A novel mechanism for disposing of effete epithelial cells in the small intestine of guinea pigs

1993;105:1059-1097

GASTROENTEROLOGY

A Novel Mechanism for Disposing of Effete Epithelial Cells in the Small Intestine of Guinea Pigs TOSHIHIKO Department

IWANAGA,

HONGXIA

HAN,

KAZUO

Bac&round: We previously showed that at the villus tips in the small intestine of guinea pigs effete enterocytes are not simply exfoliated into the lumen but phagocytosed by subepithelial macrophages, leaving only a thin apical cell portion in the epithelial lining. The aim of the present study is to investigate the fate of these apical pieces of enterocytes. Methods: The ileum of guinea pigs was perfusion-fixed and processed for transmission and scanning electron microscopic observation. Results: The apical cytoplasmic plates were found to be pushed by neighboring enterocytes and protruded from the epithelial surface, finally being pinched off into the lumen. In this process observed at the villus tips, the junctional complexes between the apical cytoplasmic plate and the adjacent enterocytes were preserved until the pinching-off of the plate. Luminal cell elements revealed a rich existence of cupshaped or spherical cell fragments covered with microvilli; nuclei were never observed in the luminal fragments. Conc/usions: The findings in the small intestine of the guinea pig are the first to account for the mechanism of the epithelial barriers being preserved while apoptotic enterocytes drop out at the tips of the villi.

E

and TSUNEO

ADACHI,

the epithelial

cells constant

of the

gut

cell renewal.

are

characterized

The epithelial

by cells

holes left after exfoliation

lial cells at the villus Our previous tine

pigs,

gated at the lamina ously engaged This hitherto

of dead epithe-

tips.‘,’

study’ showed

of guinea

that in the small intes-

macrophages

propria

in the phagocytosis unknown

are densely

aggre-

of the villus tips and vigor-

process

of effete enterocytes. contrasts

with the gen-

erally accepted view that effete epithelial cells are simply exfoliated into the lumen at the tips of villi3 The cytoplasm

of dying

enterocytes

was found

in our pre-

vious study to be fragmented presumably by the killer activity of large-granular lymphocytes within the epithelial The

lining

before

fragmentation

by resident

being

engulfed

by macrophages.’

of cells followed

by phagocytosis

cells has often been observed

of programmed

cell death

or apoptosis.“.”

in the process In our pre-

vious study,8 we also noticed that only a thin apical cytoplasm covered with microvilli remains in the epithelial

lining

been removed

after the bodies

of the enterocytes

by macrophages.

To investigate

have the fate

of these apical pieces of the enterocytes was deemed important to reveal the dynamics of the epithelial barrier in the process of the disposal of apoptotic epithelial

pithelial rapid,

FUJITA

of Anatomy, Niigata University School of Medicine. Nligata, Japan

cells. The present

phological

evidence

are processed

in the small intestine proliferate in the crypts, move towards the villi, and die at the tips of intestinal villi.‘,’

study is the first to show mor-

suggesting

without

that effete enterocytes

destroying

the barrier.

Materials and Methods

The life span of enterocytes (columnar cells or absorptive cells) is approximately 2 days in the mouse, 3 days in the rat, and 6 days in humans.-S5 Autoradiographic

Adult male guinea pigs, weighing 250-300 g, were used in this study. Ten animals were anesthetized by intra-

techniques

through

the aorta with physiological

chloride

in distilled

have

greatly

served

our

knowledge

con-

cerning the proliferation, movement, and life span of intelstinal epithelial cells. However, the precise process of the elimination of effete epithelial cells at the villus tips remains unknown. In particular, no report seems to have investigated whether the epithelial barrier is destroyed when the effete epithelial cells are, as has into the lumen, been generally accepted, “exfoliated” or it is preserved by some mechanism. A few researchers have attributed the elevated permiability of large molecules through the intestinal epithelium to

peritoneal

injection

in 0.1 mol/L performed

of

pentobarbiturate

water),

phosphate at a pressure

followed

buffer, ranging

and

saline

perfused

(0.85% sodium

by 2.5% glutaraldehyde

pH 7.4. The perfusion from

was

80 to 85 cm H,O,

which was slightly lower than normal blood pressure in the guinea pig (arterial pressure: 100-120 cm H,O). Segments

Abbreviations used in this paper: CTL, cytotoxic T lymphocyte; LGL, large granular lymphocytes; SEM, scanning electron microscopy: TEM, transmission electron microscope. [I? 1993 by the American Gastroenterological Association 0016~5085/93/$3.00

1090

IWANAGA

of the ileum,

ET AL.

