A Mucosal IgA-Mediated Excretory Immune

System In Vivo
Janet K. Robinson, Thomas G. Blanchard, Alan D. Levine,
Steven N. Emancipator and Michael E. Lamm
This information is current as
of March 4, 2015. J Immunol 2001; 166:3688-3692; ;
doi: 10.4049/jimmunol.166.6.3688
http://www.jimmunol.org/content/166/6/3688

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A Mucosal IgA-Mediated Excretory Immune System In Vivo1

Janet K. Robinson,* Thomas G. Blanchard,† Alan D. Levine,‡ Steven N. Emancipator,* and

Michael E. Lamm2*
The capacity of mucosal IgA Abs to serve as an excretory immune system in vivo was investigated. Mice expressing a transgenic
TCR were immunized intragastrically with the cognate Ag to elicit a vigorous mucosal IgA Ab response. Soon after i.v. challenge,
Ag was detected within the epithelial cells of the small intestinal crypts and to a lesser degree within the epithelial cells higher up
the villi, paralleling the gradient in expression of the polymeric Ig receptor and the transport of its ligand, oligomeric IgA. Uptake
of Ag into the epithelial cells occurred only from the basolateral aspect and only when Ag complexed to IgA Ab could be present
in the lamina propria. The results support the concept that local IgA Abs can excrete Ags from the body by transporting them
directly through mucosal epithelial cells, using the same mechanism that transports free IgA into the mucosal secretions. The

Downloaded from http://www.jimmunol.org/ at University of Michigan on March 4, 2015

Journal of Immunology, 2001, 166: 3688 –3692.

