Morphometric and cytochemical analysis of lysosomes in rat Peyer's patch follicle epitheliumTheir reduction in volume fraction and acid phosphatase content in M cells compared to adjacent enterocytes.код для вставкиСкачать
THE ANATOMICAL RECORD 216:521-527 (1986) Morphometric and Cytochemical Analysis of Lysosomes in Rat Peyer’s Patch Follicle Epithelium: Their Reduction in Volume Fraction and Acid Phosphatase Content in M Cells Compared to Adjacent Enterocytes R.L. OWEN, R.T. APPLE, AND D.K. BHALLA Department of Medicine, University of California, (R.L. O., D.K.B.)and Veterans Administration Medical Center, (R.L.O., R. T A . , D.K.B.)San Francisco, California 94121 ABSTRACT M cells are specialized epithelial cells over lymphoid follicles in Peyer’s patches which take up viruses, bacteria, and antigenic macromolecules from the intestinal lumen. Unlike ordinary enterocytes which sequester pinocytosed material in lysosomes, M cells transport such material across the epithelium to antigen-processing areas in lymphoid follicle domes, suggesting a difference in lysosomal activity or a different route for movement of endocytic vesicles. Ileal Peyer’s patches in rats were examined by electron microscopy to identify lysosomes by acid phosphatase activity. Acid phosphatase was found in dense bodies in enterocytes but not in M cells. Stereological analysis showed the volume fraction occupied by dense bodies in M cells to be 16 times less than in enterocytes ( P < .0005), even though the volume fractions of cytoplasm occupied by mitochondria in M cells and enterocytes were not significantly different. The small volume fraction of dense bodies and the absence of acid phosphatase activity in M cells thus correlate with absence of lysosomal degradation of luminal microorganisms during transport into lymphoid follicles by M cells and may provide not only a complete array of microbial antigens for initiation of immune responses, but also a route through the mucosal barrier for microorganisms which can evade local containment mechanisms. Lymphoid follicles of Peyer’s patches consist of aggregates of lymphoid cells that are separated from the gut lumen by a layer of epithelial cells. This follicle-associated epithelium (F‘AE) transports material from the intestinal lumen into the lymphoid tissue. Specialized cells in the follicle epithelium t h a t are actively engaged in uptake and transport of particulate material have been referred to as M cells (Owen and Jones, 1974) or as FAE cells (Bockman and Cooper, 1973). M cells exhibit morphological specializations, which include reduced numbers of microvilli of variable size and shape, attenuated cytoplasmic extensions to adjacent cells, and invagination by and close association with lymphocytes, macrophages, and plasma cells. Structural specializations of M cells appear to be related to their role a s antigentransporting units. Uptake and transport from the intestinal lumen to the lymphoid tissues, primarily by M cells, has been demonstrated for horseradish peroxidase (Owen, 1977; von Rosen et al., 19811, India ink and ferritin (Bockman and Cooper, 19731, cholera toxin (Shakhlamov et al., 1981; Owen et al., 1986b), bacteria (Bockman and Boydston, 1982; Owen et al., 1986a), and reovirus (Wolf et al., 1981). During transport M cells largely, if not completely, fail to sequester and degrade luminal material as is characteristic for other epithelial cells. In this report we explore the mechanism by which 0 1986 ALAN R. LISS, INC. M cells carry out such transport. These findings are discussed in relation to antigen transport, initiation of immune reactions, and Peyer’s patches as a route across the intestinal barrier for infectious agents. MATERIALS AND METHODS General Preparation Male Sprague Dawley rats weighing 270-290 g were purchased from Charles River Breeding Laboratories (Portage, Michigan). Nonfasted animals were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal sodium, 50 mg/ml, Abbott Laboratories, North Chicago, IL), 0.1 ml per 100 g body weight. Peyer’spatches were fixed initially by injecting 2.7% glutaraldehyde and 0.8% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, into the intestinal lumen. Ileal segments containing Peyer’s patches were removed, pinned flat in petri dishes lined with paraffin, and fixed further by immersion in the same fixative for 4 hours. The tissues were rinsed thoroughly with 0.1 M phos, phate buffer, postfixed with 2% aqueous 0 ~ 0 4 dehyReceived April 14, 1986; accepted July 8, 1986. Address reprint requests to Robert L. Owen, M.D., Cell Biology and Aging (151E), V.A. Medical Center, 4150 Clement Street, San Francisco. CA 94121. 