5 cm apart from the ileo-cecum

cut into

small pieces,

postfixed

in 1% 0~0,.

dehydrated

GASTROENTEROLOGY

through

ded in Araldite conventional

junction,

fixed for an additional After

a graded

(Nisshin

methods

fixation,

and

the specimens

series of ethanol

EM; Tokyo,

and observed

were

2 hours

Japan)

were

and embedaccording

by a transmission

to elec-

tron microscope. For

scanning

hyde-fixed

electron

materials

microscopy

as mentioned

were conductive-stained scribed mens frozen pact.

and transferred

at the stage in liquid

through

used. They

to isoamyl

of immersion

nitrogen

All specimens

acetate.

in isoamyl

and fractured

were

method a graded

critical

Some speci-

voltage

using

cm in length, iological

l-2

and

and injected

mL in volume.

the precipitate

ing 2.5% glutaraldehyde tates were processed Araldite embedding.

an

Five minutes

solution.

For the measurement each group were chosen length of 10 microvilli graphs and statistically

phys-

later, the at 3000 was dis-

was fixed for 1 hour by gently

as pellets

After fixation,

for conventional

add-

the precipimethods

of

of length of microvilli, 20 cells in under an electron microscope. The in each cell was measured on microevaluated as the mean f SEM.

of

In the small

large

tips.

Their

intestine

gathered

distribution

and

pigs, propria

ultrastructure

mac-

of villus have

been

report.8 Briefly, the aggregation of macrophages were recognized in all villi examined from the duodenum to the terminal ileum. The macrophages were ultrastructurally characterized by possessing phagosomes and residual bodies of various sizes (Figure 1). The phagosomes contained fragments of enterocytes undergoing different stages of digestion; mitochondria, Golgi apparatus, and rough endoplasmic reticulum of the enterocytes could thus be identified. It was noteworthy that not a single portion of the striated (microvillous) border of the enterocytes could be recognized in the phagosomes. No cellular components derived from entero-endocrine cells, goblet cells, described

in our previous

of guinea

at the lamina

intercellular

elles; these bodies

in other

regions

of

action

of

(LGLs),

the

basal

cyto-

into membrane-

containing

were believed

present

con-

tips, but

to the cytotoxic

bodies

macrophages to form of their phagosomes.

were

of villus

was fragmented

cytoplasmic

by ex-

cells (Fig-

spaces

recognized

lymphocytes

of enterocytes

wide

(compare

some

organ-

to be incorporated

by

the above-mentioned contents Skinlike apical cytoplasm was

in the epithelial

lining

in spite of

plexes

area on the top of the intestinal

with

surrounding

Figure

intact

consisting

84)

and

enterocytes of zonula

tightly via

villi

connected

junctional

occludens

and

to com-

adherens

and macula adherens (desmosomes) (Figures 2 and 3). The apical cytoplasmic plate, frequently with lowered electron

density,

was

covered

were markedly larger (1513 those of intact enterocytes (920 k 46 nm) (Figure lar in thickness not form mal

striated

border,

with

3). The microvilli

arrangement

The usually

microfilament these bundles

microvilli

apical

were irregu-

and branching, characteristic

cytoplasm

called bundles

that

_t 89 nm) in length than in the top of the villus

and, by diverging

the tight

microvilli.

crovilli;

Macrophages and Ultrastructure Epithelium at Villus Tips were

large-granular

frequently

of the epithelial

owing

villi

spaces caused

in the epithelium

Probably

in the

at the tips of intestinal

such spaces were never

bounded

recognizable

intercellular The

observed

the villi. plasm

were

and fragmentation

1, aster&.r).

tained

Results

rophages

cavation ure

lining

remarkable

a rather

with warmed

fibroblasts

No. 4

the loss of the major part of the cells (Figures 2 and 3). The cytoplasmic plates were scattered, not grouped, in

of

intraluminal fluids were collected and centrifuged rpm for 5 minutes. After the supernatant fluid charged,

ob-

under

were starved for 48 hours and anesthegas. Intestinal loops of the ileum, 3-4

were prepared

saline,

imliquid

of 10 kV.

