in pancreatic secretions. Secondly, they are bound as particu lates. Dr. Sommers interpreted this in a negative way. H e stated that heparin and the proteolytic enzymes do not get out of the cell as qui ckly as hi stamine does, and it is necessary to overstimulate the cell for t heir release. This is partly true, but the fact is that these agents do get out with relatively minor stimulation and t hen remain locally in t he tissue. Histamine comes out and disappears because it is a small molecule that diffuses readily. The proteolytic enzymes which are bound to the granule matrix remain and therefore can give rise to a prolonged inflammatory response. In following up some of these possibilities in t he rodent mast cell, we have looked into some of the possible roles the chymotrypsin -like enzyme can play in inflammation. We have observed that t his enzyme of mast cells rapidly cleaves bradykinin. As Dr. Sommers suggested, it is very likely that in humans and dogs, it will be possible to show that the trypsinlike enzyme of mast cells is capable of activating or producing bradykinin from the circulating globulin precursor. I would now like to review several misconceptions, which have been perpetuated in the literature for some t ime. Although fixation of t he mast cells for the electron microscope has been notoriously difficult, I
849
t hink today there is general agreement by the people who are actively studying mast cells that there is a limiting membrane around every mast cell granule, and this membrane seems to be important in keeping t he loosely bound histamine on the granul e. During stimulation and secre tion of t he cell , t he perigranular membrane undergoes changes that we believe are impo1 tant in releasing histamine. The question of leucine aminopeptidase in mast cell activity has been raised. The histochemical substrates t hat have been used to demonstrate this enzyme activity do not demonstrate the classical leucine am inopeptidase, but act ually a very specific aryl amidase, which seems to have no relation to leucine aminopeptidase. And t hen t here is Riley's hypothesis, which he presented at the New York Academy of Science several years ago, that heparin has something to do with the formation of ground substance. To date, every t racer study that has been clone in labeling mast cell heparin indicates t hat there is no possibility that this heparin or any of its parts is incorporated into ground substance. · Several years ago, we observed an unusual cell in t he r at colon which we hav~ had great difficulty identifying. We believe that it is probably a type of intraepithelial mast cell which shows the histochemical reaction for the chymotrypsin enzyme. I mention t his to learn if someone might have observed t his same type of cell in 1mman material, and to suggest a possible role for these cells in inflammatory lesions of the bowel. DR. B. J . HAVERBACK (Los Angeles): I would like to make a few comments about t he pharmacologically active amines that were brought up in this discussion. In con ~ sidering the gastrointestinal tract, t here are at least two varieties of histamine; one type is found in the mast cells, and the ot her in the gastrointestinal mucosa. These two sites handle histamine quite differently. For ex~ ample, if tritiated histamine is admini s~ tered to an animal, the labeled amine · is taken up by the gastrointestinal mucosa but not by t he mast cells. Also, gastrointestinal mucosal histamine is not released by 48/ 80, but histamine in mast cells is N-
850
DISCUSSION
leased readily by this substance. We have studied histamine and serotonin metabo-lism in patients with both ulcerative colitis and regional enteritis. Also, the level of · ·histamine has been studied in the mucosa of the colon in patients with ulcerative colitis. There were no abnormalities of blood platelet serotonin levels in patients with either ulcerative colitis or regional enteritis. In addition, there were no abnormalities in the level of urinary 5-hydroxyindoleacetic acid. Finally, we found no significant variation in serotonin levels in mucosal tissue of the intestine in patients with acute disease. We determined levels of histamine in blood of patients with these two diseases and found that they were quite normal. Recently, we have set up a spectrophotofluorometric method for urinary histamine and found that urine histamine excretion was normal in patients with both diseases. Levels of histamine in the colonic mucosa of patients with ulcerative colitis were normal. However, in 4 or 5 cases of regional enteritis the level of histamine in diseased mucosa was considerably elevated. In contrast to the level of 6 p.g of histamine per gram of mucosal tissue in normal human ileum, levels as high as 30 to 40 p..g per g of tissue were found in mucosa from involved ileum. Interestingly, in 1 patient with Crohn's disease of the colon, the histamine level in colonic mucosa also was markedly elevated. Although no conclusions can be drawn from 4 cases, it would be interesting to extend the measurements of histamine in mucosal tissue of many other patients with regional enteritis. DR. J . S. TRIER (Madison, Wise.): Just a word about Paneth cells. I certainly agree that we are very ignorant about their function, and would like to emphasize that while it is said in the literature that proteolytic enzymes are allegedly produced by the cell, when one measures the contents of celi-free filtrates of the intestinal lumen, after exclusion of pancreatic and biliary secretion, the only two enzymes that have
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been identified are enterokinase and amylase. So in reality we really have very little data as to Paneth cell function, what the nature of their granule is, and what they secrete. There is good evidence that in fasting man and certainly in fasting animals, these cells rapidly secrete their product. By radioautographic studies with labeled leucine, we have been able to demonstrate in the mouse that in the fasting animal the turnover rate for the secretion of the Paneth cell is about 8 hr. I would like to ask Dr. Sommers two questions. First, I would like to know what he means by "ordinary inflammation" of the colon? Secondly, I would like to know if he has seen any transition forms between Paneth and goblet cells, because we have never seen these in rather extensive studies of the small intestinal crypts? DR. SoMMERS: Ordinary inflammation of the colon was defined sigmoidoscopically by Dr. Bercovitz in people having a recent nonspecific diarrhea either thought to be food poisoning, mild salmonellosis, or possibly a viral inflammation. The mucosa was hyperemic and edematous. It was not ulcerated. Therefore, you could not say it was normal, but these people recovered and had no further bowel problem. In relation to the Paneth cells, I have looked, but I have never seen a transition between them and goblet cells. DR. MARVIN BACANER (Minneapolis): If I understand you correctly, you said that cortisone stimulated mast cell production and granule release. If you are going to tie mast cell formation into some etiological connection with ulcerative colitis, how does this conform with the fact that cortisone is one of the most useful treatments of this disease? DR. SoMMERS: Cortisone has so many different simultaneous actions that it is hard to isolate one from another. In treatment, my understanding would be that the anti-inflammatory action of cortisone probably outweighs most other effects.
GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co.
MAST CELLS AND PANETH CELLS IN ULCERATIVE COLITIS SHELDON
C.
SoMMERs,
M.D.
Department of Pathology, Columbia University College of Physicians and Surgeons, Francis Delafield Hospital, New York, New York
In the last 5 years . it has become clear that the tissue mast cells and eosinophils are particularly important in nonspecific ulcerative colitis. A primary biological factor that EngeF believed to characterize individuals who are susceptible to develop ulcerative colitis evidently involves the basophil granulocytes, eosinophils, and mast cells in the connective tissue of the large intestine. Forty years ago, Buie 2 recognized four sigmoidoscopic sequential stages in the activation of ulcerative colitis: (1) diffuse hyperemia, (2) mucosal edema with hyperemia and friability, (3) miliary mucosal abscesses , and (4). ulcers formed from the ruptured abscesses. Crypt abscesses have been regarded until recently as the earliest lesions characteristic of ulcerative colitis. 3-5 Newer evidence supports Manson-Bahr's6 belief that ulcerative colitis begins in the submucosa and affects the mucosa secondarily. A review of mast cell structure and functions will be helpful in understanding Buie's stages 1 and 2 in the development of ulcerative colitis. Tissue mast cells normally contain granules heavily stained with polychrome dyes, such as the ·wright and Giemsa methods. Mast cells are quite inconspicuous with hematoxylin and eosin stains. The cytoplasmic granules are periodic acidSchiff (PAS) positive, sudan black B positive after periodic acid and alcian blue-safranin positive at pH 1.0. 7-9 Toluidine-blue metachromasia characterized the mast cell granules, as well as intravital tetrazolium binding to acid groups. 9 - 10 DiAddress requests for reprints to: Dr. Sheldon C. Sommers, Department of Pathology, Columbia University College of Physicians and Surgeons, Francis Delafield Hospital, New York, New York 10032.
amine methods stain sulfated mucosaccharides that occur in a variety of cells with coarse cytoplasmic granules, including the polymorphonuclear leukocytes, immature eosinophils, mast cells, Paneth cells, and enterochromaffin cells; all except the last possess strongly basic proteins histochemically. 11 Toluidine-blue and diamine staining reflect a concentration of heparin in the mast cell granules, which appear by electron microscopy to develop in the hyaloplasm_12 The heparin-containing granule is a polysaccharide-protein ionic complex that incorporates considerable sulfate. 13 · 14 Mast cells are rich in histamine, 13 p.g per 106 cells, which is apparently bound to the granules electrostatically. 14 • 15 Human mast cells synthesize histamine but not 5hydroxyJryptamine ....(5JIT) (serotonin) ,1 6 alHiO'U:'gh ~ -mouse mastocytoma cells apparently do so. 17 Chymase proteolytic enzymes also are localized in the granules.14· 18 Dopamine, glucuronidase, leucine aminopeptidase, ascorbic acid, and a variety of other biologically active substances occur in mast cells. 15 • 17 - 19 Histamine, serotonin, heparin, and chymase are considered the most important in ulcerative colitis. Mast cells were named by Ehrlich 9 because they appeared well fed. They preferentially absorb and store for variable periods, by electrostatic binding or otherwise, various substances such as metal cations, basic dyes/ 0 iodine, and, in particular, 5-HT, both in human and animal tissues, to the level of 1 p.g per 106 cells. 15 · 2 1 - 24 The local concentration of 5-HT determines the extent of its uptake by mast cells. 22 The process is evident with 5-HT labeled either by 14 C or 3H. 23 The pres841
842
SOMMERS
ence of 5-HT in mast cells is best demonstrated by fluorescence microscopy. 22 · 24 In summary, the mast cell is the chief connective tissue storehouse of histamine and heparin, a depot for 5-HT, and a source of proteolytic enzymes. Discharge of the mast cell contents is selective, so that histamine is preferentially secreted and heparin retained, unless the stimuli are severe. Thus in human mastocytomas, symptoms from histamine release are common and from heparin, rare. 10· _25 · 26 Free histamine is readily released and spreads fast, so that the fixed connective tissues become activated, according to Riley. 27 Later heparin is slowly released, followed by the phagocytosis and digestion of metachromatic material by activated cells which utilize it to synthesize ground substance. ! Many different stimuli produce mast cell secretion, including autolysis, distilled :water, mechanical friction, synthetic : amines like compound 48/80, dextran, polyvinyl-pyrrolidone (PVP), antigens, ovomucoid, bacterial toxins, snake venom, tissue extracts, alkaloids, antihistaminics, surface active agents, turpentine, ultraviolet light, etcY· 28 -30 Various metal ions, sulfhydryl or amine reagents, and oxidative inhibitors prevent degranulation. In anaphylaxis the guinea pig mast cells were significantly damaged and released histamine and slow reacting substance, except for the intestinal mast cells. 31 Soluble antigen-antibody complexes were ineffective. In rats histamine release required the activation of a chymase to initiate the inflammatory response in antigen-antibody reactions. 32 -34 Immunological injury in rodents, with mast cell degranulation, was followed by increased connective tissue viscosity and a "gelatinous state." 35 Delayed type allergic reactions in guinea pigs also depended on the liberation of biologically active substances from the mast cells.30 Mast cell granules discharged by nonspecific stimuli have an increased avidity for metals, like lead, and a secondary attraction for calcium and phosphate that initiates intense calcification, the calcergy phenomenon of Selye. 37
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Endocrine factors alter the mast cell population and activities. Cortisone increased both the number of mast cells and their degranulation in experimental granulomas of rats and human or rat gastric biopsies. 38 -40 Estrogen increased the total mast cells, their granule discharge and regranulation in human oral biopsies. Estrogen thus prevented the edema that ordinarily followed surgical injury and increased the connective tissue acid mucopolysaccharides and vessel wall metachromasia.41 Lindholm 42 · 43 described the embryogenesis and cell density of mast cells in the human and animal gastrointestinal tract. In childhood the mast cells increased to an average of 2000 per mm 3. In adults still greater numbers were present in the esophagus and rectum. Hamsters had more mast cells than rats, and the smallest numbers occurred in the large intestine. Kraft and Kirsner, 44 and Norris, Zamchek, and Gottlieb 45 investigated mast cells in the human gastrointestinal tract. Cell counts were highest in the mucosa of the appendix, stomach, and duodenum, and in the submucosa of the large intestine, jejunum, and ileum. By electron microscopy the human gastric mucosa contained two types of mast cells, spindle shaped and rounded. The second type was more common ; the first type was chromaffin positive and disappeared after corticoid administration.46 Note again the local absorption of 5-HT (serotonin) by mast cells. The relevance of the foregoing information to ulcerative colitis was first pointed out by McGovern and Archer. 47 Based on an analysis of 32 surgical and 20 autopsy specimens, they concluded that the fundamental reactions were typical of histamine-release phenomena, with a depletion of granules from the mast cells found in abundance in the human colonic submucosa and muscular coats. They considered that psychosomatic influences would liberate more acetylcholine in the colon and provoke an increased mast cell degranulation. The pathological consequences were congestion and edema-most prominent in the submucosa-muscle spasm, and hypertrophy.