522 R.L. OWEN, R.T.APPLE, AND D.K. BHALLA Fig. 1. An M cell (M) characterized by short, variably shaped microvilli and thin cytoplasmic extensions encloses lymphocytes and is bound by adjacent enterocytes (E) over a rat Peyer’s patch follicle. Several dense bodies of varying shape and sizes (arrows)are seen in the apical cytoplasm of enterocytes but not in the M cell. x 10,850. a) LYSOSOMES IN PEYER’S PATCHES drated through graded alcohols and propylene oxide, and finally embedded in Epon 812. Sections were cut on a Porter-Blum MT-2 ultramicrotome and observed and photographed with a Philips 201 electron microscope (Philips Electronic Instruments, Inc., Mahwah, NJ). Acid Phosphatase Activity For cytochemical localization of acid phosphatase, Peyer’s patches were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 2 hours, rinsed several times with 0.1 M cacodylate buffer, and then sectioned a t a thickness of about 50 pm by using a tissue sectioner. The sections were rinsed with 0.1 M cacodylate buffer and incubated for varying time periods a t different temperatures in acid phosphatase medium containing either beta-glycerophosphate, disodium salt or cytidine monophosphoric acid, sodium salt (CMP) (Sigma Chemical Co., St. Louis, MO) as the substrate (Novikoff, 1963; Novikoff and Yam, 1978). Optimal results were obtained when CMP was used as the substrate and incubation was carried out for 1 hour a t room temperature. These incubation conditions were routinely used in the subsequent experiments. Controls were carried out by incubating sections in the medium lacking substrate. All reactions were stopped by adding 0.1 M cacodylate buffer containing 5% sucrose. The specimens were osmicated and prepared for electron microscopy as described above. Morphometric Procedures For morphometry, distal Peyer’s patches from five rats were prepared for electron microscopy as described above. Sections from 20 different Peyer’s patch follicles were observed and photographed a t a magnification of 7,000 x . Mature M cells were identified by short or irregular microvilli and underlying lymphocytes. Because of the relative infrequence of M cells, all identified mature M ceils were photographed for study. Over 100 micrographs of M cells, adjacent enterocytes, and 71 lymphocytes in close association with M cells were printed a t a final magnification of 18,000x . The proportion of cytoplasm (volume fraction) occupied by dense bodies within outlined M cells and adjacent enterocytes was measured by superimposing a coherent double-lattice test grid with a 16:l correspondence of small squares (0.5 cm on each side) to large squares (2.0 cm on each side) on the micrographs and counting the grid intersections lying over cytoplasm and over dense bodies (Williams, 1977). To check for the possible introduction of bias by the necessarily nonrandom selection of M cells, we used the same method to determine the volume fraction occupied by a control organelle, the mitochondrion. Volume fractions (Vf)were calculated by using the formula: Vf = Points over specific organelle Points over cell cytoplasm Relative volumes of nucleus and cytoplasm of M cellassociated lymphoid cells were also determined by the same method. Data was analyzed by using a Monroe 1860 programmable calculator (Litton-Monroe, Orange, NJ), incorporating Student’s T test for statistical significance. RESULTS The fine structure of M cells corresponded to that previously reported (Owen, 1974; Owen and Bhalla, 523 1983), revealing variably shaped and spaced microvilli, absent or disorganized terminal web, moderate numbers of ribosomes, endoplasmic reticula, and mitochondria. Although specific morphologic features of M cells allow their distinction from surrounding