Electron Microscopic Observation Cellular Elements in the Lumen Five animals tized with halothane

were

by mechanical

point-dried

evaporation-coated with gold-palladium, co,, served by a Hitachi S-450 LB SEM (Tokyo, Japan) accelerating

deseries

acetate

showed

stantly

glutaralde-

were

by the tannin-osmium ” dehydrated

by Murakami,

of ethanol

(SEM),

above

or subepithelial macrophages. The epithelial

Vol. 105,

extending

are called

of nor-

beneath

the terminal

the

web, con-

from

“rootlets”

did

the mi-

of the core

filament bundles in the microvilli proper.‘* At the basal end of the rootlets, an electron-dense zone extended horizontally and appeared to interconnect rootlets; this cross-linking zone has been shown to be composed of fine cross-linking fibrils.‘* These structures in the terminal

web were clearly

recognizable

due to the pale

cytoplasm (Figure 3). The apical cell pieces further contained small amounts of cell organelles, including mitochondria and endoplasmic reticulum as well as a few large lipid droplets (Figure 3). The skinlike apical pieces frequently extruded into the lumen, forming a domed or polypous protrusion (Figure 4, asteti~k~). Such protruded cytoplasmic bodies were still provided with intact junctional complexes for adjacent enterocytes. Some of them were connected to the epithelium by only a narrow stalk, suggesting the process of dropping off (Figures 4, 5s). At such a stage, the cell pieces were still covered by long, irregu

DISPOSING OF EFFETE EPITHELIAL CELLS IN INTESTINE

October 1993

1091

Figure 1. Electron micrograph showing a villus trp of the guinea pig ileum. *Intercellular spaces can be seen within the eprthelium, possibly because of the apoptosrs of enterocytes and the subsequent engulfing by macrophages. The subepithelial region is occupied by macrophages (M) containing large phagosomes (P). L, lym-

phocyte (bar z 5 pm). lar rnicro\~illi. displayed

The

irregular

rootlets

in the terminal

arrangement,

especially

web also

of lymphocytes

in direc-

4). The irregular

tion. Intact enterocytes containing nuclei were always present underneath these pinched cytoplasms; no opening or discontinuity in the epithelial lining was recognizable under the electron microscope (Figures 4 and 5). Occasionally, spherical cytoplasmic bodies with a microvillous border were recognized close to but free from the surface of the epithelium at the villus tips (LGgure SC). Morphological changes in the epithelial lining at the tips of villi were closely related to the infiltration

into the epithelium

lymphocytes, cytoplasmic

(Figures

the cytoplasm of apoptotic enterocytes 2). The lymphocytes contained round, granules,

300-600

1, 2, and

usually large in size, possessed processes and interdigitated with

nm in diameter,

(Figures 1 and electron-dense

in the paranuclear

portion; they were thus identified as LGLs (Figure 2). Besides the granules, clear vesicles of various size were recognizable in the cytoplasm (Figure 2); the rod-cored vesicles that are known to be another feature characteristic

of IGLs

Electron Luminal

in the rat could

Microscopic Observation Cell Elements

The precipitates

of the fluid collected

the intestinal lumen contained with the microvilli in question sometimes phocytes,

not be identified.”

of by rinsing

cytoplasmic fragments (Figure 6). They were

intermingled with small numbers of lymred blood cells, and various fragments of a

cellular nature. Of note, nuclei were never observed in the luminal cell fragments equipped with a tuft of microvilli. The shaped,

Figure 2. A thin aprcal cytoplasm is formed at the villus tip. It IS connected with adjacent intact enterocytes via junctional complexes (arrows). A large lymphocyte (L) containing electron-dense granules (arrowheads) is located under the skinlike apical cytoplasm and, at the basal part of the cell, interdigitates with an enterocyte cytoplasm. E. enterocyte (bar = 1 pm).

microvillous hemispherical,

cell fragments or spherical

were

either

cup-

in shape and var-

ied in their volume of cytoplasm; a rich cytoplasm contained a small number of organella such as mitochondria and vesicular elements, and also lipid droplets, but never a nucleus (Figure 6). The microvilli were further elongated (1784 f 89 nm) and surrounded the entire circumference of the round cytoplasmic pieces, whereas they were restricted to the convex surface in cup-shaped and hemispherical ones

1092

IWANAGA

ET AL.