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MAST AND P ANETH CELLS
843
Rebuck and associates 48 · 49 contributed numerous than normal, 56 evidently disa valuable clue by showing in ulcerative charge excess histamine to initiate the excolitis patients that skin windows with aggerated flare and wheal reaction of Mirdiphtheria toxoid had increased basophil vish.57 The stimulus may be mechanical, leukocytes, a persisting neutrophil leuko- physical, chemical, or immunologicaL 58 cyte response, lakes of metachromatic ma- Certain stimuli, like endotoxin, are ineffecterial, and a delayed lymphocyte-macro- tive.59 Histamine, beyond its excessive lophage reaction. The neutrophil persistence cal production and release, might also enwould correlate with prolonged mainte- counter an altered local responsiveness, nance of an alkaline pH in the inflamed binding, or metabolism, 60 but nothing is connective tissue, according to Menkin. 50 known of this in ulcerative colitis. The sulWolf-Jurgensen et aL 51 observed basophil fated mucopolysaccharides of ground subexudation more commonly in Schick-nega- stance are increased.6 1 tive patients. They related it to a delayed Beyond the effects of histamine the hypersensitivity reaction. chronic hyperemia and edema of the coJuhlin 52 and Shelley and Parnes53 found lonic mucosa apparently reflect the disthe relative or absolute blood basophil charge of serotonin downward from the leukocyte counts to be increased in active argentaffin mucosal cells and its temporary ulcerative colitis but normal during remis- storage in adjacent mast cells, as alluded to sions. Basophil leukocyte counts are . re- previously.22 • 46 The persistence and severduced after corticosteroids. McAuley and ity of the edema are probably attributable Sommers 54 and Hiatt and Katz 55 reported to the greater potency of 5-HT.6 2, Ga The increased mast cells in the lamina propria, resulting vasodilation is largely venular. 64 submucosa, and muscularis of surgical Blotchy hypervascularity is demonstrated specimens of ulcerative colitis. In biopsies by microangiography. 65 The biological of active ulcerative colitis Bercovitz and tragedy in ulcerative colitis is that abnorSommers 56 found mast cells in the lamina mally large numbers of mast cells are propria increased to 8.5 times normal and available to store and to release serotonin 1.7 times the number present in ordinary close to where the bulk of it is formed. 66 As concerns Buie's stage 3, or crypt abinflammation. Submucosal mast cells were increased 2- or 3-fold. Sequential biop- scess formation, EngeJl pointed out that sies in 3 patients demonstrated mast -cells ulcerative colitis never occurred spontanein the lamina propria to decrease in re- ously in animals. Mecholyl led to experimissions from 8.7 to 3.7%, from 1.8 to mental colonic vasodilation, vascular stasis, 0.4%, and from 3.8 to 1.9% of the total and hemorrhagic necrosis with ulceraconnective tissue cells. In a subsequent tions in some dogs, but not a true ulceraexacerbation the last case had a rise to tive colitis. 67 Crypt lesions or crypt ab15.0%. Submucosal mast cells changed scesses have now been observed in at least three experimental situations: (1) irradisimilarly. The dysfunctional sigmoidal changes ated rats, particularly when adrenalectomay now be visualized in ulcerative colitis mized,68 (2) irradiated mice treated with in Buie's stage 1 of hyperemia and stage 2 homologous lymphocytes, 69 (3) neonatally of edema. Mirvish57 observed that light thymectomized hamsters. 70 All three situpressure on the mucosa in remissions of ul- ations have involved inadequate regeneracerative colitis produced a characteristic tion of the crypt epithelial cells and either abnormal response involving (1) immedi- insufficient or abnormal trephocytic lymate development of a red edematous patch phocytes. Rebuck's 49 observation of the at least 2 mm high, (2) formation in 10 sec inhibited lymphocyte-macrophage reto 6 min of a soft, sharply demarcated sponse in skin windows may be pertinent. wheal, and (3) obscuring of the superficial In biopsies of ulcerative colitis epithelial network of arterioles . and, sometimes, nuclear fragmentation seems to be the punctate hemorrhages.· The submucosal earliest evident pathological change.5, 5G In mast cells, which are about 3 times more biopsies crypt abscesses occur less com-
844
SOMMERS
monly and may be regarded as secondary phenomena. 71 In stage 4 the mucosal ulcerations develop. M ast cells in the mucosa are now 8 t imes more numerous than normal and twice as numerous as the average found in ordi na ry acute or chronic inflammation.56 In the submucosa a pproximately half the mast cells are completely degranul ated, and the total population is approxim ately twic e normal and 1.5 times the average in acute or chronic infl amma ti on.56 Comp let e mast cell degra nul ation occurs a bout bYice as commonly in ulcerative colit is as in ordinary infl ammation , ''"it h a consequent release of 5-HT, heparin, and chymase. 56 · 72 · 73 It should be recalled again that the mast cell secretory pro cess is selective, hist amine first and serotonin second . H eparin and proteolytic enzymes are discha rged only after drastic stimuli.'2· 73 Since heparin interferes with plasma coagulation, and fibrin blo ck age of the venules and lymphatics is an important mechanism th at localizes infla mmation, a more widespread reaction results. Three or more proteolytic enzy mes a re released in act ive ulcerative colitis : (1) a mast cell a lkaline protease or chym ase wit h optimal activity at pH 8.9 or higher, and possibly also a tryptase,73 (2) a protease with mucinoly ti c properties, active at acid pH 6.1 ,74 and (3) a cy tolyt ic or acantholy ti c factor most active at neut ral pH of 7.2 to 7.8, described by Stought on. 75· 76 The wide pH range of the proteolytic enzymes released would fur t her exacerbate t he inflammation and increase t he associated t issue necrosis. Eosinophils are t he most notably increased mucosa l connective t issue cells in biopsies of ac tive ulcerative colit is, 30 times norm al a nd 6 times t he number present in ordinary inflammation. 56 They also occur in large numbers in cy tological prepa rations taken from the sigmoid mucosal surface77 and in the abnormal inflammatory sequence in skin windows of ulcerative colitis patients.49 The excessive local exudation of eosinophils is explained by t heir at t ra ction to tissue n ecrosis. A st atistically significant increase of the blood eosinophils also occurs during rel apses of
