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Light and electron microscopy of intestinal ferritin absorption. Observations in sensitized and nonsensitized hamsters (Mesocricetus auratus)

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Light and Electron Microscopy of Intestinal Ferritin
Observations in Sensitized and Nonsensitized Hamsters (Mesocricefus aurafus) ’
Department of A n a t o m y , U n i v w s i t y of T e n n e s s e e Medical U n i t s ,
Memphis, Tennessee
The intestinal absorptive cells of adult hamsters which have been
pretreated with intraperitoneal injections of horse ferritin or subcutaneous injections
of ferritin-adjuvant mixture show a marked absorption of this protein when it is
introduced into ligated segments of the intestinal lumen for periods up to three hours.
Ferritin is concentrated in the enteric surface coating, intracytoplasniic vesicles
in the supranuclcEr area. and the Golgi apparatus. Large aggregates are localized in
macrophages, fibrocytes, and endothelial cells of the lamina propria. Individual
molecules are also present in these cells, plasma cells, intercellular spaces and capillary
Immunological methods haev been used
in the study of intestinal absorption of
proteins since Uhlenhuth’s original contribution in 1900. Most of these studies
have utilized the precipitin test, anaphylactic reaction, and Prausnitz-Kustner
method as tools for the detection of foreign
proteins in blood or lymph. Alexander,
Shirley, and Allen (’36), and Wilson (’62)
have reviewed many such experiments.
The present study is one in which the effect of sensitization on the absorptive
process is investigated at the fine structural level.
An indication that the state of sensitization may determine the degree of reaction
to intestinally absorbed protein is evident
in the observations of IIettwer and Kriz
(’25, ’26). These investigators sensitized
guinea pigs with intraperitoneal injections
of 0.01 ml horse serum. When 1.3 ml of
the same protein was injected into the
unligated intestine 21 days later, no reaction occurred. The same amount injected into loops of intestine 10 cm or
shorter, however, resulted in anaphylactic
symptoms in the experimental animals.
If 84 days were allowed to elapse between
the sensitizing dose and the shocking injection, as little as 0.4 ml horse serum in
the unligated intestine caused anaphylaxis. Hettwer and Kriz attributed this
difference to an increased sensitization,
with a smaller amount of protein being reANAT. REC.,155: 603-622.
quired to produce anaphylactic symptoms.
A n enhanced absorption from the intestinal lumen would also explain the altered
minimal dosage.
Enteral absorption of intact proteins
might logically lead to further immune
phenomena, including the production of
specific precipitins, alteration in the reaction to the same protein, and hypersensitive reactions. DuBois et al. (’11) reported
that the ingestion of antigenic protein by
normal or marasmic infants led to the appearance of specific precipitins in the
blood, and in many cases to cutaneous hypersensitiveness, while Wells and Osborne
(’11) showed that continuous feeding with
a vegetable protein rendered guinea pigs
“immune” to it, so they could not be sensitized to that specific substance. Hettwer
and Kriz (‘25) were able to sensitize guinea
pigs to horse serum by direct injection of
the serum into a temporarily clamped loop
of small intestine. Normal infants developed precipitins for cow’s milk, egg albumin, sheep serum, or almond 8 to 25 days
after ingestion of these varied proteins for
the first time (Anderson et al., ’25).
Ferritin has been used to study various
immunological phenomena, including the
secondary response (Wellensiek and Coons,
’64), the Arthus reaction (Sabesin et al.,
’61), and the precipitin reaction (Easty
1 Supported by N.S.F. grant GB4159 and U.S.P.H.S.
grant FR-05423.
and Mercer, '58). It was chosen, therefore,
as the material best suited for direct visualization of any alteration in the response
of the intestine to protein after the host
had been sensitized to that protein. The
first observations using that method of approach constitute the subject of this report.
Adult golden hamsters of both sexes
were obtained from a commercial supplier.
The 43 animals used were divided into
four groups : ( 1) non-sensitized animals
without intraluminal ferritin; ( 2 ) nonsensitized animals with intraluminal ferritin; ( 3 ) sensitized animals without intraluminal ferritin, and ( 4 ) sensitized
animals with intraluminal ferritin.
