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Research Communications |
a School of Pharmaceutical Sciences, University of Nottingham;
b Department of Human Anatomy and Cell Biology, Queen's Medical Center, Nottingham; and Laboratories of
c Cell Signaling and Molecular Recognition, The Babraham Institute, Cambridge CB2 4AT, U.K.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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as
controls. No sign of bacterially induced damage to the epithelial
barrier was observed. Further confocal microscopy analysis of the FAE
surface showed that a significant increase in the number of cells that
showed both morphological and functional features of M cells took place
within pneumococci-treated PP tissues. These data provide the first
direct evidence that the FAE-specific antigen sampling function may be
manipulated to improve antigen and drug delivery to the intestinal
immune system.—Meynell, H. M., Thomas, N. W., James, P. S., Holland, J., Taussig, M. J., Nicoletti, C. Up-regulation of
microspheres transport across the follicle-associated epithelium of
Peyer's patch by exposure to Streptococcus pneumoniae
R36a.
Key Words: M cell • enterocyte • mucosal immunity • FAE • antigen delivery
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been suggested that the FAE represents a highly dynamic tissue that is able to modify its epithelial and lymphoid cell populations in response to certain antigenic stimuli. Mice showed an increased number of M cells and intraepithelial lymphocytes (IEL) after transfer of SPF mice to a conventional animal house (5) or feeding with living attenuated Salmonella (6) . More recently, a culture system that reproduces the main characteristics of FAE and M cells by cultivation of murine PP-derived lymphocytes with human intestinal cells has been established (7) . Taken together, these data suggest an important role for the immune system in shaping both morphology and function of epithelial cell population of the FAE.
However, direct evidence of the possibility of manipulating in vivo the immunologically relevant antigen sampling function of the FAE was still lacking. The importance of improving antigen delivery to the mucosal immune system is well recognized (8) and the identification of molecules and mechanisms that may augment mucosal antigen uptake will greatly affect the methods of oral delivery of drugs and vaccines.
We previously established that a short-term (1 h) exposure to a nonintestinal bacterium, Streptococcus pneumoniae R36a, induced marked alterations of the FAE morphology and architecture, including a massive passage of lymphoid cells from the subepithelial lymphoid tissue to the FAE, and, more important, an apparent increase of the area showing the morphological characteristics of M cells (9 , 10 ). We reasoned that the marked increase of the M cell area or numbers might lead to an enhanced ability of the FAE to transport luminal antigens to the mucosal immune system.
We decided to address this problem by evaluating the ability of the FAE of PP to bind, internalize, and transport polystyrene latex particles and the numbers of FAE sampler cells after exposure to pneumococci.
Nonionic polystyrene latex microspheres show a unique specificity to
and are transported by rabbit M cells (11
,
12
); as such, they are an excellent tool to carry out
quantitative studies of antigen uptake in the FAE as well as being a
functional marker of intestinal M cells. Isolated rabbit ileal loops,
each containing one PP, were stimulated with a saline suspension of
S. pneumoniae R36a (Gram-positive, nonintestinal),
Escherichia coli DH5 (Gram-negative, intestinal), or
sterile saline. The loops were then returned to the abdominal cavity
for 3 h, after which the contents were aspirated. The loops were
then filled with saline containing fluorescent latex microspheres for
45 min before removing the tissues. The numbers of particles were
enumerated in three different compartments: 1) bound to the
FAE surface, 2) internalized by the FAE, and 3)
translocated to the subepithelial lymphoid tissue. When
pneumococci-treated Peyer's patch tissues were examined, we observed a
marked increase in the numbers of particles either bound or transported
by the FAE compared to tissues exposed to saline solution or to the
intestinal microrganism E. coli DH5. Additional studies
enabled us to demonstrate that the increased ability of the S.
pneumoniae-treated tissues was due to the rapid appearance
of cells within the FAE that showed both morphological and functional
features of M cells.
We believe that these findings will help to define basic aspects of the biology of the FAE and could prove useful in the design of novel and effective strategies for oral delivery of vaccines and drugs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Streptococcus pneumoniae strain R36a was grown from stock
cultures frozen in 20% DMSO at -80°C. Frozen cells were plated onto
tryptic soy agar with 5% sheep blood (DIFCO Lab., Surrey, U.K.) and
the growing colonies were inoculated in tryptic soy broth (DIFCO Lab.).