Vol . 51 , No . 5, Part 2
ulcerative colitis.78 It is claimed that eosinophils pha gocytize antigen-antibody complexes and t hat their granules are a source of profibrinolysin.' 9 • 80 Explanations of t he relationship of eosinophils to ulcerative colitis await a better underst anding of their functions. Their a bundance suggest s less than adequat e corticoid levels.' 8 l'l'!ucosal plasma cells in biopsies of active ulcerative colit is are increased up to 15 t imes norma l and 4 times the number found in ordinary infla mmation. 56 This may reflect an antibody reaction to incidental bacteri al infection or t he form at ion of aut oant ibodies t o mucus. 80· 81 Ulcerative colit is t hus involves an abnormal inflammatory process attribut able t o a n excess of histamine-releasing mast cells t hat may also absorb 5-HT from the adj acent rich source of serotonin. The sub sequent release of 5-HT would considerably exacerb at e colonic hy peremia and edema . Complete mast cell granule discharge adds the furth er deleterious effects of heparin and a wide spect rum of proteolytic enzymes. The colonic contents do not seem to init iat e ulcerative colitis, which has occurred for example in an a rtifi cial vagina m ade 17 year s before from the sigmoid colon. 82 Ulcerative colitis no longer appears t o be a primary disease of t he ground substance or connective ti ssue fibers, as had been thought .83 · s4 P anet h cells are ordinarily r are in the colon but are increased t o 200 times normal in ul cerative colitis, proba bly through metaplasia of t he regenerating epithelium.85-87 However , very few occurred in sigmoid or rectal biopsies.85 One study of ulcerative colit is also reported the a r gent affin serotonin-containing mucosal cells to be increased,85 and anot her described them as decreased, with a cellular serotonin concentration only one-fifth normal. 87 P a neth cell metaplasia appeared correlated wit h the duration and degree of colitis. Other inflammatory intestinal diseases during regeneration show increased P an eth cells in the mucosal crypts.ss A lack of knowledge of the P aneth cell hampers interpreting its relationship t o ulcerative colitis. These cells are normally
November 1966
MAST AND P ANETH CELLS
845
most numerous in the small intestine but ulcerative colitis are as yet poorly underare also common in gastric intestinal stood. Ulcerative colitis, furthermore, inmetaplasia. They are characteristically as- volves several processes that parallel allersociated with a condensed eosinophilic mu- gic or hypersensitivity diseases, but how cus,80 which is one histological characteris- close the relationship is remains unclear. tic of ulcerative colitis. 90 Paneth cells are Summary further thought to secrete special proteolytic enzymes that are stored in distincIn nonspecific ulcerative colitis, mucosal tive coarse protein-polysaccharide gran- hyperemia and persistent edema are atules .91-95 Their lysozyme activity ordinarily tributable to local excesses of histamine reis high and increases after irradiation. 96 leased by an increased colonic population In fine structure Paneth granules resemble of mast cells. Mast cell storage and release the prosecretory granules of serous glands, of serotonin produced in nearby argentaffin IYith transition to goblet cells. 97 The fea- mucosal cells are likely exacerbating factures described suggest that Paneth cell tors. Severe stimuli may release mast cell metaplasia is a secondary process in ulcer- granules that contain heparin and proteoative colitis. lytic enzymes, exaggerating and spreading The deranged inflammatory reactions in the associated inflammation and necrosis. ulcerative colitis provide a partial explana- Eosinophils are particularly numerous in tion of the disease. Its etiology remains the inflamed mucosa. Blood basophil and unknown. Not all ulcerative colitis pa- eosinophil leukocytes are reportedly intients have manifested an increased baso- creased when colitis is active. phil leukocyte reaction or a measurable Paneth cell metaplasia appears to be a excess of mast cells in the colon wall. The secondary phenomenon of mucosal regenervasculitis type of ulcerative colitis, which is ation in ulcerative colitis, which may have less common and clinically often fulminant, a poorly defined relationship to altered resembles periarteritis nodosa localized to lymphocytes. The abnormal inflammatory the large intestine. This type probably has process in ulcerative colitis is a newly apa quite different pathogenesis. 9 Concerning preciated characteristic of the disease. the common superficial recurrent cryptREFERENCES abscess type of ulcerative colitis, however, our knowledge has recently considerably 1. Engel, G. L. 1961. Biologic and psychologic features of the ulcerative colitis patient. increased. Gastroenterology 40: 313-322. Improved fluorescence methods are now 2. Buie, L. A. 1926. Chronic ulcerative colitis. available to identify serotonin in the coJ. A. M. A. 87: 1271-1274. lonic argentaffin cells and mast cells during 3. Warren, S., and S. C. Sommers. 