Download functional differentiation of enterocytes in the follicle

J. Cell Sri. 55, 147-156 (1982)
147
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FUNCTIONAL DIFFERENTIATION OF
ENTEROCYTES IN THE FOLLICLE-ASSOCIATED
EPITHELIUM OF RAT PEYER'S PATCH
MICHAEL W. SMITH 1 AND GABRIELLE SYME1
1
Agricultural Research Council Institute of Animal Physiology,
Babraham, Cambridge CBz \AT, U.K.
1
Department of Physiology, The University, Sheffield S10 zTN, U.K.
SUMMARY
The ability of the follicle-associated epithelium (FAE) of rat Peyer's patch to accumulate
valine has been measured using a new technique of autoradiographic analysis. Maximum
uptake of valine was achieved by enterocytes present in the dome region of the FAE. Valine
uptake was not seen in enterocytes present on the lower slopes of the FAE or in follicleassociated crypts. The ability of the FAE to absorb valine was generally much less than that
seen in enterocytes present on adjacent villi. The main reason for this discrepancy lay in the
apparent inability of the FAE to develop a second phase of amino acid transport similar to
that seen in villus enterocytes. It is suggested that this failure results from some unexplained
interaction taking place between the FAE and its underlying lymphoid tissue.
INTRODUCTION
Enterocyte differentiation in the intestinal mucosa is a well characterized phenomenon. Cells born in the crypts of Lieberkuhn rapidly acquire the morphological
features of Paneth, goblet, endocrine or columnar absorptive cells, and the final
functional differentiation of the absorptive cells takes place as they migrate along
the upper regions of individual villi (Kinter & Wilson, 1965). Recently it has been
shown that the epithelium covering lymphoid follicles in the intestine (the follicleassociated epithelium, or FAE, of intestinal Peyer's patches) is renewed in the same
way and with a time course similar to that determined for surrounding villi (Smith,
Jarvis & King, 1980). There are, nevertheless, significant differences between the
patterns of differentiation seen in these two tissues, the most obvious being the
presence of antigen-transporting cells in the FAE (Bockman & Cooper, 1973; Owen
& Jones, 1974) and the relative lack of goblet cells in the FAE compared with the
villus eipthelium (Smith et al. 1980; Bockman & Cooper, 1973; Owen & Nemanic,
1978; Chu, Glock & Ross, 1979). The possibility exists that cells formed in the
FAE might have their pattern of differentiation determined, in part, through close
contact with underlying lymphoid tissue (Smith & Peacock, 1980).
The ability of villus enterocytes to take up different amino acids, which is a late
feature of functional differentiation, can now be studied at the cellular level by applying quantitative autoradiography to glutaraldehyde-fixed tissue (King, Sepulveda
& Smith, 1981a, b). The purpose of the present work was to use this analytical
148
M. W. Smith and G. Syme
technique with rat FAE to test whether lymphoid tissue might also affect the way
amino acid transport function becomes expressed in enterocytes during the last
stages of differentiation.
MATERIALS AND METHODS
Animals
Rats used in the present work were of the Sheffield strain. Bred in Sheffield they were
weaned at 21 days of age onto diets containing either 20 or 5 % protein, the carbohydrate
content of the low protein diet being increased so that both diets remained isoenergetic
(Syme, 1982). These animals had free access to tap water containing 3 fiM-Kl, to compensate
for the lack of iodine in the diet, from the time of weaning.
Experimental
Animals were anaesthetized with an intraperitoneal injection of Sagatal (0-15 ml 100 g"1
body weight) and the small intestine was removed. Pieces of mid-jejunum, 10 cm long, were
then cut open for mounting mucosal-face upwards in an uptake apparatus similar to that
described previously (Sepulveda & Smith, 1978). This tissue was pre-incubated for 10 min
in Na-free bicarbonate saline (Krebs & Henseleit, 1932), gassed with 9 5 % O , + s % C O t at
37 CC, before being used to measure vaJine uptake. Incubation with 1 mM-L-[3,4(n)-3H] valine
(Amersham International Ltd, Amersham, U.K.; sp. act. 100 fid ml"1), for 45 s in Nacontaining bicarbonate saline, was stopped by washing with phosphate-buffered saline
(Dulbecco 'A', pH7"3; Oxoid Ltd, Basingstoke, U.K.) containing 4 % glutaraldehyde and
2 % sucrose.