GASTROENTEROLOGY

Vol. 105,

No. 4

Figure 3. Thin apical cytoplasm of an enterocyte is bridged between intact enterocytes (E); junctional devices (arrows) rncludrng, at least, zonula occludens are recognizable between the cytoplasmtc plate and adjacent enterocytes. Microvilli tn the skinlike cytoplasm are elongated and irregular in shape. LD. lrpid droplets (bar = 1 urn).

(Figures 6 and 7). Each microvillus possessed much longer rootlets in the terminal web region than that of

usually

the intact

Dome-shaped or polyplike extrusions were found scattered on the top area of the villi (Figure 8A) tending to be located in a small pit or furrow of the epithe-

enterocytes

within

the epithelial

lining

ure 6, arrows and Figure 7). The cross-linking were now unclear or unrecognizable. The ments normal

in the core of microvilli structure

the terminal

in extending

web region

Scanning

Electron

also differed and penetrating

(Figure

(Fig-

structures microfilafrom

the

far into

7).

Microscopy

of Villus Tips

intestinal

appeared

lium. The extrusions

cause of the convexity microvilli

slightly

void of microvilli

but

basically

Figure 4. Two dome-shaped protrusions of the apical cytoplasm of enterocytes (*) are seen at a villus tip. One of them (left) IS stall connected to the epithelium by a narrow stalk (arrow). L, lymphocyte; M, macrophage (bar = 5 pm).

flat;

goblet

cells

that

were smaller

cal faces of epithelial

SEM were few in the

pigs.

in size than the api-

cells and were covered,

entirely

or partially, with elongated, irregularly arranged microvilli. The microvilli diverged from each other be-

SEM observation could overview the free surface of the intestinal villi. The epithelial sheet was waved

as holes under

villi of guinea

knobbed

tapered

of their base (Figure

toward

like drumsticks.

the tip, whereas A humplike

was sometimes

84.

Some

others

were

elevation

de-

seen on the dome.

It

October 1993

DISPOSING OF EFFETE EPITHELIAL CELLS IN INTESTINE

1093

Figure 5. Villus tips of the guinea pig ileum showing various processes of detaching of the apical cytoplasm. (A, B) Intact epithelial cells are always lined underneath the cytoplasmic fragments pinching off. (C) A cytoplasmic fragment with irregular microvilli appears to have just been detached from the epithelial lining (bar = 2 pm).

was occasionally

clear that the hump represented

a

secondary, cytoplasmic projection of the dome, but in many cases it appeared to be some exogenous body attached to the dome. Observation of perpendicularly fractured surfaces

showed the profile of the skinlike apical cytoplasm of enterocytes to be a flat or convexed plate covered with long microvilli (Figure SC). It bridged the gap between intact enterocytes. A large empty space, most probably due to the loss of the apoptotic enterocyte

1094

GASTROENTEROLOGY Vol. 105, No. 4

IWANAGA ET AL.

cytoplasm,

was usually

observed

under

piece. This space was often partially trating

cells if they remained

fracture were

and

several

elongated 84.

microvilli

These

the TEM epithelial

further

surface

apical

procedures. cell pieces

were compactly

fragments images

were believed in Figures

tissue There

in which

arranged

(Figure

to correspond

of the microvillous shown

skinlike by infil-

at the place during

preparation

bouquetlike

the

occupied

bodies

to

on the

3-5.

Discussion Disposal of Effete Epithelial Cells It has long been cells at the villus exfoliated evidence the guinea

plasm

Recent

are simply

morphological

from us suggests that in the small intestine pig, the major part of enterocyte by macrophages

The cells display

as well as pyknosis

apoptotic

that effete epithelial

tips of the small intestine

into the lumen.‘,‘4,‘5

is phagocytosed propria.8

believed

bodies,

before

located

fragmentation

of

cytoplasm

in the lamina of their cyto-

to form membrane-bounded being

engulfed

by macro-

Figure 7. fragment filaments form very

phages.8 intestinal

Closer view of an rntralumrnal cell fragment. The globular is covered with long, radially arranged microvilli. The core of mrcrovilli penetrate deeply into the apical cytoplasm to long rootlets (arrow) (bar = 1 urn).