Cadmium-free horse ferritin ' (100 mg/
m l ) was utilized for introduction into the
intestinal lumen (animals with intralum i n d ferritin). Approximately 0.2 ml ferritin was introduced, under chloral hydrate anesthesia, into a ligated segment
of jejunum or ileum about 2 cm long.
In each case, the amount of ferritin introduced into the intestinal lumen was
enough to fill it without causing undue
distention. Care was taken to preserve the
vascular supply to this segment. After 1
to 3 hours the animal was sacrificed and
the intestinal segment containing ferritin
was cut into a rectangle and immersed in
in 1-2% osmium tetroxide in Millonig's
buffer ('62) for 1 to 2 hours. Fixation was
followed by a one hour rinse of 3% formalin in the same buffer. Small pieces of
a suitable size for embedding were then
dehydrated through graded alcohols into
two changes of propylene oxide, and embedded in Dow epoxy resin 334 (Winborn, '65). Sections were cut on a PorterBlum microtome with glass knives, and
viewed either unstained or stained with
lead citrate (Reynolds, '63; Venable and
Coggeshall, '65). Some were doubly stained
with lead citrate and uranyl acetate (Watson, '58). Kodak contrast plates and duPont Cronar film were used in a n RCA
EMU 3F or Hitachi HU-11A electron microscope.
For sensitization of two groups (sensitized animals), cadmium-free horse ferritin was administered at weekly intervals by
one of two routes: intraperitoneal injec-
tions of 0.5 ml undiluted ferritin, or subcutaneous injections of 0.25 ml ferritin
emulsified with 0.25 ml complete Freunds
adjuvant (Difco Laboratories, Detroit,
Michigan). After 1 to 4 sensitizing doses,
a week was allowed to elapse before intraluminal administration of ferritin.
Portions of the intestinal segments from
all treatment groups were fixed in Bouin's
fluid, embedded in paraffin, and submitted
to the Prussian blue reaction (Gomori,
'36) for localization of relatively large deposits of iron with the light microscope.
Serum samples were collected from 15
animals by aspiration of heart blood at
sacrifice. Serum and ferritin were placed
in wells cut in 2% agar in phosphate-huffered saline which had been placed on
microscope slides. The appearance of precipitin lines was taken as evidence of humoral antibody.
Circulating antibodies are detected in
the serums of animals sensitized by both
routes. Precipitin lines form against undiluted ferritin (100 m g i m l ) after two or
more subcutaneous injections of ferritinadjuvant mixture and after four intraperitoneal injections (figs. 1, 2). Faint precipitin lines occur between horse ferritin
diluted to 10 mg/ml and serums from
animals which had received two intraperitoneal sensitizing doses. Therefore, all animals which had received a t least two
doses by either route were considered sensitized systemically. Serums from five nonsensitized hamsters exhibited no precipitin
Prussian blue reaction
Sections of intestine from non-sensitized
animals without intraluminal ferritin are
negative for the Prussian blue reaction
(fig. 3 ) . Non-sensitized animals with intraluminal ferritin show heavy reaction
products in the lumen (fig. 4 ) but not in
columnar absorptive cells or lamina propria. Sensitized animals without intraluminal ferritin show a n abundance of blue
Pentex, Inc., Kankakee, Illinois. Ferritin was pre-
cipitated without cadmium to eliminate the possibility
of toxicity.
Dow epoxy resin 334 was kindly supplied by
Mr. Dan Moffett, Vaughn. Inc., Memphis, Tennessee.
Figures 1 and 2
Photographs of microslide diffusion plates.
Fig. 1 Ferritin solution was placed in the middle well (F). Serums placed i n the peripheral wells
were from animals receiving the following treatments: a and b were not sensitized; c received three
intraperitoneal injections; d received three subcutaneous injections.
Fig. 2 Ferritin solution was placed in the middle well (F). Wells a and c contained serum from
a n animal given one subcutaneous injection of ferritin. Wells b and d contained serum from an animal given four intraperitoneal injections.
reaction product in the lamina propria
(fig. 5 ) , but the epithelium and lumen
are unreactive.
Sensitized animals with intraluminal
ferritin show the largest amounts of iron
in the intestinal mucosa. It is most apparent in the lumen and in macrophages
of the lamina propria (figs. 6-8). Lesser
amounts are seen in the endothelium of
sub-epithelial capillaries and in extracellular locations. The amount of Prussian
blue-reactive material increases with successive sensitizing doses, occurring first
and in greater concentration around the
crypts (fig. 6). Deposits in the villus core
typically are more sparse (figs. 7, 8). A
slight Prussian blue reaction is detectable
in some of the epithelial cells of the crypts.