Most of the fixed tissue consisted of intestinal villi but it was, on occasion, possible to
detect the presence of Peyer's patches in localized areas. It was on this material that further
analyses of valine uptake were carried out.
Autoradiography
Pieces of glutaraldehyde-fixed jejunum containing Peyer's patches, taken from the uptake
apparatus after contact with tritiated valine for 45 s, were transferred to fresh glutaraldehydecontaining medium for 2 h before being washed twice in phosphate-buffered medium to remove
fixative. The subsequent dehydration, embedding in glycolmethacrylate, cutting of 3-/*m
sections, coating with photographic emulsion and development 12 days later, was as described
previously for rabbit ileum (King et al. 1981 a, b).
In further experiments designed to measure cell renewal time in villus mucosa, tritiated
thymidine ([6-'H]thymidine; Amersham International Ltd, Amersham, U.K.) was injected
intraperitoneally into rats (1 fid g~l body weight). These animals were killed 2, 10, 20, 30 or
40 h later and the mid-jejunum removed for fixation in phosphate-buffered saline containing
4 % glutaraldehyde and 2 % sucrose. The subsequent processing of these tissues for autoradiography was identical to that described above.
It was routine practice to incubate all sections, following development and fixation, in
silver intensifier solution (IN-5) for a period of 1 min to increase the size of silver grains.
This improved the accuracy of subsequent analyses.
Microdensitometry
Scanning microdensitometry of unstained autoradiographs was carried out at a final
magnification of x 1000 (x 10 eyepiece together with a x 100 oil-immersion objective) using
a Vickers M85 microdensitometer (Vickers Instruments, York, U.K.). Scanning took place
in discrete steps of 5 /*m moving from the centre of the FAE or the tips of adjacent villi
towards their respective crypts. Errors in density reading, which could have arisen from
scanning of goblet cells instead of enterocytes, were avoided by moving the scanning spot
Enterocyte differentiation in rat jejunum
149
laterally into other areas of the epithelium. This operation was rarely needed in the FAE,
however, where the number of goblet cells was found to be low compared with that found
in the villus mucosa.
Quantification of results
Readings of optical density were converted into intracellular concentrations of valine by
comparison with a known arginine standard, made up in gelatin and cut at the same section
thickness as used for tissue. The water content of mucosal scrapings was found to be
80-3 ± 4-5 % (mean ± S.E. of 9 determinations). Gelatin standards were made up as a 20 % (w/v)
solution in order to correspond to tissue water content. The specific activity of valine presented
to the tissue was identical to that of arginine used as standard (100 fid ml"1). Optical
densities obtained from tissue and gelatin sections were each corrected for losses arising
from incomplete retention during glutaraldehyde fixation (66 and 54-5 % retention determined
for valine and arginine, respectively, using methods of analysis described previously; King
et al. 1981a, b). Final calculation of intracellular valine concentrations was done by dividing
corrected tissue optical densities by that obtained for the standard.
RESULTS
Autoradiographic localization of valine uptake by rat follicle-associated epithelium
Tritiated valine at a concentration of 1 mM was presented to the luminal surface
of rat mid-jejunum for a period of 45 s, as described in the previous section. A typical
autoradiograph of sectioned tissue taken in the region of a Peyer's patch is shown
in Fig. 1 A. Radioactivity is seen to be concentrated in the dome region of the FAE
and in the upper half of surrounding villi. These villi appear to take up significantly
greater amounts of valine than does the FAE.
A companion section of the same piece of tissue has been stained with haematoxylin and eosin to show the fine structure of this region of the intestine (Fig. 1 B).
The almost spherical lymphoid follicle is covered by a layer of epithelium (the
FAE). This epithelium is continuous with that covering adjacent villi. Previous
work in the mouse shows this FAE to be derived from cells located in crypts situated
around each follicle (Smith et al. 1980). Similar crypts are present around the edge
of rat lymphoid follicles (Fig. 1 B).
The rates used in these experiments had been maintained on a diet containing
5 % protein for a period of 4 weeks prior to experiment. The effect of diet on the
pattern of amino acid uptake by the FAE is dealt with in a separate section.
Quantitative analysis of valine uptake by rat FAE
Microdensitometry was carried out on autoradiographs of rat FAE prepared in
a similar way to that shown in Fig. 1 A, values of optical density being converted to
intracellular valine concentrations as described in Materials and Methods. The
results obtained are compared with those obtained from surrounding villi (Fig. 2).