This

apoptotic

villi, because

process apoptotic

unique

to the

bodies formed

seems

in epi-

thelia are generally believed to be either extruded into the lumen or, more commonly, phagocytosed by resident epithelial cells.‘” Noteworthily, the phagosomes of the macrophages in the guinea pig villi contained a variety

of enterocyte

fragments

but never

the apical,

microvillous portion of the cell. It was unlikely that the microvillous portion might be more rapidly digested than other organellae in phagosomes. This rid-

Figure 6. Electron macroscopic observation of lumrnal cellular elements obtained by rinsing the lumen of the ileum. Numerous cytoplasmic corpuscles with long mrcrovilli are found with contaminated blood cells. Nuclei were never observed in the luminal fragments with microvillr; small mitochondria and vesicular elements are present in them. Note that microvillous microfilaments deeply penetrate the cytoplasm as indicated by arrows. L, lymphocyte (bar = 2 urn).

dle was solved when we recognized, both with the TEM and SEM, that only an apical piece of the cytoplasm was left in the epithelial lining, to be subsequently pinched off into the lumen (Figure 9). This finding was supported by finding numerous microvillous bodies in the luminal spaces. It has been supposed that the epithelial barrier is tentatively broken when effete epithelial cells are, as has been conjectured, simply exfoliated into the lumen.’ The present study, however, indicates that the

October 1993

DISPOSING OF EFFETE EPITHELIAL CELLS IN INTESTINE

1095

Figure 8. SEM observation of villus tips. (A) Dome-shaped protrusions covered with irregularly arranged microvllli are dispersed. (C) A skinlike apical cytoplasm with long microvilli is bridged between enterocytes. *A large empty space is seen under the skinlike piece. (6) A small apical cytoplasm with a bouquetlike tuft of microvilli is located at the free surface of the eplthelium. It seems to have been extruded into the Iumen in the following stage L, lymphocyte (bar = 5 pm).

apical

cytoplasm

for the epithelial in the guinea

of epithelial barrier,

pig intestine.

cells, which

survives

is essential

for a certain

Its separation

period

from the epi-

thelium was shown in this study to take place after surrounding epithelial cells had closed the upper portion

of the defect

to form

new junctional

complexes

beneath evidence

it (Figure 9). The p resent study thus offers that the epithelial barrier in the villus tips is

never destroyed during the renewal of epithelial cells, at least in the guinea pig. One unexpected finding was that microvilli of enterocytes ing their

are elongated and branched after largely losbasal cytoplasm. This morphological change

is reminiscent

Figure 9. A schematic drawtng showing a process of pinching off of enterocyte apical cytoplasm.

of streocilia,

which

are believed

to be

modified microvilli. The elongation of microvilli may be caused by the uncontrolled growth of microfilament bundles in their core. This change could be related to an altered architecture of the cytoskeleton in the terminal web, which was shown in the present observation as disappearance or unclearness of the cross-linking structures between the rootlets of the core filament bundles.

1096

IWANAGA

GASTROENTEROLOGY

ET AL.

Where Is the Apical Cytoplasm The present fete epithelial and their

elongated,

were dispersed finding

SEM observation

cells, identifiable

contrasts

with

that effete epithelial

showed

by their

irregularly

over a broad

Exfoliated? that ef-

domed

extending

microvilli,

area of the villus

the previously cells drop

shape

tip. This

prevailing

view

off in a group

at one

point of the summit of the intestinal villi.2,3 The newly proposed process seems more reasonable in the following two whole

points.

villus

number

First,

because

gradually

lial barrier

may

surrounding

towards

its tip,

the

of the villus.

be much

from

of a

cells must gradually

enterocytes

of are isolated

area

decreases

of its epithelial

closer to the summit

the surface

Second,

more

when

easily

be reduced the epithe-

preserved

each other,

than

of effete epithelial

be attributed

to the gene expression

ming) for apoptosis

in the enterocytes.

have been reported

cial type of T cells bearing to detect and destroy involvement

graphical

damaged

relation

a spe-

and to be able

epithelial

cells.‘6*‘7 The

in the apoptosis

of epithe-

both by our present and our preobservations’ showing a topobetween

intraepithelial lymphocytes. vealed lymphocyte-mediated cells.‘8-20 The similarities toxic T lymphocyte