This diffuse bluing is not demonstrable in
black and white prints (fig. 6). More discrete
deposits of iron are made evident by use of
a red filter (compare fig. 7 with fig. 8).
Electron microscopy
The columnar absorptive cells of the
non-sensitized animals receiving no intraluminal ferritin possess a striated border
(fig. 9) which consists of numerous microvilli usually layered with an enteric sur-
face coat. Below this area a prominent
terminal web is evident. The Golgi complex (fig. 9) appears above an irregularly
outlined nucleus. The lateral apposition
of adjacent cell membranes is marked by
junctional complexes in the apical region,
interlocking digitations and dilated intercellular spaces (fig. 9 ) in the middle portion and simple cell membrane appositions
in the basal areas. Both smooth and rough
forms of the endoplasmic reticulum and
abundant mitochondria appear throughout
the cytoplasm. Ribosomes and cytoplasmic fibrils add to the density of the cytoplasmic matrix. The epithelial cells rest
on a basement membrane, hereafter referred to as boundary membrane (Low,
'62) and the latter forms the outer limits
of the core of the villus (fig. 9). Filling
the core of the villus are lacteals, blood
capillaries, fine connective tissue elements,
smooth muscle cells and a plethora of leucocytes. When compared with the following groups, these animals are notably lacking in any significant amount of ferritin
in the lumen or in the intestinal mucosa.
Following intraluminal injection of
horse ferritin in non-sensitized animals,
electron microscopic studies show particles
Figures 3-8 Light micrographs of intestinal sections submitted to the Prussian blue reaction with
Safranin 0 counterstain.
Fig. 3 Non-sensitized animal without intraluminal ferritin. No reaction product is present in the
intestinal lumen ( L ) , epithelium, or lamina propria ( P ) . x 525.
Fig. 4 Non-sensitized animal with intraluminal ferritin. Prussian blue reaction product (arrow)
is evident in the lumen. No reaction product appears i n the epithelium or lamina propria (P). x 525.
Fig. 5 Sensitized animal without intraluminal ferritin. No reaction product is present in the intestinal lumen ( L ) or epithelium. Blue reaction product (arrows) appears only i n the lamina propria.
X 525.
Fig. 6 Sensitized animal with intraluminal ferritin. Prussian blue reaction product (arrow) appears i n the intestinal lumen ( L ) . Although not evident i n this black and white print, there is a diffuse bluing of the cytoplasm of the epithelial cells of the crypt. Large concentrations of iron (arrows)
are evident in macrophages surrounding the crypt epithelium. x 475.
Fig. 7 Sensitized animal with intraluminal ferritin. Reaction product appears in lumen (L) and
in core of villus (arrows). X 760.
Fig. 8 Same section as in figure 7, photographed with red filter to emphasize location of blue reaction product (arrows). X 760.
of fcrritin within and without the intestinal villus (figs. 10-12). Outside the
villus the enteric surface coat is the most
conspicuous area where the various forms
of the 50-60 A iron cores of the ferritin
molecules are seen (figs. 10, 11). Ferritin
appears most heavily concentrated where
the surface coat is prominent. Ferritin is
seen in the intermicrovillus spaces and
within invaginations at the bases o i the
microvilli. It is contained in small vesicles and multivesicular bodies in the area
of the terminal web (figs. 10, 11). Occasional ferritin particles also are apparent
within the cytoplasmic matrix without a
delimiting membrane. Scattered membrane bound ferritin particles are present
in the region immediately below the terminal web and above the Golgi complex,
but no identifiable ferritin is present in
the lamellar or vesicular portions of the
Golgi system. However, accumulations of
electron-dcnse material are present in this
system; the specific identity of this substance is obscure. Ferritin has not been
observed in the perinuclear or basal regions of the columnar cells in this study.
However, it is readily visualized throughout the interstitial core of the villus beneath the columnar cells (fig. 12). It is
present in the narrow space intervening
between the boundary membrane and the
cell membrane of the basal surface of the
columnar cells and between the former
boundary membrane and the one surrounding the endothelium of the blood capillaries (fig. 12). Similarly, particles are
definable in the space between the latter
boundary membrane and the outer endothelial cell membrane and within the lumen of this capillary (fig. 12).