Radioactivity in the FAE remained close to background up to about 210/4m from
the base of follicle-associated crypts. Valine uptake then began to increase steadily
reaching maximum intracellular concentrations of about 5 mM at or shortly before
the apex of the follicle had been reached. Uptake of valine by surrounding villi
M. W. Smith and G. Sytne
B
*
Fig. i
•
«
*
«
Enterocyte differentiation in rat jejunum
01
0-2
0-3
Distance from crypt—villus junction (mm)
0-4
Fig. 2. A comparison of the ability of different areas of follicle-associated and villus
epithelium to transport and concentrate valine during a 45-s incubation at 37 °C.
Each value represents the mean of determinations carried out on 15 villi (—) or
4 follicles (--) respectively. The concentration of valine in medium presented to the
jejunum is shown by the hatched area.
remained negligible up to 160/fin from the base of villus-associated crypts. There
was then a gradual increase in valine uptake, similar to that seen in the FAE, causing
the intracellular concentration to double over the next 50 fim. of villus surface. This
was followed by a further rapid increase in absorptive function leading to a sixfold
rise in intracellular valine concentrations as the enterocytes migrated the last 90 /tm
to the villus tip. A similar two-stage increase in amino acid absorption has been
reported recently for rabbit ileum (King et al. 1981 b). All these results were obtained
using pieces of mid-jejunum taken from rats maintained on a diet containing 5 %
protein.
Fig. 1. Serial sections of rat mid-jejunum showing the location of tritiated valine
(A) and the general histological structure (B) of a rat lymphoid follicle and surrounding
villi. Sections cut at 3 ftm were either processed for autoradiography (A) or stained
with haematoxylin and eosin (B), as described in the text. Arrows show the position
of follicle-associated crypts. Bar, 500 fim.
M. W. Smith and G. Syme
152
Effect of diet on valine uptake by rat FAE
Weanling rats kept for 4 weeks on a diet containing 20% protein produce villi
that are approximately 50% longer than those found in litter-mates fed an isoenergetic
diet containing 5% protein (Syme, 1982). These changes take place without affecting
the size of associated lymphoid follicles (follicle diameter, about 500 /tm irrespective
of diet). The following experiments were carried out to compare the ability of FAE
taken from rats fed 20 % protein to take up valine with that seen using tissue taken
from rats maintained on a low protein diet.
10
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10
i
20
i
30
40
50
Time(h)
Fig. 3. Kinetics of cell replacement in rat mid-jejunum. Rats were killed after
intra-peritoneal injection of tritiated thymidine. Processing of tissue and measurement of cell position on sectioned villi was as described in the text. Values give
means ±s.E. of 10 separate determinations. Open arrow, time needed to completely
replace mid-jejunum mucosa (51 h).
Valine uptake by the FAE of tissues taken from rats fed 20% protein began, as
with the animals fed on low protein diets, to reach levels that are significantly different
from background some 200 fim. from the base of follicle-associated crypts. There
was then a gradual increase in uptake as enterocytes continued to migrate towards
the apex of the FAE. At this point, however, the total uptake of valine.was only
about one fifth of that recorded using tissue taken from the animals on the low
protein diet. This reduction in uptake was accompanied by an actual 12% increase
in valine uptake measured at the tips of villi taken from rats maintained on the
high protein diet.
It is concluded from these experiments that changes in diet can alter the capacity
Enterocyte differentiation in rat jejunum
153
of the FAE to absorb amino acids and that this can take place without noticeably
affecting the general pattern of enterocyte differentiation. It should be pointed out,
however, that the amount of valine entering the FAE of tissues taken from rats
maintained on the high protein diet was so low that it made detailed analysis
difficult. It was decided for this reason to carry out all subsequent experiments on
rats maintained on the low protein diet.
Thymidine-labelling experiments
The process of differentiation whereby a structurally complete enterocyte begins
to express absorptive properties has been described up till now in terms of cell
distance from point of origin. Knowing the cell turnover time for villus epithelium,
however (and assuming the same cell turnover time to apply to the rat FAE as it
does in the mouse; Smith et al. 1980), allows one to relate amino-acid-absorptive
function to the age of cell involved. In order to do this, tritiated thymidine was
injected intraperitoneally into a group of rats, individual animals then being killed
at known times afterwards. Pieces of mid-jejunum were processed for autoradiography
and the highest position of a cell containing label was measured and expressed
15
20
25
30
35
40
45
50
Enterocyte age (h)
Fig. 4. Relation between age of enterocyte and its ability to take up tritiated valine
during a 45-8 incubation at 37 CC. Enterocytes were studied in follicle-associated
epithelium (—) and in surrounding villi (
). Each value gives the mean of
determinations carried out on 4 follicles and 15 villi.