ordinary enterocytes, the two cell types shared several common features. Both were oriented along the basal lamina and formed tight junctions at their apical margins. Most of the cytoplasmic organelles in M cells were identical in size, shape, distribution, and density to those in the enterocytes, but dense bodies of variable form and size which were observed in the apical cytoplasm of enterocytes were rare in M cells. In enterocytes dense bodies were usually uniform but granules with light interior and dense periphery or vice versa were also observed (Fig. 1).Incubation of tissue slices in the acid phosphatase medium containing CMP as the substrate resulted in the deposition of variable amounts of black precipitation over dense bodies in Peyer’s patch follicle enterocytes but not in M cells (Fig. 2). We found, as previously observed (Nuvikoff and Yam, 19781, that CMP used as a substrate produced relatively fewer diffusion artifacts and more specific reaction product deposits then did beta-glycerophosphate. In most observed M cells no dense bodies were present. When any structures consistent with dense bodies were observed in mature fully developed M cells, no acid phosphatase reaction product was seen despite its presence in structurally similar dense bodies in surrounding enterocytes. Following cytochemical confirmation of dense bodies as the principle Peyer’s patch follicle epithelial organelles rich in the major lysosomal enzyme, acid phosphatase, the distribution of dense bodies was analyzed by using stereological techniques. The fractions of cytoplasm occupied by mitochondria and dense bodies were expressed as mean volume fractions (Table 1).The volume fraction containing mitochondria was not significantly different in M cells from that in adjacent enterocytes. The volume fraction containing dense bodies was, however, at least 16 times less in M cells than in enterocytes and this difference was statistically significant. Rare multivesicular bodies were seen in M cells. All darkly stained membrane-bound organelles in a n M cell were counted as dense bodies; some may have been vesicles filled with darkly staining luminal material or glancing sections through mitochondria membranes so that this decision would, if anything, overestimate M cell lysosomes. M cells were generally irregular in outline and unlike adjacent enterocytes, their cytoplasm almost always surrounded one or more lymphoid cells, which occupied a position intermediate between the apical membranelike bands of cytoplasm and the basal nucleus of the M cell. The lymphoid cell exhibited round, elongate, or irregular forms of both nucleus and cytoplasm (Fig. 1).The majority of them measured between 6 and 10 pm in diameter. Their cytoplasm contained abundant ribosomes and moderate amounts of endoplasmic reticulum. Occasionally, these cells displayed highly developed endoplasmic reticulum suggestive of developing plasma cells. Vesicles of variable sizes and multivesicular bodies were frequent. Dense bodies or well-developed Golgi elements were only occasionally seen. The nuclei often had large euchromatic areas and prominent nucleoli. Small lymphocytes with thin rims of cytoplasm were generally absent. Although cytoplasmic volume varied from cell 524 R.L. OWEN, R.T. APPLE, AND D.K. BHALLA Fig. 2. Peyer’s patch follicles processed for acid phosphatase activity reveal granular reaction product deposits over dense bodies (arrows) in the enterocytes (E). An M cell (MI lacking dense bodies is devoid of acid phosphatase activity. Apical vesicles in the M cell are free of granular deposits seen over the dense bodies. ~9.700. 525 LYSOSOMES IN PEYER'S PATCHES TABLE 1. Grid intersections and volume fractions of cytoplasm occupied by organelles' Dense bodies Point intersections Volume (small squares) fraction' M cells (107 cells) Columnar cells (102 cells) 30 814 0.0008 f 0.0002 SD = 0.0022 0.0156 f 0.0015 SD = 0.0149 (P < ,0005) Mitochondria Point intersections Volume (large squares) fraction 265 451 0.126 f 0.008 SD = ,081 0.138 f 0.010 SD = 0.100 (P< .2) Cytoplasm point intersection (large squares) 2,090 3,478 'Dense bodies occupy a significantly smaller fraction of the cytoplasm in M cells than in adjacent enterocytes. Mitochondria occupy almost the same fraction of cytoplasm in both cell types, indicating that the observed paucity of dense bodies does not represent displacement of cell organelles from the M cell apex. 