An additional passage in broth was made; the early log phase
pneumococci were harvested by centrifugation (3000 rpm/20 min) and
washed in cold phosphate-buffered saline (PBS). Bacteria were adjusted
to the desired concentration. The concentration was measured based on
previous calibration and then confirmed by plating the pneumococci onto
blood agar plates. The same protocol was used to grow E.
coli strain DH5. In this case, the bacteria were grown in LB
broth (DIFCO Lab.).
In vivo stimulation of Peyer's patch
Rabbits were anesthetized by inhalation of Fluothane
(Mellinckrodt Vet., Uxbridge, U.K.) and kept under deep anesthesia
throughout the experiment. The muscle and eye reflexes were checked at
10–15 min intervals, and the dose of anesthetic delivered through a
facial mask varied accordingly. The abdominal cavity was opened; ileal
loops measuring about 3 cm in length and containing one Peyer's patch
each were isolated by a distal and a proximal ligature. Particular care
was taken to maintain the normal blood supply. The isolated loops were
subsequently filled with 2–2.5 ml of a saline suspension containing
approximately 1 x 108 CFU of pneumococci, and control
loops were filled with a similar number of E. coli or
bacterium-free PBS. A total number of 16 rabbits was used, with three
to four patches for each animal. For each animal, two or three loops
were used for bacterial stimulation (S. pneumoniae or
E. coli) and one loop was inoculated with sterile PBS as an
additional control. The loops were then returned to the abdominal
cavity for 3 h. Care was taken to maintain the normal body
temperature throughout the experiments. After 3 h, fluid from the
intestinal loops was withdrawn by aspiration and the loops were
carefully and repeatedly washed with sterile, warm PBS. After washings,
a saline suspension containing 1 x 109 yellow-green
fluorescent latex polystyrene 0.5 µm microparticles (Polysciences,
Eppelheim, Germany) was injected into the isolated ileal loops, which
were then returned to the abdominal cavity for 45 min. The animals were
then killed by lethal dose of anesthetic and the tissues were removed.
Quantitation of microparticles adherence and uptake
Peyer's patches were removed, washed, and dissected. Care was
taken to properly wash the tissues. The mucosa tissues were pinned out
in small petri dishes containing Silgard 184 silicone elastomer (Dow
Corning 6198, Seneffe, Belgium) and vigorously washed with ice-cold
saline containing 0.5 mM dithiothreitol to remove mucus and nonadherent
particles, then dissected. Half of each Peyer's patch was immediately
frozen in isopentane, cooled in liquid nitrogen, and stored at -80°C
until used. The other half of the patch was fixed in 1% glutaraldehyde
for 2 h at 4°C and the tissues were stored in PBS at 4°C.
Binding and uptake of particles by the FAE was quantitated in
propidium-iodide stained 8–15 µm section of frozen tissue mounted
without dehydration in glycerol/PBS solution Citifluor AF-1 (Chem. Lab,
Canterbury, U.K.).
Two different approaches were used to enumerate particles bound, internalized or translocated by the FAE. Initially, particles were counted by a fluorescence microscope (Nikon Labophot, Japan). The particles were enumerated per fixed FAE length, FAE area, and subepithelial tissue area as measured by SOM v 4.26 software (BioRad, U.K.). Subsequently, a BioRad MRC 600 confocal microscope was also used. A Nikon x50 oil immersion lens was used and dual X-Y images of 20 follicles per each patch were obtained by optically sectioning the tissue at 1 µm intervals. SOM v 4.26 software (BioRad) was used to calculate FAE length, FAE area, and subepithelial lymphoid tissue area. Dual imaging was performed by excitation of the yellow-green particles at 488 nm, propidium iodide at 588 nm, and detection at 510 and 565 nm, respectively. Each image was Kalman averaged 10 times and then a maximum projection was constructed using standard CoMos v 7 software (BioRad). The number of particles bound, internalized, and translocated to FAE tissues was scored manually.
Enumeration of microsphere binding cells within the FAE
A BioRad MRC 600 confocal microscope was used to enumerate
fluorescent microparticle binding cells within FAE. Three or four
follicles from each PP were microdissected from glutaraldehyde-fixed
tissues. The isolated follicles were mounted onto a glass slide in
PBS-glycerol (1:1 v/v) solution. A coverslip was then applied to
flatten down the dome-shaped follicle for viewing it on a single focus
plane. For each follicle, two areas at the periphery of the FAE were
randomly selected using SOM v 4.26 software, observed (magnification
x300), and microsphere binding cells were manually scored. Within the
selected areas, we also measured the area of the apical (luminal) side
of individual cells binding fluorescent microspheres. The areas of 160
cells from either bacteria-treated or control PP were measured.
