Role of the Glycocalyx in Regulating Access of Microparticles to Apical Plasma Membranes of ...

15 Pages·2003·4.11 MB·English

Role of the Glycocalyx in Regulating Access of
Microparticles to Apical Plasma Membranes of Intestinal
Epithelial Cells: Implications for Microbial Attachment and
Oral Vaccine Targeting
By saerdnA ,yerF neraK .T ,acsannaiG drahci.,P ,niztleW
.JluaP ,acsannaiG Hubert ,oigge.R enyaW .I ,recneL
dna nairaM R. artueN
From the Department of ,scirtaideP Harvard Medical School and GI Cell Biology ,yrotarobaL
Children's Hospital, Boston, Massachusetts 02115
D
Summary o
w
n
lo
Transepithelial transport of antigens and pathogens across the epithehal barrier by M cells may ad
e
be a prerequisite for induction ofmucosal immunity in the intestine. Efficient transport of anti- d fro
gens and pathogens requires adherence to M cell apical surfaces. Couphng of antigen-contain- m
h
ing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed sa a ttp
means for increasing antigen uptake because the CTB receptor, ganglioside ,1MG si a glycohpid ://rup
re
present in apical membranes of all intestinal epithehal cells. To test the accessibility of entero- ss.o
cyte and M cell membrane glycohpids to ligands in the size ranges of viruses, bacteria, and par- rg
/je
ticulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit m
/a
Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to rticle
apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, -p
d
28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent f/18
4
microparticles (final diameter, 1.13 )m~p failed to adhere to enterocytes or M cells in vivo or to /3/1
0
well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound 45
/1
specifically to 1MG on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that 10
8
lacked brush borders and glycocalyx. Measurements of giycocalyx thickness by electron mi- 74
9
croscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of /10
4
5
1-~m microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithe- .p
d
lial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, f b
y g
and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system. u
e
st o
n
2
5
F
E ffective immune surveillance of foreign antigens and questered intraepithelial spaces and to the underlying lym- eb
ru
pathogens on the intestinal mucosal surface requires phoid tissue (1, 2). Antigen-sensitized, IgA-committed ary 2
transepithelial transport across the epithelial barrier to spe- lymphoblasts proliferate in these inductive sites and eventu- 0
2
3
cialized sites containing organized mucosal lymphoid folli- ally "seed" local and distant mucosal tissues with IgA
cles (1). Delivery of antigens, particles, and microorganisms plasma cells that produce protective, polymeric IgA for
into these "inductive" sites si accomplished by a distinct transport into mucosal secretions (3).
epithelial cell type, the M cell, that occurs only in the lym- The ability of M cells to endocytose samples of luminal
phoid follicle-associated epithelium (FAE) .1 M cells are contents has been exploited by microbial pathogens that
specialized for endocytosis and vesicular transport into se- use this cell sa an invasion route by selectively adhering to
M cell apical surfaces (for a review see reference 4). Selec-
tive, efficient M cell transport si also considered a desirable
snoitaiverbbA~ desu ni this :repap Av-P, ,detaoc-nidiva ,deifidom-yxobrac feature ofmucosal vaccines, a concept supported by the ef-
der tnecseroulf xetal ;selcitrap Bc-P, nitycoib ;selcitraporcim CTB, chol- fectiveness of live, genetically engineered, attenuated vac-
are toxin B ;tinubus CTB-P, detaoc-BTC ;selcitrap ,AEE sumynouE -orue cine strains of pathogens that enter the mucosa via M cells
sueap ;ninitulgga ,ME electron ;ypocsorcim ,EAF detaicossa-elcillof epi- (5, 6). The M cell surface characteristics that account for
;muileht FBBG, sutnemalif brush border ;xylacocylg ,^N ordagovA
this selectivity are unknown, however, and M cell targeting
;tnatsnoc ,AFP ;edyhedlamrofarap ,Ip cirtceleosi point; PLP, enerytsylop
xetal ;selcitraporcim P.T, room ;erutarepmet ,SBBNS cificeps number of of nonliving vaccines has proven difficult because there si
gnidnib-nitoib ;setis ,ASS cificeps ecafrus .aera little information available concerning the apical membrane
1045 j. Exp. Med. (cid:14)9 The Rockefeller University sserP (cid:12)9 0022-1007/96/09/1045/15 $2.00
Volume 481 September 1996 1045-1059
components that might serve as potential receptors on this success or failure of CTB-targeted mucosal vaccine parti-
cell type. One approach has been to package antigens in cles.
microparticles (7) since this provides protection from intes- To test the accessibility of intestinal epithelial cell mem-
tinal enzymes and takes advantage of the fact that M cells branes to particulate antigens, we studied the effect of par-
can endocytose particles up to several microns in diameter, ticle size on the ability of CTB to bind to 1MG on M cells
whereas enterocytes cannot (8, 9). Although some micro- and enterocytes. Ganglioside 1MG has been demonstrated
particles and liposomes have been shown to adhere to mu- to be the only receptor for cholera toxin in diverse cell
cosal surfaces by hydrophobic interactions and to be taken types (25) including enterocytes of rabbit small intestine
up into mucosal lymphoid tissue (10, 11), uptake is gener- (26) and enterocyte-like intestinal cell lines (27). The car-
ally inefficient because such particles are readily entrapped bohydrate head groups of 1MG protrude only 2.5 nm above
in mucus gels and many fail to reach the mucosa. For opti- the surface of the membrane lipid bilayer (28), and the 1MG