'Points over specific organelle mm3 Organelle - Volume fraction. Points over total cytoplasm mm3 Cytoplasm to cell, in many of the M cell-associated lymphocytes the that lysosomal modification in M cells is a specific inhercytoplasm occupied more than half of the cell volume. ent functional specialization. Morphometric analysis of 71 lymphoid cells revealed a From this study we have no information about the nuclearkytoplasmic ratio of 1.17 f 0.11. transcellular route or mechanism of vesicular transport in M cells. Pathways which do not involve lysosomes DISCUSSION have been described in neonatal enterocytes (AbrahamIn lymph nodes afferent lymphatics bring antigens to son and Rodewald, 1981). Association with membrane lymphoid follicles but in Peyer's patches, M cells trans- receptors during endocytosis has been postulated as a port particles and macromolecules from the intestinal mechanism for intracellular targeting by which endocylumen into Peyer's patch lymphoid follicles, which lack tic vesicles pass to the lateral or basal border of the cell afferent lymphatics. Uptake of intact reovirus (Wolf et instead of to lysosomes (Anderson and Kaplan, 1983). al., 1981) and of whole cholera vibrios (Owen et al., Vesicles formed by fluid phase and by receptor-mediated 1986a) from the intestinal lumen by M cells and trans- endocytosis may be sorted and directed to their eventual port to the spaces between M cells and lymphoid cells targets by a specialized portion of the Golgi (transretisuggest differences in the function or structure of lyso- cular [TR] Golgi, Golgi-endoplasmic reticulum-lysosoma1 systems of M cells and of enterocytes. Inefficiency some complex [GERL], Compartment for uncoupling of in sequestering and degrading macromolecules could receptor and ligand [CURL]),but the exact location and result from absence of lysosomes, lysosomes with dimin- means by which this occurs remain a subject of active ished response, normally responsive lysosomes in re- investigation (Pasten and Willingham, 1985). Studies with radioactively labeled DNA precursors duced numbers, or a n unusual transcellular route of endocytic vesicles which do not fuse with lysosomes. Our indicate that M cells are derived from surrounding enultrastructural and cytochemical studies show that M terocytes (Bhalla and Owen, 1982) in what may be a cells contain relatively few structures consistent in ap- precommitted pool over follicle surfaces (Bye et al., 1984). pearance with dense bodies, and even these are deficient Embryologically, M cell formation does not occur until in the major lysosomal enzyme, acid phosphatase. Al- lymphocytes begin to infiltrate lymphoid follicle epithethough dense bodies appear to be present in M cells, lium (Bockman and Cooper, 1975). In adult animals, M analysis by stereological techniques revealed that there cells have usually been found in association with intrais a significantly smaller volume fraction occupied by epithelial lymphocytes, suggesting that contact with lymphocyte membranes or cell products induces transdense bodies in M cells than in adjacent enterocytes. Our observations quantitate the presence of modified formation of enterocytes into M cells (Smith and Pealysosomal development in M cells and partially explain cock, 1980). Bye et al. (1984) report that immature M observations that M cells transport but do not degrade cells can be recognized by membrane particle distribumacromolecules as do enterocytes in which engulfed tion and cholesterol content prior to development of a n macromolecules are sequestered in lysosomes (Blok et invagination filled by lymphoid cells. If, as current evial., 1981).Deformation by lymphoid cells of M cell cyto- dence indicates, M cells develop from surrounding implasm into a thin apical rim raised the possibility that mature M cells there must be a point when lysosomes most M cell cytoplasmic organelles including dense bod- are still present. Because we defined M cells by