Transmission and scanning electron microscopy
Peyer's patches samples from tissues previously fixed in 1%
glutaraldehyde were postfixed in 1% osmium tetroxide for 2 h,
dehydrated, further processed according to standard protocol, and
analyzed by a Jeol JEM-100C electron microscope or an ISI-SX25 scanning
electron microscope
Immunohistochemistry
Immunohistochemistry was carried out on frozen tissue sections
(7–8 µm) by incubation with antivimentin antibody clone V9
(Biogenesis, Poole, U.K.) diluted 1:500 in PBS 1% BSA, followed by
incubation with biotin-labeled sheep antimouse immunoglobulin (Ig)
(Amersham Int., Little Chalfont, U.K.). A final incubation with
FITC-labeled Streptavidin (Serotec, Oxford, U.K.) was then performed.
More frozen tissue sections were stained by incubation with 1 µg/ml
of TRITC-labeled phalloidin (Sigma Chem., St. Louis, Mo.) for 30 min at
room temperature.
Quantitation of IEL within follicle- or villus-associated epithelium was carried out in hematoxylin/eosin-stained sections from frozen tissues by image analysis. Serial sections from the same tissues were then used to determine subsets of IEL by immunostaining with monoclonal antibody 12.AB to rabbit Ig (a gift from Dr. E. Bertelli) and T cells (L11/135), followed by incubation with sheep antimouse Ig-biotinylated antibody (Amersham Int., Little Chalfont, U.K.) Incubations with peroxidase-labeled streptavidin (Amersham Int.) and diaminobenzidine were then performed. Data were expressed as number of cells ±SEM per 100 epithelial cells.
Detection of apoptotic cells in Peyer's patch sections
The presence of apoptotic cells in PP tissue sections was
analyzed by the Apo Alert in situ apoptosis detection kit (Clontech
Lab., Palo Alto, Calif.). The assay is based on the terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling methods.
Apoptotic cells containing the fluorescent-labeled fragmented DNA were
visualized by a fluorescence microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the number of fluorescent
microparticles bound to the FAE surface; only those in direct contact
with the epithelium were considered as bound. A marked increase in
pneumococci-treated tissue was seen compared to either PBS (5.2-fold
increase, P<0.001) or E. coli (6.9-fold
increase, P<0.001) -treated tissues. A comparable increase
of internalized particles in FAE exposed to S. pneumoniae
was also seen (Fig. 1
B). Here, too, the increase observed in pneumococci-treated
PP was highly significant compared to treatment with PBS (4.7-fold
increase, P<0.001) or E. coli (fourfold
increase, P<0.001). In agreement with a previous report
(12)
, we did not find evidence of relevant paracellular
transport of microparticles within the FAE.
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Thus, it appeared that challenge with S. pneumoniae
up-regulated the ability of the FAE to bind and internalize
microparticles. Moreover, the most striking difference was observed
when we examined the number of particles translocated to the
subepithelial lymphoid tissue (Fig. 1C
). In this case, the
number of particles increased 7.1-fold as compared to PP treated with
PBS (P<0.001) and sixfold as compared to E.
coli-treated PP (P<0.001). This finding is
particularly noteworthy since the lymphoid tissue underlying the FAE of
PP is the location of initiation of mucosal immune responses. In no
case did we observe a statistically significant difference between
values from PBS- and E. coli-treated PP tissues. Tissue
sections used for microsphere enumeration were obtained by cutting
frozen tissues in different directions, enabling us to show that the
distribution of microspheres within PP tissues was independent of the
direction of section cutting. In addition, microspheres were counted in
randomly selected sections by confocal microscopy, which did not detect
significant differences as compared with fluorescent microscopy (data
not shown). A total number of 16 rabbits was used and a high number of
follicles/group (ranging between 190 and 235/group) was used to
quantitate particles bound or within PP tissues.
Morphological analysis of the FAE after exposure to bacteria
As observed by scanning electron microscope, the most striking
feature of FAE tissues treated for 3 h with pneumococci
(Fig. 2
B, D) was an almost total absence of enterocytes
compared to PBS-treated tissues (Fig. 2A, C
). In the latter,
the FAE is formed by enterocytes and M cells, recognized by
morphological characteristics, whereas in pneumococci-treated tissues,
the FAE was formed mainly by a majority cell population showing
morphological features similar to those of M cells. Thus, it appears
that the highly specialized FAE is able to rapidly modify its
morphology and, more important, function in response to certain
bacterial stimuli.