mal uptake efficiency, macromolecules or particles should binding sites in CTB pentamers are 2.3-nm-deep cavities
mimic M cell-invasive pathogens: they should be coated (deduced from its homologue, heat-labile Escherichia coli en-
with a ligand that allows passage through mucous gels and terotoxin B subunit; 29). Thus, to bind to GM1, CTB must
selective adherence to M cells. come into very close contact with the lipid bilayer. Our
The nontoxic, pentameric binding (B) subunit of cholera data show that the accessibility of 1MG to CTB is dramati-
toxin (CTB) has been successfully used to target antigens cally altered by immobihzation of the ligand on particles,
to mucosal surfaces. CTB does not bind to mucins but and that particle size determines whether the CTB binding Do
w
binds specifically to ganglioside GM1, a glycolipid present in is ubiquitous, restricted to M cells, or abolished. nlo
a
membranes of all cells (12), including apical membranes of de
d
intestinal epithelial cells (13). Binding of CTB is not M cell fro
m
specific: indeed, binding and endocytosis of CT by entero- Materials and Methods h
ttp
cytes results in the well-known secretory effect of cholera Animals and Cell Lines. Female New Zealand White rabbits ://ru
holotoxin (13). Nevertheless, mucosal immune responses weighing 1.4-3.8 kg were purchased from Pine Acres (Norton, pre
to soluble protein antigens can be dramatically altered by MA) or Charles River Laboratories (Wilmington, MA). The ss.o
rg
conjugation to CTB (14). On this basis it has been sug- BALB/c 3T3 fibroblast cell line clone A31 was obtained from the /je
m
gested that M cell-specific uptake and optimal immune re- American Type Culture Collection (tkockville, MD) and clone /a
sponses might be achieved by coating antigen-containing ,2eBB2-ocaC derived from the Caco-2 human adenocarcinoma rticle
cell line, was a gift from Dr. Mark Mooseker (Yale University, -p
microparticles with CTB since the particulate carrier d
would prevent endocytosis by enterocytes and allow en- New Haven, CT). f/184
Reagents and Particles. CTB and its FITC and biotin conju- /3
docytosis by M cells. This approach, however, would re- /1
gates (CTB-FITC, CTB-biotin) were purchased from List Biolog- 04
quire that the CTB particle complex maintain 1MG gangli- ical Laboratories Inc. (Campbell, CA). Avidin-coated, carboxy- 5/11
oside binding capacity. modified, red or green fluorescent latex particles and biocytin 087
The apical surfaces of M cells and enterocytes differ dra- were obtained through Molecular Probes, Inc. (Eugene, OR.); 49/1
matically. Apical surfaces of enterocytes are highly differen- red fluorescent uncoated latex particles were purchased from 045
tiated structures consisting of rigid, closely packed micro- Polysciences Inc. (Warrington, PA). BSA was from Boehringer .pd
f b
villi (15) whose membranes contain highly glycosylated, Mannheim (Indianapolis, IN) and avidin was from ICN Pharma- y g
setxaplrkeesds gllyarcgoe,p rotein transmembrane enzymes mu(16c).i n In like addition, glycoproteins enterocytes that ceuticals tor Laboratories (Costa Mesa, Inc. (BurlCiAn)g.am e, All lectins CA) were except purchased for Limax from flavus Vec- uest on
form a continuous 400-500-nm-thick blanket that covers agglutinin which was from EY Laboratories Inc. (San Mateo, 25 F
the tips of the microvilli (17-19). This appears to serve as a aCnAd) . the The mouse mouse anti-human anti-human dipeptidylpeptidase sucrase-isomaltase IV mAb mAb Caco DAO 3/73 7/ ebrua
size-selective diffusion barrier that excludes particles such as 219 were kindly provided by Dr. Andrea Quaroni (Cornel1 Uni- ry 2
0
bacteria and viruses, preventing their contact with the en- 2
versity, Ithaca, NY; 30). TRITC- and FITC-streptavidin were 3
terocyte plasma membrane and impeding access to the from Molecular Probes, Inc. and peroxidase-labeled streptavidin
small inter-microvillus membrane domains involved in en- was from Sigma Chemical Co. (St. Louis, MO). FITC-labeled
docytosis (20, 21). The apical surfaces of M cells, in con- goat anti-mouse IgG was from Cappel (Durham, NC).
trast, may allow closer contact of particles and microorgan- noitaraperP of the Probes. CTB was coupled to 14 nm colloidal
isms because they generally lack densely packed microvilli, gold sol prepared by the citrate-tannic acid method, and BSA was
have broad membrane microdomains from which endocy- coupled to 5 nm colloidal gold made by the modified citrate
method (31). Colloidal gold sols were adjusted 0.5 pH units
tosis occurs, are deficient in stalked glycoprotein enzymes
above the isoelectric point (pI) of the protein, and protein was
(4, 22), and usually lack the thick filamentous glycoprotein
added in low ionic strength solution to final concentrations of 5-
coat typical of enterocytes (20). If so, CTB-coated particles
50 txg protein/ml. After 15-30 rain of stirring at 4~ the colloids
would be expected to have relatively free access to GMI re-
were stabilized by addition of BSA to a final concentration of
ceptors on M cells. On the other hand, M cells do have 0.1% (wt/vol), stirred for another 51 rain, and washed twice in
apical membrane glycoconjugates (23) and some ultrastruc- 6.7 mM Na phosphate buffer, pH 7.3, by centrifugation at 48,000
tural studies have documented thick surface coats on M (14 nm gold) or 60,000 g 5( nm gold) for 60-90 rain. The washed
cells (24). Whether glycolipids are accessible or masked on colloids were stored at 4~ for up to 1 wk or in 50% (vol/vol)
M cell membranes could be the determining factor in the glycerol at -20~ for longer periods.