presence ies are mechanically relocated. Our control morphomet- of a lymphocyte-filled cavity, such transitional immaric analysis of mitochondria rules out physical dis- ture M cells would have been excluded from our analysis. Lymphocyte transformation is accompanied by recogplacement during deformation of M cells as the cause of reduction in the cytoplasmic volume occupied by dense nizable morphological changes (Douglas, 1973; Janossy bodies. These studies show that even though dense bod- et al., 1973; Biberfeld and Mellstedt, 1974; Marsh, 1975). ies are a t least 16 times less prevalent in M cells than Lymphoid cells associated with M cells have characterin enterocytes, mitochondria, which are of approxi- istics of medium-sized lymphoblasts, including inmately the same size as dense bodies, occupy the same creased volume of cytoplasm, abundance of ribosomes, proportion of the cytoplasm in the two cell types. The and presence of multivesicular bodies and endoplasmic lack of correlation between volume fraction of lysosomes reticulum. Fine structure analysis, however, does not and mitochondria in Peyer's patch epithelium implies allow their classification into T or B cell types. In studies 526 R.L. OWEN, R.T. APPLE, AND D.K. BHALLA I 4 E c Fig. 3. A diagrammatic model of uptake and transport by M cells (MI over Peyer's patch follicles. A considerably smaller volume of lysosomes is present in M cells than in adjacent epithelial cells (E). Most of the particulate antigen (*) taken up by M cells from the intestinal lumen therefore escapes lysosomal degradation and is released in the intercellular spaces, where it stimulates lymphocytes (L). Antigen taken up by enterocytes is largely sequestered and digested in lysosomes (arrowhead). of separated spleen B and T lymphocytes, we found that both cell types migrate to follicle epithelium and lodge under M cells (Bhalla and Owen, 1983). Small lymphocytes with a thin rim of cytoplasm and scanty organelles were rare. Presence of activated lymphoblasts in the meshes of M cell cytoplasm is consistent with the hypothesis that M cells take up particulate antigen from the intestinal lumen and transport it across their cytoplasm to the intercellular spaces (Fig. 31, stimulating lymphocytes prior to their migration and differentiation for immune reaction a t other locations. Intestinal luminal particles or microorganisms are pinocytosed or phagocytosed by M cells, and the resulting vesicles carry them through the epithelial barrier, releasing them into the intercellular space. If fusion of lysosomes with vesicles ever occurs in M cells, we do not know whether sequestration and destruction of vesicle contents follows as in other cells. If so, it must be relatively infrequent due to the paucity of' M cell lysosomes and their acid phosphatase deficiency. Structurally, the breach in the intestinal barrier produced by M cells over lymphoid follicles is usually com- pensated for by macrophages and other phagocytes clustered beneath the follicle epithelium (Owen et al., 1981). M cells and macrophages have structural similarities but also marked and complementary differences in function. Both can phagocytose particles and microorganisms of widely varied sizes. M cells are epithelial cells, fixed to adjacent enterocytes by apical tight junctions, desmosomes, and interdigitation of lateral membranes, but macrophages are free to migrate in and out of the intestinal epithelium and have been observed phagocytosing microorganisms within the epithelium beneath M cells (Owen et al., 1981, 1986a; Bockman and Boydston, 1982). In macrophages the process of endocytosis stimulates lysosome formation (Cohn, 1963),which we have not observed in M cells even when great numbers of transport vesicles are present. Horseradish peroxidase, a glycoprotein of 40,000 molecular weight, is carried directly from the intestinal lumen through M cells to lymphocytes within the follicle epithelium (Owen, 19171, but microorganisms and larger particles are taken up by macrophages. These macrophages, which degrade complex microorganisms and particles to LYSOSOMES IN PEYERS PATCHES antigenic constituents, are as well supplied with lysosomes as M cells are