(program-

The intraepithe-

76 receptors

of lymphocytes

lial cells is suggested vious ultrastructural

cells may not

to represent

effete

enterocytes

and

Recent studies have reapoptosis in a variety of between

(CTL)-mediated

apoptosis cytolysis

and cytosuggest

that killer cells activate an endogenous suicide program in their targets.21 The lymphocytes in the villus tips of the guinea pig are classified into LGLs, which function as CTLs. Rat LGLs in the liver and gut have been reported to contain, in addition to the dense granules, small clear vesicles with a rod core.‘3*22 This type of vesicle could not be shown in the LGLs of the intestinal villi of guinea pigs. Localization of rodcored vesicles in the LGLs has been confirmed in the rat, mouse, and humans, but no information is available in other mammals including the guinea pig (reference 14 and Kaneda, personal communication, November, 1992). Therefore, it remains unclear whether all LGLs in the guinea pig lack the rod-cored vesicles or that intestinal LGLs examined are a special type of LGLs without the vesicles. As observed

in mouse

cells, CTL-mediated

the intercellular

and target

protein,

more

perforin24-26;

contents

space

cell.2” The cytolytic shown

molecular

weight

contained

in the granules

substance

to be the pore-forming

recently,

a protein

of 40 kilodaltons

with

was found

and to cause DNA

to be

Besides the occurrence

of these cytolytic

topographically

contact

and its target has been shown for cytolysis21

ent observations plasm

close

of enterocytes

between

subthe

to be a neces-

The previous

and pres-

penetrating

the cyto-

of lymphocytes

a

fragmen-

tation.

sary condition

of

between

stances, lymphocyte

No. 4

seems to meet that condition.

Disposal of Enterocytes

in Other Mammals

The mechanism

here for the disposal

of effete enterocytes,

proposed in which

by macrophages

the cytoplasm

is phago-

and only the apical cytoplasm

is shed into the lumen, is in disagreement with the general concept concerning the fate of effete enteroit is an important question as to cytes. ‘,2 Therefore,

of lntraepithelial

The disposal

into

has been biochemically

cytosed

Functional Significance Lymphocytes

lial lymphocytes

the effector

by

in the case that

by the release of the granular

the lymphocyte

the cells to be disposed

they are grouped.

simply

sis is induced

Vol. 105,

apopto-

how extensively cies. To obtain

this mechanism the answer,

is shared

among

the morphology

spe-

of the vil-

lus tips should be carefully examined in each species. Our experiences imply that the aggregation of macrophages

rich

transportation

in phagosomes

is a reliable

of effete epithelial

propria.

28*29The density

propria

and their

sign of the

cells into the lamina

of macrophages

in the lamina

morphology differ markedly the existence species,‘* suggesting

mammalian ferent mechanisms

for the disposal

among of dif-

of effete epithelial

cells. In rats, as well as mice, which studies on cell kinetics numerous

in the lamina

have been favored

of enterocytes, propria

macrophages

of the villi.

for are

They are

so slender in shape and so poor in phagosomes that they may be not involved in the active phagocytosis of effete cells as they are in the guinea pig.28 This view is supported by our preliminary study showing that luminal

fluids

of the rat intestine

contained

much

cell

debris including nuclei. In the monkey, on the other hand, phagosome-rich large macrophages are gathered at the villus tips and contain cellular elements similar to those of epithelial cells.” An aggregation of similar macrophages is recognized also in the human intestine, as our preliminary observation indicates. The novel mechanism of the disposal of effete epithelial cells and maintenance of the epithelial barrier as observed in the guinea pig may therefore be shared by humans, monkeys, and possibly by some other mammalian species. That this particular cell disposal mechanism does not seem to take place in the popular laboratory animals

October

DISPOSING

1993

such as the rat and mouse

may account

hitherto

by researchers.

been overlooked

for why it has

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16. 17.

18.

19.

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22. 23. 24.

25.

26.

27.

28.

29.

OF EFFETE

EPITHELIAL

CELLS

IN INTESTINE

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Received January 1, 1993. Accepted April 19, 1993. Address requests for reprints to: Toshihiko Iwanaga, Ph.D., Department of Anatomy, Niigata University School of Medicine, AsahiMachi 1, Niigata 951, Japan.