H ow does the immune system deal with Ags that penetrate
mucosal surfaces on an ongoing basis or that are re-
leased directly into the lamina propria from microbial
pathogens during mucosal infections? Ags accessing the lamina
propria would immediately encounter Abs, principally IgA that
had been secreted by local plasma cells, which on a biosynthetic
basis account for the majority of the body’s total pool of Igs (1, 2).
One possibility for the IgA immune complexes forming in the
lamina propria would be to reach the circulation, where they could
interact with the mononuclear phagocyte system. An additional
mechanism for clearing circulating IgA immune complexes, oper-
ative in rodents, is transport by the liver into the bile (3–5) because
rodent hepatocytes express on their sinusoidal surface the poly-
meric Ig receptor (pIgR),3 the function of which is to transcytose
oligomeric IgA across epithelial cells. However, this mechanism is
not available in humans, whose hepatocytes do not express pIgR.
Moreover, even in rodents, removal of immune complexes by the
liver applies only to complexes that have reached the blood. An-
other, more direct means of removing mucosal Ags at the source,
before they reach the circulation, and one that would function in
humans as well as rodents would be to excrete them through the
overlying epithelium into the lumen by the same route and mech-
anism used to transport free IgA into the mucosal secretions. In
this process, oligomeric IgA binds via its Fc portion to pIgR, con-
stitutively expressed on the basolateral surface of mucosal epithe-
lial cells. Bound IgA is then endocytosed, transcytosed from ba-
solateral to apical, and released into the lumen as secretory IgA
(6). If this route also operates for IgA that is complexed to Ag, it
could serve as a means for quickly removing Ags from mucous
membranes, thereby minimizing the burden of immune complexes
Departments of *Pathology, †Pediatrics, and ‡Medicine, Case Western Reserve Uni-
versity School of Medicine, Cleveland, OH 44106
Received for publication October 18, 2000. Accepted for publication January 4, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported by National Institutes of Health Grants AI-26449,
AI-36359, and DK-46461, and a grant from the Crohn’s and Colitis Foundation of
America.
2
Address correspondence and reprint requests to Dr. Michael E. Lamm, Institute of
Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH
44106-4907. E-mail address: mel6@po.cwru.edu
3
Abbreviation used in this paper: pIgR, polymeric Ig receptor.
in the circulation. Another advantage of such a local excretory
route is that IgA, the major mucosal Ig, is relatively noninflam-
matory (7), whereas Ag or immune complexes reaching the blood
would more likely bind Abs of the phlogistic IgG class.
Earlier we demonstrated that soluble immune complexes con-
taining oligomeric IgA Abs were readily transported intact into the
apical medium across polarized epithelial cell monolayers that ex-
pressed the pIgR on their basolateral surface (8, 9). In other words,
the Fc␣-dependent mechanism used to transport IgA across epi-
thelial cells functioned the same for IgA that was complexed to Ag
by its Fab binding sites as it did for free IgA. Based on these
experiments in vitro we suggested that an IgA-mediated excretory
immune system could function in vivo. The present work was un-
dertaken to explore whether such an excretory immune system
indeed functions in vivo. The experimental design was to immu-
nize mice intragastrically to stimulate an intestinal IgA Ab re-
sponse, after which Ag was injected i.v. to generate soluble IgA-
containing immune complexes in the intestinal lamina propria.
Subsequent detection of Ag in small intestinal crypt cells was
taken as evidence of mucosal excretion by specific IgA Ab.
Materials and Methods
Mice
Transgenic BALB/c mice of both sexes expressing the ␣␤ TCR from a T
cell hybridoma, DO11.10, that recognizes a chicken OVA peptide (323–
339) restricted by MHC class II I-Ad (10) were obtained from Dr. Kenneth
Murphy (Washington University). They were maintained and bred in our
animal facility with food and water ad libitum. TCR transgenic progeny
were identified by PCR assays on DNA isolated from tail clips (11). An-
imal studies were approved by the Case Western Reserve University In-
stitutional Animal Care and Use Committee and were performed in com-
pliance with institutional guidelines.
Immunization
Chicken OVA (Sigma, St. Louis, MO) was coupled with biotin (Pierce
Chemical, Rockford, IL) according to the supplier’s instructions at a
weight ratio of 15% biotin. An average of seven biotins were coupled to
each molecule of protein. Mice were immunized five to six times at weekly
intervals with 10 mg biotinylated OVA or the control protein BSA (Sigma),
by stomach tube in the presence of 10 ␮g of the mucosal adjuvant cholera
toxin (12) in 0.5 ml of 0.2 M NaHCO3. IgA Ab titers in serum, measured
by ELISA, reached a maximum at this time. In selected mice, it was con-
firmed that specific IgA Ab was also secreted into the intestinal lumen. For
this, a segment of small intestine was rinsed with 5 ml of a mixture of
protease inhibitors (Complete Protease Inhibitor Cocktail; Roche, Gipf-
Oberfrick, Switzerland) in PBS (pH 7.2). Intestinal secretions collected