1949. Patho24 98 different stages of ulcerative colitis. • genesis of ulcerative colitis. Amer. J. Path. Histochemical studies are needed to investi25 : 657-679. gate in greater detail the formation, stor4. Lumb, G. 1958. Rectal biopsy in ulcerative age, and release of histamine, 5- HT, hepacolitis. Dis. Colon Rectum 1: 37--43. rin, and proteolytic enzymes by mast cells 5. Lumb, G., and R. H. B. Protheroe. 1958. Ulin the human large intestine. The fragmencerative colitis. Gastroenterology 34: 381407. tation of crypt epithelium that leads to 6. Manson-Bahr, P. 1943. The dysenteric discrypt abscess formation and its relation to orders, p. 629. Williams & Wilkins Comaltered lymphocytes and local mitotic inhipany, Baltimore. bition by heparin 99 are worth further in7. Feagler, J . R., and J. F. A. McManus. 1964. vestigating. The conflicting endocrine data Extraction resistance of sudan stained mast indicate that corticoids and estrogen are cells after previous acid treatment. J. Ristosomehow associated with increased mast chern. Cytochem .12 : 530-532. cells or basophils and with their augmented 8. Combs, J. W., D. Lagunoff, and E. P. Benditt. degranulation. The roles of the eosinophils 1965. Differentiation and proliferation of and plasma cells remain uncertain. Paneth embryonic mast cells of the rat. J . Cell Biol. 25: 577-592. cell functions and enzymatic processes in
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SOMMERS
9. Selye, H. 1965. The mast cells, p. 494. Butterworth and Company, \Vashington, D.C. 10. Koenig, H. 1965. Intravital staining of lysosomes and mast cell granules by tetrazolium salts. J. Histochem . Cytochem. 13: 411-413. 11. Spicer, S. S. 1965. Diamine methods for differentiating mucosubstances histochemically . .T. Histochem. Cytochem. 13: 211-234. 12. Fernando, X. V. P ., and H . Z. Movat. 1963. The fine structure of connective tissue. III. The mast cell. Exp. iVIolec. Path. 2: 450-463. 13. Watson, IY. C., and J. S. Kennedy. 1960. Regeneration of the tissue mast cell: An autoradiographic study. Brit. J. Exp. Path . 41: 385-388. 14. Lagunoff, D., lVI. T. Philips, 0. A. Iseri, and E. P. Benditt. 1964. I solation and preliminary characterization of rat mast cell granules. Lab. Invest. 13 : 1331-1344. 15. u,·nas, B. 1964. The acute inflammatory response: Release processes in mast cells and their activation by injury. Ann. N. Y. Acad. Sci . 116: 880-890. 16. Zimmer, J. G., B. M. McAllister, and D. J. Demis. 1965 . Mucopolysaccharides and mastocytosis. J. Invest. Derm. 44: 33-37. 17. Lovenberg, W., R. J . Levine, and A. Sjoerdsma. 1965. A tryptophan hydroxylase in cell-free extracts of malignant mouse mast cells. Biochem. Pharmacal. 14: 887-889. 18. Ende, N., Y. Katayama, and J. V. Auditore. 1964. Multiple proteolytic enzymes in the human mast cell. Nature (London) 201: 1197-1198. 19. Glick, D ., and S. Hosoda. 1965. Histochemistry. LXXVIII. Ascorbic acid in normal mast cells and macrophages and in neoplastic mast cells. Proc . Soc. Exp. Bioi. Med. 119 : 52-56. 20. Jones, D. J., M . G. Buse, and W. C. Worthington, Jr. 1964. Mast cell localization of radioiodine in the rat. J. Histochem. Cytochem .12: 856-857. 21. Ende, N., and E. I. Cherniss. 1958. Mast cells, histamine and serotonin. Amer. J. Clin. P ath. 30: 35-36. 22. Enerback, L. 1963. Serotonin in human mast cells. Nature (London) 197: 610-611. 23. Furano, A. V., and J . P . Green. 1964. The uptake of biogenic amines by mast cells of the rat. J. Physiol. (London) 170: 263-271. 24. Van Orden, L. S., I. Vugman, and N .J. Giarman. 1965. 5-Hydroxytryptamine in single neoplastic mast cells: A microscopic spectrofluorometric study. Science 148: 642-644. 25. Mutter, R . D ., M. Tannenbaum, and J. E.
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Vol. 51, No. 5, PaTl 2
Ultmann. 1963. Systemic mast cell disease. Ann. Intern. Med. 59: 887-906. Braunsteiner, H. 1964. Mastocytosis. German Med. Monthly 9: 422--424. Riley, J. F. 1962. Histamine and heparin in mast cells. Why both? Lancet 2: 40-41. Schmid, H. 1965. Die Granula der Gewebsmastzellen der Ratte bei Autolyse. Virchow Arch. Path . Anat. 339: 168-170. Valtonen, E. J., J. J anne, and l\1. Siimes. 1964. The effect of the erythemal reaction caused by ultraviolet irradi ation on mast cell degranulation in the skin. Acta · Dermatovener. 44 : 269-272. Johansson, H., B. \Yestin, and B. Tornberg. 1964. The m ast cell and regional circulation in the skin of t he rat in turpentine-induced local inflammatio n. Acta Dermatovener. 44: 342-348. Boreus, L., and N. Chakrayarty. 1960. Tissue mast cells, histamine and "slow reacting substance" in anaphylactic reaction in guinea pig. Acta Physio l. Scan d. 48: 315-322. Mota, I. 1961. M echanism of action of antigen-antibody complexes : Their effect on mast cells. Nature (London) 191: 572- 573. K eller. R., and I. Beeger. 1963. Anaphylaxis in isolated rat mast cells. I. Effect of peptidase substrates and inhibitors. Int. Arch . Allerg. 22 : 31-44. Draper, L. R., and D . E. Smith. 1961. A function for tissue mast cells. Science 134 : 842. Goldgraber, M., and R. M. Lewert. 1965. Immunological injury of mast cells and connective tissue in mice infected with Strongyloides ratti . J. Parasit. 51: 169-174. Raab, W. G. 1965. Role of mast cells in allergy of delayed type. Nature (London) 206 : 518-519. Selye, H., G. Gabbiani, and N. Serfimov. 1964. Histochemical studies on the role of the mast cell in calcergy. J. Histochem. Cytochem. 12 : 563-569. Romani, J. D. 1953. Action des glycocorticoides sur les mastocytes dans le tissue conjunctif du granulome experimental chez le rat. Compt. Rend. Soc. Bioi. 147: 17711773. Siurala, M., T . Rasanen, and M. Sundberg. 1959. The effect of ACTH upon the tissue eosinophils and mast cells in the human gastric mucosa. Acta Path. Microbial. Scand. 47: 337-341. Rasanen, T. 1961. The role of the adrenals on the mucosal mast cells and tissue eosinophils in the gastric wall of rat. Acta Physiol. Scand. 52: 162-166.