1046 Barrier Function of Intestinal Cell Glycocalyx
Table 1. Dynamic Light gnirettacS Data were derived from their diffusion coefficients in solution, which
were used to calculate hydrodynamic diameters and molecular
D* DH :~ SrM masses. The diffusion coefficients of the protein components
were determined by dynamic light scattering in aqueous solution
at 780 nm. Since the solubility of CTB-biotin (<1 mg/ml in 20
10-13m2/s mn kD
mM Na phosphate buffer, pH 7.5) was below the detection limit
CTB 634 + 22 7.4 + 2.4 70.1 + 7.2 of the light scattering device, CTB was analyzed in place of
CTB-FITC 722 (cid:127) 26 6.4 (cid:127) 1.6 51.5 (cid:127) 4.8 CTB-biotin. The proteins were dissolved in 40 mM Na phos-
Avidin 662 (cid:127) 17 7.0 + 1.4 63.5 (cid:127) 4.3 phate buffer, pH 7.5 (CTB, GTB-FITC, BSA), or PBS (avidin)
at concentrations of 2 (CTB, CTB-FITC) or 5 Izg/ml (avidin,
BSA 613 (cid:127) 9 7.2 (cid:127) 1.4 66.3 + 2.2
BSA), centrifuged twice at 13,000 g for 10 rain, filtered through a
20-nm Anotop 10 filter (Whatman, Hillsboro, OR.), and injected
Data are given sa mean values +- SD, calculated from 6 to 91 indepen- into a dp-801 molecular size detector (Biotage, Inc., Charlottes-
dent experiments. ville, VA) equipped with a personal computer. Data were re-
*Diffusion coefficient at 24~ (297.15 K) (CTB, CTB-FITC, avidin) trieved and analyzed using the Biotage data logging software
or at 21.7~ (294.85 K) (BSA) in 40 mM Na phosphate buffer, pH 7.5 package and the QuattroPro 2.0 program (Borland International,
(CTB, CTB-FITC, BSA), or PBS (avidin), sa determined by dynamic Scotts Valley, CA). The dynamic light scattering data are summa-
light scattering. rized in Table .1 The calculated hydrodynamic diameters of avi-
*Hydrodynamic diameter, calculated from the Stokes-Einstein equa-
din and CTB pentamers were almost identical to those deter-
tion: D H = ,Dq~rl"6/Tk2 where k si the Boltzmann constant, T si the D
absolute temperature, 1~ si the viscosity of water (1.019 X 01 -3 mined crystallographically (33, 34), and their molecular masses own
calculated from diffusion coefficients were similar to those re- lo
Nsm-2), and D si the diffusion coefficient. a
d
Molecular mass, calculated from D H by the empirically determined al- ported by others (12, 35). This confirmed the globular shape of ed
gorithm for globular proteins: M r = (0.7745 X DH) .624"2 avidin and CTB in solution, and allowed us to use a "tight pack- fro
m
ing on a sphere" model for calculations ofligand number, protein h
lraeysecr ent thickness, microparticle and final probes. particle size of the colloidal gold and fluo- ttp://rup
re
CTB-P were prepared by coupling biotinylated CTB to avi- The diameters of the colloidal gold particles were determined ss.o
din-coated, carboxy-modified, red fluorescent latex parfcles (Av-P) on EM photographs of dispersed gold sols and tissue sections as rg/je
of 1 m~b nominal size. To avoid aggregation of Av-P and CTB- described previously (36). The gold particles used for coating m
/a
bloiaodt in, was both calculated of which according are multivalent, to the modelt he maximum described CTB below surface and wreistpehct ively. CTB and The BSA diameters were 14.0 of all _-- latex 1.3 nm particle and 5 cores, nm in the diameter, specific rticle-p
d
coupling was done with CTB-biotin in eightfold molar excess. surface area (SSA), and the specific number of biotin binding sites f/1
8
Thus, particles and CTB-biotin were mixed to final concentra- (SNBBS) for Av-P and the parent particles of Bc-P were pro- 4/3
tions of 0.4% (vol/vol) particles and 400 ~g/ml CTB-biotin in 1 ml vided by the manufacturers: polystyrene latex microparticles (PLP /10
4
20 mM Na phosphate buffer, pH 7.5, 30 mM NaCl, and 400 IxM diameter 0.99 Izm --- 4.4%); Av-P (diameter 1.09 Ixm --- 5.3%, 5/1
1
NAN3. The particle/protein mixture was rocked at 1.5 rpm for 72 h SSA 52,176 cm2/g, SNBBS: 1.39 nmol/mg); and parent particles 08
7
at 4~ in the dark, centrifuged at 500 g, and the supernatant was of Bc-P (diameter 0.977 ham -__ 2.6%, SSA 58,311 cm2/g, SN- 49
removed. Particles were repeatedly washed by resuspension in BBS 1.79 nmol/mg). /104
5
PBS containing 250 b~g/ml gentamicin and centrifugation, until To calculate the final diameters and ligand loads of the particu- .pd
fsrteer eptavidin-peroxidase CTB-biotin was no as longert he detection detectable system by GM1-ELISA (32). The result- using lsapthee re probes, (gold it or was latex assumed core) covered that the completelpy robe consists by one of a or central more f by gu
e
ing CTB-P were stable for at least 3 mo at 4~ layers of tightly packed small spheres, i.e., globular protein mole- st o
Euonymus sueaporue agglutinin (EEA)-coated microparticles cules (avidin, GTB, BSA, or EEA). In case of a multilayer, each n 2
5
(EEA-P) were prepared by mixing Av-P and an estimated eight- protein molecule of the outer layer would be positioned in the Fe
b
fold molar excess of EEA-biotin to final concentrations of 0.4% triangular gap formed by three occupants of the underlying layer. ru
a
(vol/vol) particles and 875 Izg/ml EEA-biotin in 1.2 ml PBS Assuming an avidin/biotin binding ratio of 1:4, the number of ry 2
containing 5 mM Hepes, 3 mM NAN3, 50 ~M CaCl ,2 and 5 laM avidin layers (r/layer) on Av-P and the parent particles of Bc-P was 023
MnCl .2 Control biocytin microparticles (Bc-P) were prepared by calculated by the equation:
mixing excess biocytin with avidin-coated, carboxy-modified,
green fluorescent latex microparticles of 1 ,al m nominal size at fi- 3f~ SNBBS
reyaln = -~- (cid:141) N A X ~ X nidivad 2 (for dparticle>>davidin ) (1)
nal concentrations of 0.4% (vol/vol) particles and 1 mM biocytin
in 1.5 ml PBS. Coupling and washing conditions were the same
as described for CTB-P. Microparticles were resuspended in PBS where N a is the Avogadro constant and davidin is the hydrody-
containing 250 Izg/nfl gentamicin, quantitated in a hemocytome- namic diameter ofavidin. Using equation )1( r/layer was calculated
ter, adjusted to 5 (cid:141) 901 particles/ml, and stored at 4~ in the to be 1.6 for Av-P (monolayer plus 60% filled second layer) and
dark. 1.95 for the parent particles ofBc-P (,~double layer). Since a par-
noitanimreteD of eborP retemaiD dna dnagiL .daoL As the final tially filled avidin layer has the same effective thickness sa a com-
hydrodynamic diameters of protein-particle conjugates could not pletely filled layer, there must be two avidin layers for both parti-
be analyzed directly, the diameters of the soluble and solid probe cle types. The total thickness (or height) of the avidin coats )~aoch(
components were measured separately and the final sizes and was then calculated by the equation:
ligand loads were calculated on the basis of these measurements.