deficient. The complementary lysosomal systems of M cells and macrophages thus allow for lymphocyte stimulation in the follicle by antigens derived from a wide size range of luminal macromolecules, particles, and microorganisms transported by lysosome-deficient M cells but for containment within follicle domes by lysosome-rich macrophages. Macrophages cannot confine all microorganisms carried from the lumen by M cells. Scarring and narrowing of the terminal ileum occurs in intestinal tuberculosis when Mycobacterium tuberculosis localizes in Peyer’s patches, and intestinal perforation may take place when Salmonella typhi enters Peyer’s patches in typhoid fever. Reoviruses have been shown to distribute widely beyond the intestine after M cell transport from the lumen (Wolf et al., 1981). Dissemination of infective agents can be anticipated whenever the breach in the intestinal epithelial barrier produced by lysosome-poor M cells cannot be compensated for by macrophages, especially when genetic or nutritional lysosome deficiency exists (Popov et al., 1979; Quie and Mills, 1979). 527 Cohn, Z.A. (1963) The fate of bacteria within phagocytic cells. 11. The modification of intracellular degradation. J.Exp. Med., 117:43-53. Douglas, S.D. (1973)Human lymphocyte growth in uitro: Morphologic, biochemical, and immunologic significance. Int. Rev. Exp. Pathol., 10:41-114. Janossy, G . ,M.F. Greaves, M.J. Doenhoff, J. Snajdr (1973)Lymphocyte activation. V. Quantitation of the proliferative responses to mitogens using defined T and B cell populations. Clin. Exp. Immunol., I4r581-595. Marsh, M.N. (1975) Studies of intestinal lymphoid tissue. I. EIectron microscopic evidence of ‘blast transformation’ in epithelial lymphocytes of mouse small intestinal mucosa. Gut, 26:665-682. Novikoff, A.B. (1963) Lysosomes in the physiology and pathology of cells: Contributions of staining methods. In: Ciba Foundation Symposium on Lysosomes. A.V.S. Reuck and M.P. Cameron, eds. J. and A. Churchill Ltd., London, pp. 36-77. Novikoff, P.M., and A. Yam (1978) Sites of lipoprotein particles in normal rat hepatocytes. J. Cell. Biol., 76:l-11. Owen, R.L., and A.L. Jones (1974) Epithelial cell specialization within human Peyer’s patches: An ultrastructural study of intestinal lymphoid follicles. Gastroenterology, 66:189-203. Owen, R.L. (1977) Sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of Peyer’s patches in the normal unobstructured mouse intestine: An ultrastructural study. Gastroenterology, 72t440-451. Owen, R.L., C.L. Allen, and D.P. Stevens (1981) Phagocytosis of Giardia muris by macrophages in Peyer’s patch epithelium in mice. Infect. Immun., 33:591-601. ACKNOWLEDGMENTS Owen, R.L., and D.K. Bhalla (1983) Lympho-epithelial organs and lymph nodes. In: Biomedical Research Applications of Scanning This study was supported by the Veterans AdministraElectron Microscopy. G.M. Hodges and K.E. Carr, eds. Academic tion and by NIH grant AM 33004. Press, London, Vol. 3, pp. 79-169. Owen, R.L., N.F. Pierce, R.T. Apple, and W.C. Cray, Jr. (1986a) M cell LITERATURE CITED transport of Vibrio cholerae from the intestinal lumen into Peyer’s patches: A mechanism for antigen sampling and for microbial Abrahamson, D.R., and R. Rodewald (1981)Evidence for the sorting of transepithelial migration. J. Infect. Dis. 253: 1108-1118. endocytic vesicle contents during the receptor-mediated transport, Owen, R.L., F.T. Koster, and T.H. Ermak (198613) Autoradiographic of IgG across the newborn rat intestine. J. Cell Biol., 92r270-280. localization of uptake of cholera toxin in Peyer’s patches in nonAnderson, R.G.W., and J. Kaplan (1983) Receptor-mediated endocytoimmune rats. Clin. Res., 34t101A (abstract). sis. Mod. Cell Biol., 1:l-52. Pastan, I., and M.C. Willingham (1985) The pathway of endocytosis. Bhalla, D.K., and R.L. Owen (1982) Cell renewal and migration in In: Endocytosis. I. Pastan and M.C. Willingham, eds. Plenum Press, lymphoid follicles of Peyer’s patches and cecum-An autoradioNew York, pp. 1-44. graphic study in mice. Gastroenterology, 82:232-242. Popov, A.A., I.T. Apostolov, and L.P. Krustev (1979) Lysosome activity Bhalla, D.K., and R.L. 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