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A typical feature of S. pneumoniae-treated tissues
was a marked increase of the intraepithelial lymphocytes (IEL)
population (9
, 10
, 13
). A
quantitative and qualitative immunohistochemical study showed that the
total number of FAE-associated IEL in pneumococci-treated tissues
markedly increased (Fig. 3
) and that Ig+ B cells were preferentially recruited
within the FAE. The relative percentage of B cells within the FAE
increased from 35% in normal (PBS-treated) tissues to 54% in
pneumococci-treated tissues. In contrast, the percentage of
FAE-associated T cells decreased from 48% to 33%. The percentage of
the non-Ig+, non-T cell population (NP) of intra-FAE cells
did not show a great variation in control tissues (13%) compared to
the stimulated ones (17%).
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In some cases bacteria may penetrate and lyse epithelial cells, thereby
disrupting the protective barrier overlying the mucosal surfaces
(14
, 15
). It would then be possible that, in
our experimental model, pneumococci-induced ulcerations of the FAE
allowed the increased entry of latex microparticles. With this in mind,
we assessed the integrity of the epithelial barrier after the challenge
with living pneumococci. A TEM microphotograph of a portion of the
pneumococci-treated FAE is shown in Fig. 4
. No signs of cell lysis (Fig. 4A
) or alteration of
cell-to-cell junctions (Fig. 4B
) were observed.
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Furthermore, the distribution of F-actin in bacteria-challenged PP
tissue, as determined by staining with TRITC-labeled phalloidin, did
not undergo detectable alteration compared to control (Fig. 5
A, B). In addition, we determined the level of
bacterially induced apoptosis in the FAE (Fig. 5C, D
). The
presence of scattered apoptotic cells was detected within the villus
submucosa. We failed, however, to observe apoptotic cells within either
the FAE or subepithelial lymphoid tissue of both control and stimulated
tissues. The integrity of the epithelial barrier was further confirmed
by immunostaining with antivimentin antibody. In contrast to other
species such as human, mouse, and rat, rabbit M cells do express
vimentin (16)
, an intermediate filament protein that is
typical of nonepithelial cells of mesenchymal origin.
Immunohistochemical analysis revealed that many vimentin-positive cells
were present within the FAE of both control and pneumococci-treated
tissues (Fig. 5E, F
). In this case, however, we did
not detect alterations of the FAE after exposure to S.
pneumoniae R36a. Together, these results demonstrated that the
increased ability of the FAE in binding, internalizing, and
translocating polystyrene microparticles after exposure to S.
pneumoniae R36a did not depend on bacterially induced damages to
the intestinal epithelial barrier.
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Enumeration of microsphere binding cells within the FAE
Two possible mechanisms for increased particles uptake were
envisaged. One was that the pneumococci-induced passage of lymphoid
cells into the FAE (9
, 10
, 13
)
led to an increase in the cell body size and therefore to an improved
transport capability of individual M cell. Alternatively, the
morphological and functional changes might be due to an increased
number of antigen sampler cells inhabiting the FAE.
We addressed this issue using two different approaches. First,
the area of the apical (luminal) side of various individual microsphere
binding cells was determined in both control and bacteria-stimulated PP
(Fig. 6
). In control tissues, cells showing the typical M cell ability of
binding microspheres exhibited an area of 104–189 µm2
(n=160, mean=143, 75±45) whereas in pneumococci-treated
tissue the area ranged from 102–215 µm2
(n=160, mean=151.74±39). This indicated that epithelial
cells with the ability to bind latex microspheres did not undergo major
changes in area size after exposure to pneumococci. Second, enumeration
of microsphere binding cells was carried out in microdissected
follicles. The isolated follicles were placed onto a glass slide and
then flattened by the application of a coverslip. This method allowed
viewing of the dome-shaped follicle on a single focal plane. The number
of microparticle binding cells was enumerated in each follicle within
two randomly selected areas of the periphery of the FAE. The values are
shown in Fig. 7
. A significant increase took place in pneumococci-treated FAE as
compared to either PBS (P<0.02) or E. coli
(P<0.02) -treated tissues.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The main finding of this work is that a short-term exposure to S.
pneumoniae R36a enhances antigen transport across the FAE by
increasing the number of sampler cells. Interaction of bacteria with
FAE does not always determine changes in the FAE cell population, as
evidenced by the absence of response to challenge with E.
coli DH5. This difference might be explained on the basis of
inherent differences in the cell wall composition and in
bacteria-derived or induced factors released upon interaction with host
cells. This may be important considering that genetically engineered
microorganisms have been successfully used as vaccine-delivery vehicles
in experiments of oral/nasal immunization (17
,
18
). The notion that certain bacteria may have an
intrinsic ability to up-regulate mucosal antigen uptake may have a
great effect in the choice of living vectors for the delivery of
heterologous antigens. Additional experiments using formalin-fixed
bacteria showed that dead bacteria were not able to induce any changes
of the FAE morphology and function (C. Nicoletti et al., unpublished
observation). This would suggest that molecules actively released or
induced by certain living microorganisms may regulate FAE-specific
functions.