The diameters of the solid particles were readily determined by
hcoat=(l+(nlayer-1)(cid:141)215 (for >~reyaln 1)(2)
electron microscopy (EM). The sizes of the protein components
1047 Frey et al.
To obtain the final probes, Av-P were reacted with excess CTB- tion of 25% (wt/vol) urethane in PBS (10 ml/kg), and the intes-
biotin or EEA-biotin, whereas the colloidal gold was coated with tines were exposed by laparatomy. For in vivo studies, three to
CTB or BSA. Under the conditions used we assumed that only five jejunal/ileal segments 2-3 cm in length and containing a
one layer of these proteins could bind and the thickness of the Peyer's patch were ligated, and 500 ID of probe solution was in-
added monolayer was identical to the diameter of the protein jected into the lumen. Probes applied in vivo included CTB-
molecule. Thus the total hydrodynamic diameter hd of a particu- FITC at 1 mg/ml, CTB-P, Av-P, and PLP at 901 particles/ml,
late probe is described by the equation: and a 1:1 mixture of CTB-P and Bc-P at 5 (cid:141) 801 particles/ml
each, all in PBS containing 50 p~g/ml gentamycin. The ligated
loops were returned to the abdominal cavity and excised 60 min
d h = elcitrapd + 2 x )3( later. Loops were opened and mucosal surfaces washed exten-
.=
sively with cold PBS. The entire tissue was immersed in freshly
where dparticle is the particle diameter as measured by EM, taoch is depolymerized 3% (wt/vol) paraformaldehyde in PBS (PFA-
the total height of a particular protein coat, i is the summation in- PBS), and mucosal samples were dissected in fresh fixative.
dex, and m is the total number of protein coats coupled onto the Colloidal gold probes aggregated rapidly after injection into li-
particle surface. As the particle core diameters and the hydrody- gated loops in vivo. Thus, these probes were applied to mucosal
namic diameters of the proteins were measured independently, explants ex vivo. Jejunal/ileal segments containing Peyer's patches
the Gaussian error propagation rule applies and was used for the were excised from anesthetized rabbits, the mucosal surface was
calculation of the final standard deviations. rinsed with PBS, the muscularis externa was stripped off, and the
Us!ng the final hydrodynamic diameters of Av-P, colloidal mucosa was cut into pieces of 3 (cid:141) 3 (cid:141) 1 mm. Mucosal explants
D
gold, and the protein ligands, the ligand load on the surface of a were placed in 100 p,l of oxygenated HBSS containing 0.5% (wt/ ow
n
particle, ,~kuceon,n was calculated using the equation: vol) BSA and 10 ODs50 of CTB-gold or BSA-gold. After incuba- loa
d
tion at room temperature (RT) for 1 h, the tissues were rinsed in ed
PBS and fixed in a solution containing 2.5% (wt/vol) glutaralde- fro
m
elucelomn s ---- ~ X pd r~ + i )4( hyde, 2% (wt/vol) formaldehyde, 4 mM CaCI ,2 and 2 mM h
ttp
MgC12 in 0.1M Na cacodylate buffer, pH 7.4. For application of ://ru
where dpartid e is the particle diameter (including the height of the CTB-FITC, explants were placed in 200 lxt oxygenated, high pre
avidin coat in the case of Av-P) and ietorpd n is the hydrodynamic glucose (25 raM) DME (GIBCO BRL, Gaithersburg, MD) con- ss.o
diameter of the ligand. Final probe characteristics are summarized taining 250 Ixg/ml CTB-FITC and incubated for 30 or 45 rain at rg
/je
in Table 2. 15~ in the dark. Explants were then washed five times with 1.5 m
/a
fasted Application overnight of seborP (water ot ad Rabbit libidum), Intestinal anaesthetized Mucosa. Rabbits by i.p. injec-w ere vmoll ving PBS rabbaintsd fixed were immediately performed in in PFA-PBS.ac cordance All with procedures the Guide- in- rticle-p
d
f/1
8
4
/3
/1
0
Table 2. Physical and Chemical Properties of the seborP 45
/1
1
0
8
Probe Size* Mean (cid:127) SD CTB load* Surface property~ 74
9
/1
0
4
5
nm eborp~selucelom .p
d
CTB-Probes f by g
u
CTB-FITC 6.4 + 1.6 1 Hydrophilic, low positive charge pI"-~7.8 est o
CTB-gold 28.8 + 5.0 ~30 Hydrophilic, low positive charge pI~7.8 n 2
5
CTB-P 1,130.0 + 58.0 ,'-'80,000 Hydrophilic, low positive charge pI~7.8 F
e
b
Control Probes rua
BSA-gold 19 llA n/a Hydrophilic, low negative charge pI~5.3 ry 2
0
2
Av-P 1,115.4 ___ 58.0 n/a Hydrophilic, high positive charge pD~ 3
Bc-P 1,002.0 (cid:127) 26.0 n/a Hydrophilic, high positive charge pI"-'10.5
PLP 990.0 (cid:127) 44.0 n/a Hydrophobic, not charged
EEA-P ~1,130.0 +- 60.0(cid:1)82 n/a Hydrophilic, high negative charge pI~4.5
All computations are based on the model of dense packing of globular proteins on the particle surfaces and were calculated sa described in Materials
and Methods.
* Stokes diameter. Computed from the diameter of the particle core sa determined by EM and the hydrodynamic diameter of the proteins of the sur-
face coats sa measured by dynamic light scattering (Table .)1
~:Number of CTB molecules per individual particle. Computed from the diameter of the particle core sa determined by EM and the hydrodynamic
diameter of the proteins of the surface coat sa measured by dynamic light scattering (Table .)1
~Hydrophobicity, -philicity, and presumed charge of the probe surface at pH 7.3-7.5. In brackets, pI of the proteins displayed on the probe surface.
ploT B 7.8 (12); plBs^ 5.3 (51); plavidin 10.5 (52); plEE A 4.3-4.7 (45).
oNII standard deviation saw calculated for the size of the gold particle core.
(cid:1)82 size of the EEA-P which was prepared from the same particle stock sa the CTB-P.
n/a, not applicable.
1048 Barrier Function of Intestinal Cell Glycocalyx
lines for Animal Experimentation established by Harvard Medical were then washed three times, fixed, washed again, and mounted
School and Children's Hospital. on glass slides as described above. Some cell monolayers that were
cipocsorciM Analysis of Probe Binding dna Cell Smface -cetihcrA exposed to rnicroparticles were subsequently labeled with soluble
ture. For analysis of the binding and uptake of fluorescent pro- CTB, lectins, or antibodies as described below. In this case, fixed
teins and microparticles in mucosal tissues, samples were prepared cells were incubated for 51 min in 2 ml of 50 mM NH4C1 in PBS
for cryostat sectioning. Small tissue blocks were soaked for 2 h in to quench free aldehydes and washed again before staining. In
15% (wt/vol) sucrose in PBS followed by infiltration for 10 rain some experiments with EEA-P, the coverslips were fixed and
in OCT compound (Miles Scientific, Naperville, IL). They were quenched before exposure to microparticles.