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MAST AND PANETH CELLS
41. Schiff, M., and H. F. Burn. 1961. The effect of estrogens on mast cells and enzymes in connective tissue metabolism in humans. Laryngoscope 71: 765-780. 42. Lindholm, S. 1959. Mast cells in the wall of the alimentary tract. A quantitative study on human fetuses and man. Acta Path. Microbial. Scan d. (Suppl .) 132: 1-73. 43. Lindholm, S. 1960. The occurrence of connect ive tissue mast cells in the alimentary canal wall of hamster and rat. Acta. Path. Microbial. Scand. 48 : 328-334. 44. Kraft, S. C., and J. B. Kirsner. 1960. Mast cells and the gastrointestinal tract . Gastroenterology 39 : 764-770. 45. Norris, H. T ., N. Zamcheck, and L. S. Gottlieb . 1963. The presence and distribution of mast cells in the human gastrointestinal t ract at autopsy. Gastroenterology 44: 448-455. 46. Hibbs, R. G., G. E. Burch, and J. H. Phillips. 1960. Electron-microscopic observations on the human mast cell. Amer. Heart J . 60: 121-127. 47. McGovern, V. J., and G. T. Archer. 1957. The pathogenesis of ulcerative colitis. Aust. Ann. Med. 6 : 68-74. 48. Priest, R. J., J . vV. R ebuck, and G. T. Havey. 1960. A new qualitative defect of leukocyte function in ulcerative colitis. Gastroenterology 38: 715-720. 49. Rebuck, J . W., J . M. Hodson, R. J. Priest, and C. L. Barth. 1963. Basophilic granulocytes in inflammatory tissue of man. Ann. N . Y. Acad. Sci.103: 409-426. 50. Menkin, V. 1950. Newer concepts of inflammation, p. 23-30. Charles C Thomas Company, Springfield, Ill. 51. Wolf-Jiirgensen, P., P. Anthonisen, and P. Riis. 1965. Cutaneous abacterial inflammatory reaction in patients with ulcerative colitis. Gut 6: 221-224. 52. J uhlin, L. 1963. Basophil leukocytes in ulcerative colitis. Acta Med. Scand.173: 351-359. 53. Shelley, W. B., and H . M. Parnes. 1965. The absolute basophil count. J. A. M. A. 192 : 368-370. 54. McAuley, R. L., and S. C. Sommers. 1961. Mast cells in nonspecific ulcerative colitis. Amer. J. Dig. Dis. 6 : 233-236. 55. Hiatt, R. B., and L. Katz. 1962. Mast cells in inflammatory conditions of the gastrointestinal tract. Amer. J. Gastro ent. 37: 541545. 56. Bercovitz, Z. T ., and S. C. Sommers. 1966. Altered inflammatory reaction in ulcerative colitis. Arch. Intern. Med. 117 : 504-510.
847
57. Mirvish, L. 1960. The mucosa of the rectosigmoid in ulcerative colitis. S. Afr. Med. J. 34 : 732-735. 58. Kirsner, J. B. 1965. The immunologic response of the colon. J . A. M. A. 191: 809814. 59. Gustafson, G. T., and S. Cronberg. 1963. Effect of endotoxin on mast cells and the extension of the local Shwartzman reaction to the hamster. Acta Path. Microbial. Scand. 59 : 21-31. 60. Code, C. F., M. M. H urn, and R. G. Mitchell. 1964. Histamine in human disease. Mayo · Clin. Proc. 39 : 715-737 . 61. H akkinen, I. P. T. 1961. Histamine induced changes in the sulphate metabolism of the duodenum and prepyloric region of the stomach : An experimental study with special reference to ulcer formation. Acta Physiol. Scan d. (Suppl .) 177: 1-76. 62. Rowley, D. A., and E. P. Benditt. 1965. 5-Hy~ droxytryptamine and histamine as mediators of the vascular injury produced by agents wlfich damage mast cells in rats. J. Exp. Med. 103: 399-411. 63. Majno, G., and G. E. P alade. 1961. Studies on inflammation. I. The effect of histamine and serotonin on vascular permeability: An· electron microscopic study. J. Biophys. Biochem. Cytol. 11 : 571-605. 64. Majno, G., G. E . Palade, and G. I. Schoefl, 1961. Studies on inflammation. II. The site of action of histamine and serotonin along the vascular tree. A topographic study. J. Biophys. Biochem. Cytol. 11 : 607-627. 65. Spjut, H. J., and A. R. M argulis. 1965. Microangiographic patterns of chronic ulcerative colitis. Dis. Colon R ectum 8: 215-221. 66. Udenfriend, S. 1959. Biochemistry of serotonin and other indoleamines. Vitamins Hormones 17: 133-154. 67. Wener, J ., H. E. Hoff, and M. A. Simon. 1949. E xperimental production by Mecholyl of ulcerative colitis. Gastroenterology 12 : 637647. 68. Sommers, S. C., and S. W an·en. 1955. Ulcerative colitis lesions in irradiated rats. Amer. J. D ig. Dis. 22 : 109-111. 69. de Vries, M . J . 1962. P atho genesis of the intestinal lesions in radiation chimeras. Transplantation 30: 13-20. 70. H ard, R. C., C. Martinez, and R. A. Good. 1964. Intestinal crypt lesions in neonatally thymectomized hamsters. Nature (London) 204- : 455-457. 71. M atts, S. G. F. 1961. The value of rectal -hi-