In our model we have used fluorescent polystyrene microspheres as particulate antigen to quantitate mucosal antigen uptake. The utilization of polystyrene microparticles as a rabbit M cell marker offers several advantages over conventional biochemical markers, such as presence of vimentin (16) and a low level of alkaline phosphatase (19 , 20 ). First, our approach did not allow us to use the intermediate filament vimentin as a marker to detect newly arising M cells within the FAE in view of the brevity of the experimental procedure. Also, low expression (or the absence) of alkaline phosphatase in M cells may not be an optimal marker to identify these cells in rabbits due to the high variability in the content of such brush border enzyme in epithelial cells of rabbit PP (2, 12; C. Borghesi, personal communication).
The results described here indicate that certain bacterial stimuli may induce, within 3 h, de novo formation of cells populating the FAE that show the morphological and functional features of intestinal M cells. It may be that challenge with pneumococci provides the ultimate stimulus to predetermined M cells, already inhabiting the FAE, to rapidly mature and differentiate into fully operational M cells. Alternatively, it is possible that the newly arising M-like cells may derive from fully or partially differentiated enterocytes upon interaction with immune cells and/or soluble mediators. The latter hypothesis would be in accord with evidence of in vitro and in vivo lymphocyte-induced M cell formation recently provided by Kerneis et al. (7) . Here, the ability of lymphocytes to induce M cells in vivo was assessed 9 days after the injection of freshly isolated PP-derived lymphoid cells into the duodenal submucosa. Remarkably, in our experiments the increase in number of cells with the morphological and functional features of M cells took place only 3 h after challenge with pneumococci.
We have previously demonstrated that a peculiar feature of pneumococci-treated PP tissues was a marked increase in the number of IEL (9 , 10 , 13 ) It is noteworthy that no variation of the total number of IEL was seen in the epithelium of villi interspersed among Peyer's patch follicles. The latter demonstrates that the FAE and villus-associated epithelium markedly differ in their ability to rapidly modify their intraepithelial cell population in response to bacterial challenge.
In contrast to E. coli DH5, S.
pneumoniae R36a is transported by M cells (10)
. One
could therefore argue that the endocytotic process is exploited by the
microparticles to penetrate the epithelial barrier simply as
passengers. We tend to exclude such a possibility on the basis of two
considerations. First, at the end of the stimulation time and before
the injection of microparticles, the intestinal loops were carefully
washed by repeated injection of saline solution. Even though this
procedure does not rule out the possibility of bacterial contamination,
it should minimize the risk. Second, killed pneumococci, when used as
challenging microorganism, were normally endocytosed by FAE-M cells,
but in such a case we failed to detect any increase of mucosal antigen
uptake.
These results provide definitive evidence of possible manipulation of the immunologically relevant antigen sampling function of the FAE in the gut and demonstrate the highly dynamic nature of this tissue. The ability of the FAE to rapidly modify its permeability properties in response to certain bacteria, bacterially derived or induced molecules may be exploited to enhance absorption of orally delivered biologically active compounds.
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ACKNOWLEDGMENTS |
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and E. Bertelli for the monoclonal antibody 12.AB. We are also indebted
to P. J. Kilshaw, C. Borghesi, and J. P. Kraehenbuhl for
critical review of the manuscript and to G. Morgan and A. D.
Philips for transmission and scanning electron microscopy. The valuable
help of D. C. Lowe with bacteria and I. Janson with laboratory
animals is also acknowledged. Work supported by contract CSA 3710 from
the Ministry of Agriculture, Fisheries and Food, U.K. |
FOOTNOTES |
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1 Abbreviations: FAE, follicle-associated
epithelium; IEL, intraepithelial lymphocytes; Ig, immunoglobulin; PBS,
phosphate-buffered saline; PP, Peyer's patch.
Received for publication October 13, 1998.
Revision received December 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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