Section from animals which received
sensitizing doses but no intraluminal injection (figs. 13, 14) exhibit aggregations
and individual molecules of ferritin in the
lamina propria. The pattern of distribution within the lamina propria is similar
to that described below for sensitized animals with intraluminal ferritin. However,
the intestinal lumen and the epithelial
cells (fig. 13) are notably devoid of ferritin.
Sensitized animals receiving intraluminal ferritin display the highest concentrations of ferritin within the intestinal mu-
cosa (figs. 15-22). Ferritin molecules are
concentrated above and between microvilli in the enteric surface coating (figs.
15, 1 7 ) . Ferritin of similar concentration is localized in invaginations of the
surface membrane and in more electron
dense vesicles beneath the surface (figs.
15, 1 7 ) . Deeper in the apical region,
larger, more complex bodies are observed;
the density of these vesicles is irregular,
and some resemble multivesicular bodies
(fig. 17). Large accumulations of extremely electron dense material appear in
the Golgi area (figs. 16, 18). Individual
particles comparable in size and density
to ferritin molecules are observed between epithelial cells, but are difficult to
distingiush from similar particles observed
in non-treated animals, even in unstained
Ferritin occurs as circumscribed dense
aggregates and as dispersed individual
particles in macrophages within the lamina
propria (figs. 19, 20). A membrane usually is detectable around the aggregates.
Frequently, as described by Miller and
Palade ('64), a clear rim separates the
bounding membrane from the closely
packed ferritin molecules (fig. 19). Similar aggregates and individual particles
of ferritin are observed in fibrocytes of the
lamina propria. Only individual molecules are localized within plasma cells.
Individual particles usually are scattered
at random in the intercellular spaces. As
shown in figure 21, however, an area of
moderate electron density occasionally js
present around groups of ferritin molecules.
Aggregates and individual molecules are
observed within endothelial cells underlying the epithelium (fig. 22). Individual
molecules also are present in capillary lumens. There is no evidence of specializations in the cell membrane of the endothelium for passage of these molecules,
since ferritin is no more concentrated in
areas of vesicles than elsewhere.
The intestinal mucosa of the golden
hamster appears similar to that of other
species (Dalton, '51: Palay and Karlin,
'%a; Hampton and Quastler, '61a, b;
Deane, '64). Since the detailed account
of the intestinal villus by Palay and Karlin
('59a, b ) more information concerning the
apposition of adjacent cell membranes has
been brought into focus by Farquhar and
Palade ('63) and Brightman and Palay
('63). In the light of these studies, it
seems apparent that the apical epithelial
cell apposition not only presents an adhesive appearance, but offers a "barrier"
to direct intercellular aborption. The observations in the present report agree with
these findings and indicate that absorption
of ferritin occurs by invagination of the
cell surface or pinocytosis (Graney, '64).
No evidence is available to show passage
of ferritin through the zonulae occludcntes
of the junctional complexes to the intercellular spaces. This is in keeping with
.other tracer studies (Miller, '60; Farquhar
and Palade, '63) dealing with experimental
hemog Io binuria.
Since circulating antibodies to horse
ferritin were produced by the hamsters in
these experiments, the ferritin must be
considered a foreign protein. Foreign protein in the amounts observed in the intestinal epithelium after intraluminal injection has not been demonstrated before
in adult animals. Clark ('59) described
the absorption of considerable amounts of
proteins and colloidal materials by the
jejunum and ileum in rats and mice up to
18 days after birth, but not beyond.
Graney ('64) detected pinocytotic uptake
of ferritin by cells in the terminal ileum of
15-day-old suckling rats. The present study
indicates that ferritin is absorbed in ligated intestinal segments of non-sensitized
adult hamsters but in smaller amounts
than those observed after previous sensitization.
The mechanism for the increased absorption is difficult to interpret. The experimental animals are certaiiily not iron
deficient, at least in the usual sense. Ferritin from different species is immunologically cross-reactive (Fine and Harris,
'63; Richter, '64). Although it seems unlikely, such a cross reaction could conceivably alter the effective level of iron and
thus alter absorption.