mounted in Cryo-Gel embedding compound (Instrumedics, gnilebaL of Cultured Cells with Soluble Toxin, Lectins, dna Anti-
Hackensack, NJ), frozen rapidly on the cryostat quick freezing .seidob The microparticle-labeled, fixed and quenched Caco-2
holder, and 4-8-p~m sections were cut at -18 to -20~ in a Mi- cells were incubated in 0.2% (wt/vol) gelatin (Sigma Chemical
notome cryostat (International Equipment Company, Needham, Co.) in PBS for 30 min at R.T to block nonspecific protein bind-
MA). Sections were mounted on glass slides with Moviol (Calbio- ing sites, and stained with either 10 p~g/ml lectin-FITC conju-
chem-Novabiochem Corp., San Diego, CA) containing 2.5% gates, 10 Ixg/ml CTB-FITC, or 1:100 dilutions of antibodies in
(wt/vol) 1.4-diazabicyclo-2.2.2octane (Sigma Chemical Co.) PBS/gelatin for 15-16 h at 4~ Cells labeled with antibodies
(Moviol-DABCO) and photographed with a Zeiss Axiophot mi- were washed three times for 10 rain in PBS/gelatin and stained
croscope (Carl Zeiss, Inc., Thomwood, NY) equipped for epifluo- with 2 p,g/ml FITC-labeled goat anti-mouse IgG for 90 rain at
rescence using T-Max 400 film (Eastman Kodak, Rochester, NY). RT. Coverslips were washed, mounted, and examined as de-
For EM of tissues exposed to colloidal gold probes, glutaralde- scribed above. In some experiments, washed, fixed, and quenched
D
hyde/formaldehyde-fixed rabbit Peyer's patch mucosal samples Caco-2 cells not exposed to microparticles were labeled with 10 ow
n
were processed as previously described (20, 21). For EM visual- p,g/ml biotinylated lectins, stained with 10 ~g/ml streptavidin- lo
a
d
ization of cell surface glycocalyx, rabbit Peyer's patch tissue and FITC or -TRITC, and processed as above. e
d
2~BB2-ocaC monolayers were fixed by a simultaneous osmium- Analysis of elcitraporciM ecnerehdA dna Uptake. Uptake of fluo- fro
m
glutaraldehyde procedure described by Bye et al. (24). Ultrathin rescent microparticles by the FAE of rabbit Peyer's patches was h
ttp
sections were stained with uranyl acetate and lead citrate, and ex- quantitated by counting particles in 8-~m cryostat sections of ://ru
amined with a 100CX electron microscope 0EOL, Peabody, MA). mucosal tissue. To avoid artifactual displacement of free luminal p
re
Cell Culture. BALB/c 3T3 fibroblasts were grown in high microparticles during sectioning, mucosal surfaces were vigor- ss.o
glucose (25 mM) DME supplemented with %01 (vol/vol) calf se- ously washed before fixation so that luminal material and loosely rg
/je
rum (Hyclone Laboratories, Inc., Logan, UT), 2 mM glutamine, adherent particles were eliminated. Only particles that were in di- m
/a
25 100 mM U/ml Hepes, penicillin/100 and 3.7 g/liter p~g/ml NaHCO3 streptomycin (Sigma (GIBCO Chemical BR.L), Co.) ruenctd erlying contact lymphowiidt h the FAE,f ollicle, or werleo cated counted. within the For epitheliumea ch Peyer's or rticle-p
d
at 37~ in a humidified atmosphere containing %01 (vol/vol) patch, an average of 65 representative follicle sections were quan- f/1
8
CO .2 2~BB2-ocaC cells were cultured in the same medium devoid titated, and microparticle uptake was expressed as the average 4/3
of Hepes but with the addition of 10 txg/ml human transferrin number ofmicroparticles per section of follicle for a given Peyer's /10
4
(Boehringer Mannheim) in a humidified atmosphere containing patch. Microparticle binding to cultured fibroblasts and Caco-2 5/1
1
5% (vol/vol) CO .2 For immunocytochemical and particle bind- cells was quantitated from photographs of at least three randomly 08
7
ing studies, 3T3 fibroblasts or Caco-2 cells were seeded onto selected nonoverlapping regions of each cell monolayer viewed 49
round (13-mm diameter) glass coverslips (Bellco Glass, Vineland, en face. Microparticle binding was averaged for each experiment /10
4
5
NJ) in 24-well tissue culture plates (Costar, Cambridge, MA). and expressed as particles per mm .2 .p
d
The fibroblasts were used for experiments at 12-13 d (6-7 d after .scitsitatS Statistical analysis of microparticle binding and up- f b
confluence) and the Caco-2 cells at 24 d 12( d after confluence). take studies was performed on a Macintosh Ilsi computer (Apple, y gu
e
noitacilppA of CTB and Lectin elcitraporciM seborP ot stsalborbiF Cupertino, CA) using the StatView II program (Abacus Concepts, st o
dna lanitsetnI Cells In Vitro. BALB/c 3T3 fibroblasts were washed Berkeley, CA). Mixed probes of test and control particles, and n 2
5
gently five times with 2-3 ml prewarmed (37~ PBS containing differentiated and undifferentiated areas of Caco-2 cells, were F
e
b
0.9 mM CaC12 and 0.5 mM MgC12 (CM-PBS), and 500 1~ of a considered paired samples. The results of independent experi- ru
a
probe mixture was added to each well of the 24-well plates. Mi- ments were treated as unpaired samples. Differences in binding or ry 2
croparticles were applied as 1:1 mixtures of 5 (cid:141) 701 particles/ml uptake among particle types at a significance level of/>95% were 02
3
each of CTB-P (or Av-P) and Bc-P in DME containing 50 p,g/ml calculated by two-tailed Student's t test.
gentamicin. To test competition of CTB-P binding by free CTB,
coverslips were preincubated for 5 rain with 250 1,p of 2 or 20
Ixg/ml CTB in DME before addition of 250 1*I of 1:1 mixtures of
Results
01 s particles/nil each of CTB-P and Bc-P in DME. After incuba-
tion for 60 rain at 37~ coverslips were washed three times for Characterization of the Probes. To test the accessibility of
10 rain by gently adding and aspirating 2 mi CM-PBS, fixed in intestinal cell membrane glycolipids to particulate ligands in
1.5 ml PFA-PBS for 2 h, washed in PBS followed by distilled wa-
the size ranges of viruses, bacteria, and particulate mucosal
ter, and mounted on glass slides with Moviol-DABCO. Cell
vaccines, CTB was used as model ligand in the form of
monolayers were examined and photographed en face with a Zeiss
probes of three distinct sizes: CTB-FITC, CTB-colloidal
Axiophot microscope equipped for epifluorescence using Kodak
gold and red fluorescent CTB-P. Nonadherent BSA-gold
T-Max 400 film.
