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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.

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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,
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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.
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