Alternatively, the presence of a substance, such as an immune globulin, in
the intestinal lumen of a sensitized hamster might alter the barrier to the pene-
tration of a foreign protein. The amount
of precipitating antibody in the serums of
these hamsters was not correlated with
the amount of ferritin absorbed. Similar
results were obtained after intraperitoneal
injection, which resulted in small quantities of such antibody, and with subcutaneous injections of ferritin-adjuvant mixture, which yielded much higher levels.
Ilowever, Crabbe et al. ('65) have correlated the high proportion of yA-immunoglobulin-containing plasma cells in human
intestinal mucosa with the presence of this
immunoglobulin in intestinal secretions.
One might speculate that an immune
globulin of this type could operate a t a
local level in the digestive tract, thus
leading to a1tered absorption.
No ferritin is present in the intestinal
lumen or epithelial cells of hamsters which
are sensitized but given no intraluminal
injection. This would argue against interpreting the intraepithelial and intralumirial ferritin as being a state of excretion of
iron contained in the lamina propria.
The time intervals used in this study
would seem too short to permit breakdown of horse ferritin and resynthesis of
the iron moiety into hamster ferritin.
Richter ('59) followed the transformation
of colloidal iron into ferritin in mice. Only
small numbers of ferritin molecules were
identifiable four hours after intraperitoneal
injection, whereas by the sixth day ferritin
was abundant in close proximity to deposits of the injected iron compounds.
The system used in this investigation
allows the "outside" of the animal, a n absorptive layer, and the necessary elements
of the immune reaction to be viewed in
one electron microscopic field during absorption and reaction to a foreign substance. It would seem, then, a valuable
system for investigating the interplay between intestinal contents and these elements (Abrams et al., '63).
We gratefully acknowledge the excellent
technical assistance of Mrs. Sue Little.
Abrams, G. D., H. Bauer and H. Sprinz 1963
Influence of the normal flora on mucosal morphology and cellular renewal in the ileum; a
comparison of germ-free and conventional
mice. Lab. Invest., 12: 355-364.
Alexander, H. L., K. Shirley and D. Allen 1936
The route of ingested egg white to the systemic
circulation. J. Clin. Invest., 15: 163-167.
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1925 The intestinal absorption of antigenic
protein by normal infants. Proc. SOC. Exp.
Biol. Med., 23: 180-182.
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fine structure of ependyma in the brain of
the rat. J. Cell Biol., 29: 415439.
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and colloidal materials by columnar absorptive
cells of the small intestine in suckling rats
and mice. J. Biophys. Biochem. Cytol.. 5: 4150.
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mucosa as a major source of plasma cells
containing vA-immunoalobulin.
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Dalton, A. J. 1951 Electron micrography of
epithelial cells of the gastrointestinal tract and
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Deane, H. W. 1964 Some electron microscopic
observations on the lamina propria of the gut,
with comments on the close association of
macrophages, plasma cells and eosinophils.
Anat. Rec., 149: 453-474.
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1925 The development of cutaneous hypersensitiveness following the intestinal absorption of antigenic protein. Proc. SOC.Exp. Biol.
Med., 23: 176-180.
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microscopic studies of the antigen-antibody
complex. Immunology, 1: 353364.
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Biol., 17: 375-412.
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and immunological studics of horse and human
ferritin. Clin. Chim. Acta, 8: 794-798.
Gomori, G. 1936 Microtechnical demonstration
of iron. Am. J. Path., 12. 655-663.
Graney, D. 1954 The uptake of ferritin by
pinocytosis i n intestinal lining cells of suckling
rats. Anat. Rec., 148: 286 (Abstract).
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electron microscopic study of the reaction of
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and casein administered by gavage. Anat.
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1961b Combined autoradiography and
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H3-thymidine. J. Biophys. Biochem. Cytol., 20:
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tract as determined by the direct anaphylaxis
test. Am. J. Physiol., 73: 539-546.
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mousc kidney. J. Biophysic. and Biochem.
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1959b An electron microscope study of
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at high pH as a n electron-opaque stain i n
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1964 Electrophoretic and serological
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Anaphylaxis. J. Infect. Dis., 8: 66-124.
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B. Saunders, Philadelphia, pp. 212-219.