Caco-2 cells on coverslips in 24-well plates were washed sa served as negative control for CTB-colloidal gold. As neg-
above. 500 l~p of DME containing 10 s CTB-P or EEA-P/ml, 50 ative control for CTB-P we used the parent red fluorescent
Izg/ml gentamicin, and 01 p~g/ml transfemn were added to each avidin microparticles from which the CTB-P were pre-
well, and plates were incubated for 60 min at 37~ Coverslips pared (Av-P), or when a clearly discernible internal control
1049 Frey et al.
Figure 1. Binding of CTB-
FITC to EAF and epithe- villus
lium of rabbit Peyer's patch
mucosa. Mucosal tissues were
exposed to 052 ~g/ml CTB-
FITC for 54 rain at 15~ vivo ex
,)A( or to 1 mg/ml CTB-FITC
for 06 min in vivo Cryostat (B).
snoitces were viewed by fluores-
cence microscopy. )A( Soluble
CTB-FITC labeled the entire
EAF which sniatnoc both M sllec
and enterocytes. )B( CTB-FITC
delebal eht secafrus of setycoretne
on most villi, although certain
saera were devoid of (tip label of
fight .)sulliv Bar, 05 .m,p
for CTB-P binding was required, we used green fluores- CTB-colloidal gold (total diameter 28.8 nm) for 1 h at RT
cent biocytin-quenched avidin microparticles (Bc-P) of ap- ex vivo, and analysed by EM. Many samples showed gold D
o
proximately the same size as CTB-P. As positive controls particles caught in adherent mucus but no probe on mu- wn
lo
we used microparticles coated with EEA-P that were pre- cosal surfaces. In areas where adherence of CTB-gold oc- ad
e
d
pared from Av-P, and uncoated, hydrophobic PLP. curred, it was selective for M cells (Fig. 2 A). On these fro
The probe properties that were of particular importance cells, CTB-gold bound to the entire apical plasma mem- m h
afcocerss this to study membrane were hydrgolydcyolnipaimdsi, c surface diameter, charge/hydropho- which affects bgroalnde particles including were microviUi, also present microfolds, in coated and and coated uncoated pits. CTB- ves- ttp://rup
re
bicity, which affects nonspecific binding, and ligand load, icles, implying that endocytosis had occurred (Fig. 2 B). In ss.o
which affects the avidity of specific binding (Table 2). We contrast, as shown in previous studies (36), BSA-gold failed rg
/je
assumed that the pI of the protein forming the outermost to adhere to any epithelial ceils on villi or in the FAE in m/a
layer of the probe determined the surface charge, and that spite of its smaller diameter (data not shown). Thus, when rticle
protein coats would be hydropbilic and uncoated polysty- administered as particles <30 nm in diameter, CTB had -pd
rene latex would be hydrophobic. The ligand load of each access to its ganglioside receptor on certain M cells but not f/18
4
probe is dependent on the probe size and ranged from 1 pen- on enterocytes. /3/1
0
tameric CTB molecule/probe for CTB-FITC to ~80,000 CTB Coupled to Micropartides Does Not Have Access ot Api- 45
/1
CTB molecules/probe for CTB-P. However, the potential lac Membrane Glycolipids of Intestinal Epithelial Cells In Vivo. 10
8
positive effect of ligand load on binding avidity was offset The latex particle-based probes (such as CTB-P, diameter 74
9
by the negative effect of large particle size on adherence, as 1.13 ~m) carried fluorescent markers that allowed us to /10
4
5
shown below. count individual microparticles on or in mucosal tissue. Pi- .p
d
Soluble CTB Has Access ot Apical Plasma Membranes of All lot experiments showed that uptake of CTB-P varied f by g
Intestinal Epithelial Cells In Vivo. Rabbit Peyer's patches were widely among Peyer's patches, even in the same rabbit. To ue
exposed to 1 Ixg/ml CTB-FITC (diameter 6.4 nm) 1 h in determine whether this variation was specific to the CTB-P st o
n
vivo or to 250 Ixg/ml CTB-FITC ex vivo for 30 or 45 rain probe, we tested uptake of uncoated, uncharged, hydro- 25
F
at 15~ to retard endocytosis. Analysis of r sections phobic latex particles (PLP) that had previously been eb
ru
from multiple Peyer's patches from two rabbits showed shown to bind avidly to mucosal surfaces and to be readily ary 2
that randomly distributed areas of mucosal surface were la- transcytosed by M cells (10). These were applied to multi- 0
2
3
beled but other areas were unlabeled, presumably due to ple Peyer's patches of a single rabbit and to single patches of
adherent mucus that was not removed. Within the labeled different rabbits, by injection of 5 X 108 PLP into ligated
areas, all epithelial cell surfaces of the FAE showed CTB- loops and incubation for 1 h. Washing conditions were
FITC binding (Fig. 1 A), although staining intensity varied stringent enough to remove all loosely adherent particles
from cell to cell, Similarly, the brush borders of absorptive from the mucosal surface, leaving only those that were
enterocytes on villi, both in Peyer's patches and in other tightly attached or that had been taken up into the tissue.
regions, bound soluble CTB-FITC whether exposed in li- Counting of surface-attached and endocytosed particles
gated loops at 37~ or in mucosal explants at 15~ (Fig. 1 B). confirmed that endocytic activity was relatively unifoml in
These data indicate that whereas mucus or other factors all 15-20 FAE of a given Peyer's patch, but that entire
may have impeded contact of probes with mucosal surfaces, patches varied widely in their endocytotic activity. The
once these luminal diffusion barriers were breached, plasma "high uptake patches" showed avid binding and significant
membrane 1MG of both enterocytes and M cells was acces- uptake of PLP in almost all domes, whereas in the "low
sible to CTB-FITC. uptake patches," the majority of the domes remained unla-
CTB Coupled ot Colloidal Gold Particles Binds Selectively ot beled and uptake was very low, most likely because these
M Cells. Rabbit Peyer's patch explants were exposed to patches were coated with mucus.