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DISCUSSION
Yol. 51, No.5, Part 2
opsy in the di agnosis of ulcerative colitis. 86. P aterson, J. C., and S. H . \Vatson. 1961. P aQuart. J. Med. SO: 393-407. neth cell metaplasia in ulcerative colitis. Amer. J. Path. 38: 243-249. 72. Tocantins, L. M. 1948. Historical notes on blood platelets. Blood S : 1073-1082. 87. Verity, M . A., S. M. Mellinkoff, M. Frankland, 73. Lagunoff, D ., and E . P. Benditt. 1963. Proand M. Greipel. 1962. Serotonin content and argentaffin and Paneth cell changes in ulteolytic enzymes of mast cells. Ann. N. Y. Acad. Sci. 103: 185-198. cerative colitis. Gastroenterology 43: 24-31. 74 . Warren, S., and S. C. Sommers. 1950. Proteol- 88. Conrad, M . E., F. D . Sch'i\·artz, and A. A. Young. 1964. Infectious hepatitis: A generysis in intestinal disease. Gastroenterology alized disease . Amer. J . iVIed. 37 : 789-801. 14: 522-526. 75. Stoughton, R. B. 1952. Enzymatic cytolysis of 89. H ertzog, A. J. 1937. The Paneth cell. Amer. J . P ath. 13: 351-358. epithelium by filtrates of feces from pa- \ j 90. vVarren, S., and S. C. Sommers. 1954. Patholtients with ulcerative colitis. Science 116: ogy of regional ileitis and ulcerative colitis. 37-39. 76. Fleming, W. H., E . W. Smith, and T. R. HenJ. A. M. A. 154: 189-193. drix. 1962. Acantholytic factor and ulcera- 91. De Castro, N. M., S. ·w. D a Silva., and F. A. Saad. 1959. Preliminary observations of t he tive colitis. Bull. Hopkins Hosp. 111: 285Paneth cell of the T amand1ta tetradactyla 291. Lin. Acta Anat. (Basel) 38 : 345-352. 77. Anthonisen, P ., and P. Riis. 1961. A new diagnostic approach to mucosal inflammation in 92. Selzman, H. M., and R. A. Liebelt . 1961. A cytochemical analysis of Paneth cell secreproctocolitis. Lancet 2 : 81-82. tion in the mouse. Anat. Rec. 140 : 17-22. 78. Riisa.ger, P. M. 1959. Eosinophil leukocytes in 93. Selzman, H. M., and R. A. Liebelt. 1962. The ulcerative colitis. Lancet 12 : 1008-1009. 79. Litt, M. 1964. The acute inflammatory rePaneth cell granules of the mouse intestine. sponse: Eosinophils and antigen-antibody Anat. Rec. 142: 278. reactions. Ann. N. Y. Acad. Sci. 116: 964- 94. Selzman, H . M., and R. A. Liebelt. 1962 . Paneth cell granule of mo use intestine. J. 985. 80. Koffler, D ., S. Minkowitz, W. Rothman, and Cell Bioi. 15: 136-139. J. Garlock. 1962 . Immunocytochemical stud- 95. Taylor, J . J., and R. C. Flaa.. 1964. Histochemical analysis of P aneth cell granules in ies in ulcerative colitis and regional ileitis. Amer. J. Path. 41: 733-745. rats. Arch . Path. 77 : 278- 285. 81. Harrison, W. J. 1965. Autoant ibodies against 96. Speece, A. J. 1964. Histochemical distribution of lysozyme activity in organs of normal intestinal and gastric mucous cells in ulcermice and radiation chimeras. J. Histochem. ative colitis. Lancet 1: 1346-1350. Cytochem. 12: 384-391. 82. Metz, A. 1962. Ein Fall von Colitis ulcerosa in einer Vagina art ificialis. Gastroenterologia 97. Geyer, G., and W. Linss. 1965. Histochemie und Feinstruktur der Prosekret-granula in (Basel) 98: 113-116. den Panethschen Zellen der Ma.us. N atur83. Levine, M. D., J . B. Kirsner, and A. P. Klotz. wissenschaften 52 : 14-15. 1951. New concept of pathogenesis of ulcerative colitis. Science 114 : 552-553. 98. Barter, R., and A. G. E. P earse. 1953. D etection of 5-hydroxytryptamine in mammalian 84. Streicher, M . H., H. R. Catchpole, and C. L . enterochromaffin cells. Nature (London) Pi rani. 1956. Chronic ulcerative colitis: Histochemical studies. Illinois Med. J . 110: 172: 810-811. 99. Paff, G. H., H. T. Sugiura, C. A. Bacher, and 172-176. 85. Watson, A. J ., and A. D . Roy. 1960. P aneth J. S. Roth. 1952. The probable mechanism of heparin inhibition of mitosis. Anat. Rec. cells in t he large intestine in ulcerative colit is. J. Path. Bact. 80 : 309-316. 114: 499-505.