6
CELJ5
154
M. W. Smith and G. Syme
as a fraction of the total villus height. The results obtained from these experiments
are summarized in Fig. 3.
Labelling was confined to the bottom of crypts 2 h after thymidine injection.
These labelled cells were emerging from crypts onto villi some 8 h later. Migration
of enterocytes along the whole length of villi was related linearly to the time after
thymidine injection. Extrapolation of the last part of the graph gives an estimated
time for cell renewal of 51 h. Assuming 51 h to represent the time needed for
complete cell renewal in the FAE as well as the villus epithelium, it was calculated
that enterocytes were 30 h old when they first began to absorb valine (Fig. 4). This
applied whether the enterocyte came from a villus or the FAE. The rate at which
valine entered enterocytes then began to increase at a constant rate for a period of
10 h, the intracellular concentration of valine in a 40-h-old enterocyte being about
three times that presented originally to the rat jejunum. From then on the ability
to concentrate valine diverged in the two tissues; enterocytes in the FAE increased
their capacity to transport valine at the same rate as before, while villus enterocytes
entered a final stage where uptake increased more rapidly.
DISCUSSION
There is already extensive literature supporting the idea that lymphocyte traffic
across cell monolayers can affect their gross morphology. Some of these authors
have studied the ability of lymphocytes to create a particular type of high endothelial
cell in post-capillary venules (for references, see Goldschneider & McGregor, 1968),
and endothelial cells showing this characteristic have already been identified in the
interfollicular areas of Peyer's patches taken from both rats and mice (Schoefl, 1972;
Anderson, Anderson & Wyllie, 1976; Abe & Ito, 1977). More recently it has also
been suggested that lymphocyte traffic across the FAE of mouse Peyer's patches can
affect the morphology of columnar enterocytes so that they begin to transport
antigens (Smith & Peacock, 1980; Smith & Peacock, 1982). But do lymphocytes
actually have to cross cell layers in order to produce these marked changes in cellular
structure and function? The generally accepted finding that the FAE contains fewer
goblet cells than villus epithelium (Bockman & Cooper, 1973; Owen & Nemanic,
1978; Chu et al. 1979), even though both types of tissue originate from closely
associated crypts (Smith et al. 1980), suggests that something within follicles
(possibly the lymphocyte) is capable of exerting much more specific effects on cell
differentiation than had been previously recognized. This hypothesis is strengthened
by the present finding that morphologically similar enterocytes in villus and follicular
epithelium behave quite differently when asked to perform a common absorptive
function.
The general reduction in the capacity of follicular enterocytes to absorb valine,
seen irrespective of diet, agrees with an earlier finding in rabbit Peyer's patch
showing the transmural flux of different amino acids to be significantly less than
that measured across a corresponding area of villus epithelium (Hajjar, Hawrani &
Khuri, 1972). The present work allows one to interpret this kind of result in greater
Enterocyte differentiation in rat jejunum
155
detail. We have found two phases of uptake in the villus epithelium but only one
in the FAE. It is the absence of this second phase of carrier expression that results
in the differences seen when measuring amino acid entry into these two tissues.
Why FAE enterocytes fail to show this later stage of functional differentiation
remains unclear. One suggestion could be that this aspect of enterocyte development
needs to be initiated by factors only found within the core of the villus. Another
suggestion could be that lymphocytes play a more active role in actually inhibiting
a normal process of cell differentiation programmed to take place within migrating
enterocytes. Though unproven, this latter suggestion would be consistent with the
further finding that goblet cells are less readily formed from stem cells in the crypts
of the FAE (Bockman & Cooper, 1973; Owen & Nemanic, 1978; Chu et al. 1979;
Smith et al. 1980) and that close proximity of lymphocytes to fully differentiated
enterocytes often leads to gross changes in microvillar structure (Smith & Peacock,
1980, 1982). It is hoped that the introduction of the present technique, allowing
the functional characteristics of enterocytes to be monitored in the FAE throughout
differentiation, might help to resolve some of these uncertainties in the future.
We wish to thank Mr I. S. King for preparation of tissue sections for histological and
autoradiographical analysis, and Mr A. L. Gallup for photography of intestinal tissue.
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