1050 Barrier Function of Intestinal Cell Glycocalyx
D
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rticle
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8
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Figure 2. Selective binding of CTB-colloidal gold to rabbit Peyer's ru
a
to CTB-gold h were at exposed for 1 explants Mucosal M cells. patch tkT ry 2
in vitro. )A( EM sisylana that revealed CTB-gold adhered exclu- almost 02
3
to the sively apical secafrus of had enterocytes M adjacent whereas cells,
few with gold associated particles their microvilli. )B( In M gold cells,
were particles present in clathrin-coated pits indicating and vesicles en-
transport. and docytosis Bar, 004 .mn
To eliminate the confounding factor of variability 4 A). Analysis of the high uptake patches revealed that
among Peyer's patches, a 1:1 mixture each of 2.5 (cid:141) 10 8 red CTB-P were taken up even less efficiently than Bc-P and
fluorescent CTB-P and green fluorescent control Bc-P was that both protein-coated particles were endocytosed less ef-
applied to ligated loops for 1 h. Examination of cryostat ficiently than the PLP control (Fig. 4 /3). The preferential
sections revealed that both types of particles adhered in uptake of Bc-P over CTB-P was consistent for every patch
small numbers and were endocytosed, but CTB-P were no at a ratio of 1.8 Bc-P:I CTB-P (correlation coefficient
better than control Bc-P (Fig. 3). Statistical analysis of par- 0.988). These data indicate that the CTB ligand immobi-
ticle uptake for a total of 15 patches, normalized to 2.5 (cid:141) lized on particles/>1 btm in diameter did not have access to
108 particles per loop, showed that patches were either high glycolipids in the apical plasma membranes of either en-
uptake or low uptake for all types of particles analyzed (Fig. terocytes or M cells, and that other factors such as ionic in-
1051 Frey et al.
Figure 3. Simultaneous uptake of CTB-
coated and control biocytin-quenched
microparticles into rabbit Peyer's patch
domes. Peyer's patch mucosa saw exposed
to equal numbers (2.5 X 01 s particles) of
red fluorescent CTB-P and green fluores-
cent control Bc-P for h 1 in vivo. Fluores-
cence microscopy of a representative cry-
ostat section shows that both types of
particles were taken up into the dome, but
not into adjacent villi. Control Bc-P
)neerg( were taken up in greater numbers
than CTB-P ,)der( suggesting that uptake
was due to nonspecific interaction of the
cationic particles with FAE cell .secafrus D
The particles are located primarily within ow
n
the FAE, presumably associated with M lo
a
bar, Scale cells. 001 Izm. de
d
fro
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h
ttp
teractions in case of the cationic Bc-P, or hydrophobic in- ://ru
teractions in case of PLP, influenced binding and uptake of pre
these particles by M cells. ss.o
CTB Coupled ot Microparticles Binds ot GMI on Cultured Fi- rg/je
.stsalborb To rule out the possibility that the immobiliza- m/a
tion of CTB on fluorescent microparticles masked, inacti- rticle
vated, or destroyed the ligand, we tested the ability of -pd
f/1
CTB-P to bind to a fibroblast cell line known to express 84
/3
high levels OfGM1 ganglioside (25). Live 3T3 cell monolay- /1
0
4
ers were exposed to a 1:1 mixture of 2.5 (cid:141) 107 particles 5/1
1
each of CTB-P and Bc-P for 1 h at 37~ Over 38 times 0
8
7
more CTB-P than Bc-P adhered. Blocking of 1MG sites 49
/1
with free CTB reduced binding of CTB-P to control levels 04
5
(Fig. 5). This confirmed that CTB-P were capable of bind- .pd
ing to Gm on live cells via the CTB immobilized on their f by g
u
surfaces. e
CTB-P Bind ot Apical Membranes of Undifferentiated but st on
2
Not Differentiated Caco-2 Cells. Because the fibroblast mem- 5 F
e
brane is not a valid model for the highly specialized apical bru
a
domain of intestinal epithelial cells, we tested the binding ry 2
of CTB-P to polarized monolayers of Caco-2BBe2 adeno- 02
3
carcinoma cells. These cells form a columnar enterocyte-
like epithelium with well-organized brush borders when
cultured on Transwell filters (37). When cultured on glass,
however, small "islands" of flat cells that do not develop
brush borders or express apical membrane enzymes remain
Figure 4. Quantitative analysis of CTB-P, Bc-P, and PLP uptake by within the monolayer, even long after confluence (38, and
rabbit Peyer's patch domes. Uptake of three types of m*1-1 particles by Fig. 6 A). When such monolayers were exposed to 5 (cid:141) 107
the FAE saw analyzed by counting fluorescent particles in 974 domes in
CTB-P for 1 h at 37~ binding of particles was largely re-
cryostat sections of 51 rabbit Peyer's patches that had been exposed to
stricted to the undifferentiated islands (Fig. 6 C), with
2.5 (cid:141) l0 s particles each of CTB-P and Bc-P, or 5 (cid:141) 801 PLP for 1 h in
vivo. Counts are expressed sa average particles per dome section of a
given patch, normalized to application of 2.5 (cid:141) 801 particles per patch.
)A( Particles were taken up uniformly in lla domes of a given Peyer's
patch, but there saw wide variation among patches. In some patches, lla types of control particles P-cB( and PLP) saw consistently higher than that
domes showed few or no particles (Low ,)ekatpu whereas in other patches of CTB-P (*), with clear significance for CTB-P .sv Bc-P (two-tailed,
domes were heavily labeled hgih( .)ekatpu )B( Peyer's patches showed ei- paired t test, P <0.05) and borderline significance for CTB-P .sv PLP
ther low or high uptake activity for lla types of particles. Uptake of both (two-tailed, unpaired test, t P = 0.065).