DISCUSSION OF PAPER PRESENTED BY DR. SOMMERS DR. D AVID LAGUNOFF (Seattle): There are fashions in research much as in dress, the mast "cellologist" in the company of students of inflammation finds himself somewhat in the position of a high school
girl with her skirt around her ankles. The recent lack of interest in mast cells stems from a disillusion in any role for histamine in inflammatory responses. I, therefore, would like to emphasize one
1\' ovem ber 1966
DISCUSSION
of the points D r. Sommers made, namely, that there are other substances in mast cells that qualify as potential chemical media tors \Yhich can play a role in inflammation. Chief among these are chymase and tryptase. As far as human mast cells go, the evidence is reasonable that a trypsin-like enzyme does occur in mast cells in addi tion to a chymotrypsin-like enzyme. In rodents, t here is only a chymotrypsin-like enzyme. Both these enzymes are unique in tissues in that t hey have an alkaline pH optimum, unlike t he cathepsins t hat are common constituents of cells. In fact, the alkaline pH optimum proteolytic enzymes shO\m histochemically occur only in mast cells. There are two interesting features of th e~e enzymes. First, they come packaged in an active form. They do not have to be activated like chymotrypsinogen or trypsinogen which are found in pancreatic secretions. Secondly, they are bound as particu lates. Dr. Sommers interpreted this in a negative way. H e stated that heparin and the proteolytic enzymes do not get out of the cell as qui ckly as hi stamine does, and it is necessary to overstimulate the cell for t heir release. This is partly true, but the fact is that these agents do get out with relatively minor stimulation and t hen remain locally in t he tissue. Histamine comes out and disappears because it is a small molecule that diffuses readily. The proteolytic enzymes which are bound to the granule matrix remain and therefore can give rise to a prolonged inflammatory response. In following up some of these possibilities in t he rodent mast cell, we have looked into some of the possible roles the chymotrypsin -like enzyme can play in inflammation. We have observed that t his enzyme of mast cells rapidly cleaves bradykinin. As Dr. Sommers suggested, it is very likely that in humans and dogs, it will be possible to show that the trypsinlike enzyme of mast cells is capable of activating or producing bradykinin from the circulating globulin precursor. I would now like to review several misconceptions, which have been perpetuated in the literature for some t ime. Although fixation of t he mast cells for the electron microscope has been notoriously difficult, I
849
t hink today there is general agreement by the people who are actively studying mast cells that there is a limiting membrane around every mast cell granule, and this membrane seems to be important in keeping t he loosely bound histamine on the granul e. During stimulation and secre tion of t he cell , t he perigranular membrane undergoes changes that we believe are impo1 tant in releasing histamine. The question of leucine aminopeptidase in mast cell activity has been raised. The histochemical substrates t hat have been used to demonstrate this enzyme activity do not demonstrate the classical leucine am inopeptidase, but act ually a very specific aryl amidase, which seems to have no relation to leucine aminopeptidase. And t hen t here is Riley's hypothesis, which he presented at the New York Academy of Science several years ago, that heparin has something to do with the formation of ground substance. To date, every t racer study that has been clone in labeling mast cell heparin indicates t hat there is no possibility that this heparin or any of its parts is incorporated into ground substance. · Several years ago, we observed an unusual cell in t he r at colon which we hav~ had great difficulty identifying. We believe that it is probably a type of intraepithelial mast cell which shows the histochemical reaction for the chymotrypsin enzyme. I mention t his to learn if someone might have observed t his same type of cell in 1mman material, and to suggest a possible role for these cells in inflammatory lesions of the bowel. DR. B. J . HAVERBACK (Los Angeles): I would like to make a few comments about t he pharmacologically active amines that were brought up in this discussion. In con ~ sidering the gastrointestinal tract, t here are at least two varieties of histamine; one type is found in the mast cells, and the ot her in the gastrointestinal mucosa. These two sites handle histamine quite differently. For ex~ ample, if tritiated histamine is admini s~ tered to an animal, the labeled amine · is taken up by the gastrointestinal mucosa but not by t he mast cells. Also, gastrointestinal mucosal histamine is not released by 48/ 80, but histamine in mast cells is N-
850
DISCUSSION
leased readily by this substance. We have studied histamine and serotonin metabo-lism in patients with both ulcerative colitis and regional enteritis. Also, the level of · ·histamine has been studied in the mucosa of the colon in patients with ulcerative colitis. There were no abnormalities of blood platelet serotonin levels in patients with either ulcerative colitis or regional enteritis. In addition, there were no abnormalities in the level of urinary 5-hydroxyindoleacetic acid. Finally, we found no significant variation in serotonin levels in mucosal tissue of the intestine in patients with acute disease. We determined levels of histamine in blood of patients with these two diseases and found that they were quite normal. Recently, we have set up a spectrophotofluorometric method for urinary histamine and found that urine histamine excretion was normal in patients with both diseases. Levels of histamine in the colonic mucosa of patients with ulcerative colitis were normal. However, in 4 or 5 cases of regional enteritis the level of histamine in diseased mucosa was considerably elevated. In contrast to the level of 6 p.g of histamine per gram of mucosal tissue in normal human ileum, levels as high as 30 to 40 p..g per g of tissue were found in mucosa from involved ileum. Interestingly, in 1 patient with Crohn's disease of the colon, the histamine level in colonic mucosa also was markedly elevated. Although no conclusions can be drawn from 4 cases, it would be interesting to extend the measurements of histamine in mucosal tissue of many other patients with regional enteritis. DR. J . S. TRIER (Madison, Wise.): Just a word about Paneth cells. I certainly agree that we are very ignorant about their function, and would like to emphasize that while it is said in the literature that proteolytic enzymes are allegedly produced by the cell, when one measures the contents of celi-free filtrates of the intestinal lumen, after exclusion of pancreatic and biliary secretion, the only two enzymes that have
Vol. 51, No.5, Part 2
been identified are enterokinase and amylase. So in reality we really have very little data as to Paneth cell function, what the nature of their granule is, and what they secrete. There is good evidence that in fasting man and certainly in fasting animals, these cells rapidly secrete their product. By radioautographic studies with labeled leucine, we have been able to demonstrate in the mouse that in the fasting animal the turnover rate for the secretion of the Paneth cell is about 8 hr. I would like to ask Dr. Sommers two questions. First, I would like to know what he means by "ordinary inflammation" of the colon? Secondly, I would like to know if he has seen any transition forms between Paneth and goblet cells, because we have never seen these in rather extensive studies of the small intestinal crypts? DR. SoMMERS: Ordinary inflammation of the colon was defined sigmoidoscopically by Dr. Bercovitz in people having a recent nonspecific diarrhea either thought to be food poisoning, mild salmonellosis, or possibly a viral inflammation. The mucosa was hyperemic and edematous. It was not ulcerated. Therefore, you could not say it was normal, but these people recovered and had no further bowel problem. In relation to the Paneth cells, I have looked, but I have never seen a transition between them and goblet cells. DR. MARVIN BACANER (Minneapolis): If I understand you correctly, you said that cortisone stimulated mast cell production and granule release. If you are going to tie mast cell formation into some etiological connection with ulcerative colitis, how does this conform with the fact that cortisone is one of the most useful treatments of this disease? DR. SoMMERS: Cortisone has so many different simultaneous actions that it is hard to isolate one from another. In treatment, my understanding would be that the anti-inflammatory action of cortisone probably outweighs most other effects.