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triallyl cyanurate, and similarly modified
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All electron micrographs are of tissue fixed in 1-2% osmium tetroxide
and embedded in Dow epoxy resin 334. Unless otherwise indicated, the
sections were stained with lead citrate.
9 The columnar epithelial cells ( C ) occupy the major portion of the
field. These cells display a striate border ( S ) . terminal web ( T ) ,
Golgi apparatus ( G A ) , dense bodies (arrows) and interlocking
digitations (D). The nucleus ( N ) of one cell shows three prominent
nucleoli. Dilated intercellular spaces (I) appear between two columnar cells, and a foot process ( P ) is seen in the basal region between two other columnar cells. Boundary membranes (BM) closely
appose the cell membranes of the columnar cells and endothelial
cells of a capillary (CA). A lymphocyte ( L ) occupies the intraepithelial position. A process of fibrocyte (FB) appears between the
two boundary membranes. X 5,600.
Dale E. Bockman and William B. Winborn
Non-sensitized animals with intraluminal ferritin. Ferritin molecules appear above and between the microvilli (F,), within surface invaginations (Fz), within cytoplasmic vesicles (Fs), and
within a multivesicular body (F4). Figure 10, X 90,000; figure 11,
X 95,000.
Dale E. Bockman and William B. Winborn
12 Non-sensitized animal with intraluminal ferritin. Portion of capillary underlying columnar absorptive cells. Ferritin is present in the
tissue space ( F , ) , between the boundary membrane of the capillary
and the endothelial cell (Fz), and within the capillary lumen (Fs).
x 138,000.
13 Sensitized animal without intraluminal ferritin. No fenitin is present
in the intestinal lumen ( L ) or apex of the columnar absorptive cell.
Uranyl acetate and lead citrate stain. X 35,000.
Sensitized animal without intraluminal ferritin. Portion of a macrophage i n the lamina propria, containing a n aggregate of ferritin ( F )
and individual molecules of ferritin (circles) i n the cytoplasm.
X 216,000.
Dale E. Bockman and William B. Winborn
Sensitized animal with intraluminal ferritin. High concentration of
ferritiii is present in the enteric surface coating (F1) and in the
cytoplasmic vesicles (Fz). X 10,000.
Sensitized animal with intraluminal ferritin. The Golgi ( G ) area
stands out in sharp contrast due to the presence of an electroil
opaque material in the supra- and para-nuclear areas of columnar
absorptive cells follcwing the intraluminal administration of ferritin. x 18,000. See figure 18 for higher magnification of a similar
Dale E. Bockman and William B. Winborn
17 Sensitized animal with intraluminal ferritin. A marked accumulation of ferritin (F,) is evident i n the enteric surface coating above
and between the microvilli of a columnar absorptive cell. Ferritin is
concentrated in surPace invaginations (F2) and within simple ( F J )
and complex (F4) cytoplasmic vesicles. x 47,000.
Sensitized animal with intralumirial ferritin. Accumulatioii of ferritin in the Golgi area of a columnar absorptive cell. The vesicles
toward the top (F1) are similar to apical cytoplasmic vesicles. Those
close to the lamellar portion of the Golgi complcxcs (F2) appear less
dense. X 41,000.
Dale E. Bockman and William B. Winborn
PLz4TE 6
Sensitized animals with intraluininal ferritin
Portion o f macrophage in lamina propria. Ferritin apFears as aggregates ( F ) and as individual molecules dispersed in the cytoplnsm.
Individual molecules are in the intercellular space ( I ) . Clear rims
( C ) appear betwecn aggregates of ferritin and the bounding membrane. x 43,000.
Portion of macrophage in lamina propria. Part of a membranebound aggregate occupies the upper margin. The characteristic confisuration of individual molecules (arrows) is cvident in the cytoplasm. X 229,000.
Ferritin molecules (arrows) i n the intercellular space of the lamina
propria. Moderate electron density is evident around groups of
molecules. Unstained. X 313,000.
Aggregates of ferritin (F) are located i n the capillary endothelium.
Individual molecules of ferritin appear in the intcrcellular space of
the lamina propria ( I ) , in the endothelial cell, and in the capillary
lumen (arrow). X 93,000.
Dale E. Bockman and William B. Winborn
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hamster, intestinal, mesocricetus, sensitized, ferritic, observations, microscopy, nonsensitized, light, auratus, electro, absorption
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