1052 Barrier Function of Intestinal Cell Glycocalyx
Figure 5. CTB-mediated binding of 1 m,p particles to live BALB/c
3T3 monolayers were Fibroblast exposed to fibroblasts. 2.5 (cid:141) 701 each of
CTB-P and Bc-P, or Av-P and Bc-P, for 1 h at 37~ Particle binding
saw analyzed by fluorescence microscopy. CTB-P binding saw signifi-
cantly )*( higher than binding of the Bc-P or Av-P controls. In the pres-
ence of free CTB, binding of CTB-P saw reduced to control Data levels. Do
w
in A represent the mean values of five independent experiments (two- n
lo
tailed, paired t test, P <0.05). ad
e
d
fro
m
h
2,331 + 414 particles/mm 2 on undifferentiated cells versus ttp://ru
10 + 1 particle/mm on well-differentiated cells. To deter- p
re
mine whether this selectivity was due to differences in 1MG ss.o
expression, we exposed the monolayer to CTB-FITC after rg/je
particle binding and observed that binding of soluble CTB- m/a
FITC occurred throughout the monolayer. Indeed, CTB- rticle
FITC labeling was equal or higher on the well-differenti- -pd
ated cells than on the cells in the islands (Fig. 6 B). This f/18
4
indicates that selective binding of CTB-P to undifferenti- /3/1
0
ated Caco-2 cells was due to enhanced accessibility of 1MG 45
/1
and not simply to the presence of 1MG in the apical mem- 10
8
7
brane. 49
Apical Membrane Components of Undifferentiated and Differ- /10
4
5
entiated Caco-2 Cells. To explore the basis for selective .p
d
binding of CTB-P to undifferentiated Caco2B~e2 cells, we f by g
compared the expression of glycoproteins, including stalked ue
brush border enzymes and glycocalyx, on apical plasma st o
n
2
membranes of differentiated and undifferentiated Caco-2 5
F
cells. As shown previously (38), the undifferentiated islands eb
ru
were completely negative for both dipeptidylpeptidase IV, ary 2
an enzyme that appears early during enterocyte differentia- 02
3
tion (39), and sucrase-isomaltase, an enzyme considered a
marker of terminal differentiation (40). All "differentiated"
ceils were positive for dipeptidylpeptidase IV and ~70%
Figure 6. Specific binding of CTB-coated microparticles to undiffer-
expressed sucrase-isomaltase, but all failed to bind CTB-P. entiated Caco-2 cells in vitro. Confluent, polarized, live Caco-2 cell
This indicates that membrane surface components of par- monolayers were exposed for 60 min at 37~ to 5 (cid:141) 701 CTB-P, fixed,
counterstained with CTB-FITC and viewed en face by contrast phase )A(
tially differentiated cells were sufficient to block access of
or fluorescence microscopy B( and C). )A( Morphologically distinct is-
CTB-P to GM> lands of flat, undifferentiated cells )sdaehworra( were observed within the
Enterocyte differentiation is also accompanied by the ap- monolayers of columnar Caco-2 cells. )B( Cells in all regions of the
pearance of other highly glycosylated, membrane-anchored monolayer were labeled with CTB-FITC, but labeling intensity saw
lower within the islands of cells. flat )C( Binding of CTB-P )stod( saw re-
glycoproteins that form the glycocalyx on microvillous
stricted to the islands of flat cells. Within the particle island, binding saw
membranes (19). To monitor the appearance and oligosac- higher on that cells expressed more 1MG sworra( in B and .)C bar, Scale
charide heterogeneity of these components on differentiat- 200 .mxI
ing Caco-2 ceils, we applied a battery oflectins specific for
epitopes typical of N- and O-linked complex oligosaccha-
rides. Most of these lectins bound to differentiated Caco-2
1053 Frey et al.
cells but not to flat ceils in the undifferentiated islands, and
indicated both N-linked core glycosylation (Con A:
branched N-linked hexasaccharides [41]; Lycopersicon escu-
lentum agglutinin: oligomeric N-acetylglucosamine [42]) and
O-linked glycosylation (Vicia villosa agglutinin: terminal
N-acetylgalactosamine [43]) as well as heterogeneous, com-
plex oligosaccharides as shown in a previous study from
this laboratory (38). Cells in the undifferentiated islands
were stained only by Lotus lectin, a probe that shows
highest affinity for the N-linked disaccharide fucosyl ot(1-6)
N-acetylglucosarnine, but does not bind to that epitope
when the chitobiose core and the carbohydrate antennae of
the N-linked oligosaccharide are intact (44). Taken to-
gether, the lectin-staining data demonstrated that columnar
Caco-2 cells with well-developed brush borders displayed
abundant apical membrane glycocon]ugates with branched
complex carbohydrates and mature oligosaccharide side
chains, and that this was associated with inaccessibility of Do
w
1MG to CTB-P. In contrast, cells in the undifferentiated is- nlo
a
lands lacked mature glycoconjugates and displayed only the de
d
truncated disaccharide recognized by Lotus lectin. On fro
m
these cells, 1MG was sufficiently exposed to allow CTB-P h
ttp
binding. ://ru
selcitraporciM Have Access ot Terminal Sugars of Membrane pre
setagujnococylG of detaitnereffiD Caco-2 Cells In Vitro. The lec- ss.o
rg
tin data suggested that a particle-associated ligand directed /je
m
against peripheral components of the glycocalyx itself /a
should be able to adhere to differentiated cells but not to rticle
-p
the poorly glycosylated undifferentiated cells. To test this df/1
hypothesis, we generated 1 Ixm particles coated with lectin 84
/3
EEA that recognizes complex carbohydrate epitopes (45) /10
4
that would be expected to occupy distal positions in oli- 5/1
1
gosaccharide side chains. These microparticles bound av- 08
7
4
idly to differentiated Caco-2 cells and binding was closely 9
/1
0
correlated with the density of EEA receptors, as revealed by 4
5
.p
subsequent counterstaining with EEA-FITC (Fig. 7). In d
f b
contrast, very low numbers of particles bound to cells in y g
u
the undifferentiated islands, and binding was restricted to est o
the few cells that showed low levels of the EEA receptor. n
2
5
Cross-linking of the glycocalyx by PFA fixation before ap- F
e
plication of EEA-P exposure did not affect the binding pat- bru
a
tern, confirming that the EEA receptor occupied a periph- ry 2
0
eral position on the cell surface. Thus, microparticles of the 23
same size as CTB-P but bearing a ligand directed against a
peripheral component of the epithelial cell glycocalyx
readily bound to apical surfaces of differentiated Caco-2
cells.
larutcurtsartlU Features of the Glycocalyx of Intestinal Epithe-
ail Cells In Vivo and In Vitro. On apical brush borders of
intestinal enterocytes, a thick "filamentous brush border
Figure 7. directed Ligands tsniaga etardyhobrac sepotipe of the glyco- glycocalyx" (FBBG) coats the tips of microvilli (18, 19).
xylac binding mediate of 1 mxI selcitrap to detaitnereffid Caco-2 .sllec There are conflicting views about the presence and thick-
cell Caco-2 sreyalonom were ylfeirb desopxe ,dexif for 06 min ta 37~ to
5 (cid:141) 01 v der tnecseroulf EEA-biotin by followed EEA-P dna -nidivatperts
FITC, dna dezylana en by contrast phase face )A( dna ecnecseroulf mi-
croscopy B( dna .)C The sdnalsi of detaitnereffidnu sllec ,A( )sdaehworra dna well with correlated AEE receptor ,noitubirtsid that demonstrating
dekcal eht by recognized epitopes complex carbohydrate AEE .)B( )C( EEA-coated dah selcitrap ssecca to AEE receptor setis in the Caco-2 llec
Binding of EEA-P )stod( occurred primarily on detaitnereffid-llew sllec elacS .xylacocylg bar, 002 .mxI
1054 Barrier Function of Intestinal Cell Glycocalyx