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

The Journal of Immunology
with polywicks (Polyfiltronics Group, Rockland, MA) (13) were assayed
for IgA Ab content by ELISA.
Measurement of Ab response
IgA Ab responses to biotinylated OVA were measured by ELISA. Nunc
Immuno Plates (Naperville, IL) were coated by overnight incubation at 4°C
with 10 ␮g/ml Ag in carbonate buffer (pH 9.5). Nonspecific binding sites
were blocked with PBS containing either 1% BSA or 40% soy milk. Serial
half-log10 dilutions of antisera (50 ␮l), made in either 1% BSA or 40% soy
milk, were added and the plates were incubated at ambient temperature for
90 min. Plates were then washed with PBS and incubated with affinity
purified, alkaline phosphatase conjugated goat anti-mouse IgA-specific Ab
(Southern Biotechnology Associates, Birmingham, AL) at ambient tem-
perature for 90 min. The plates were washed and developed with disodium
p-nitrophenyl phosphate (Sigma) in glycine buffer (pH 9.6). After 60 min,
ODs were read at 405 nm with a Molecular Devices Vmax plate reader
(Menlo Park, CA). For each serum sample, the least-squares regression of
the OD as a function of the log of the serum dilution was used to calculate
the titer. The titer was defined as the log of the dilution that generated an
OD equal to two SDs above the mean background OD developed with
nonimmune syngeneic control serum.
Ag challenge and tissue preparation
Five to seven days after the final intragastric immunization, the mice were
injected in the tail vein with 50 mg biotinylated OVA in PBS and sacrificed
30 min later. The small intestine was removed and the lumen was rinsed
with 20 ml of cold PBS containing 0.9 mM Ca2⫹ and 0.49 mM Mg2⫹. The
intestine was filled with OCT Compound (VWR, Bridgeport, NJ), coiled,
snap frozen in 2-methyl butane, and stored at ⫺70° before sectioning.
Microscopy
Cryostat sections (7– 8 ␮m) of coiled small intestine were air-dried, fixed
for 1 min in acetone, air-dried, and stored at ⫺20°. For detection of bio-
tinylated OVA Ag, sections were hydrated in PBS, exposed to avidin-
biotinylated HRP complex (Vectastain Elite ABC; Vector Laboratories,
Burlingame, CA) for 30 min, rinsed in PBS, exposed to diaminobenzidine,
and rinsed in PBS. Sections were counterstained with hematoxylin, cleared,
and sealed with a coverslip. For detection of mouse IgA, hydrated frozen
sections were exposed to peroxidase-conjugated goat Ab (Southern Bio-
technology Associates), rinsed, developed with diaminobenzidine, coun-
terstained, cleared, and sealed with a coverslip. Slides were numbered ran-
domly by one investigator and examined and scored in blinded manner by
another on a scale of 0 (no staining) to 3 (prominent, dense small intestinal
crypt cell staining easily observed at low power) by direct visual observa-
tion with a Leica DMLB microscope (Deerfield, IL). The semiquantitative
peroxidase staining intensity scores for the amount of biotinylated OVA in
the small intestinal crypt epithelium of the individual mice were subjected
to one-way ANOVA, stratified by mouse group. Post hoc comparisons
among the groups used Scheffe’s test and Fisher’s protected t test. In ad-
dition, the distribution of individual mice scored “positive” (intensity score
⬎0) or “negative” (intensity score ⫽ 0) in each group was assessed by ␹2
FIGURE 1. Schematic drawing of small intestinal
mucosa. Shown is the relationship of the epithelial cells
lining the crypts to the lamina propria (which contains
numerous IgA-secreting plasma cells, not illustrated)
and the crypt lumens, which are continuous at the crypt
openings with the main intestinal lumen. IgA Abs that
have been transcytosed across the crypt epithelial cells
enter the crypt lumens, from which they reach the main
intestinal lumen. In the current experiments, immune
complexes containing biotinylated OVA and specific
IgA Abs were present in the lamina propria and in the
epithelial cells of the crypts, where maximal pIgR-me-
diated transcytosis occurs (see Fig. 3). Arrows show the
direction of transport of IgA immune complexes from
the lamina propria across the epithelium into the crypt
lumens.
3689
contingency analysis in a two (level) by three (group) design, with Fisher’s
correction for small numbers applied. Photographs were made with a Di-
agnostic Instruments digital camera.
Results and Discussion
D011.10 transgenic mice expressing a TCR for OVA were immu-
nized intragastrically with biotinylated OVA to stimulate a muco-
sal IgA Ab response. This response, as monitored by serum IgA
Ab, reached an ELISA titer of 1600. In contrast, control transgenic
mice immunized instead with BSA did not manifest Ab to biotin-
ylated OVA (t ⫽ 5.6; p ⬍ 0.001 vs mice immunized with biotin-
ylated OVA). The transgenic mice were used because they make a
more vigorous IgA Ab response than wild-type BALB/c mice after
intragastric immunization with biotinylated OVA and, thus, are
capable of excreting more Ag through intestinal epithelial cells.
Immunized mice were then injected i.v. with a bolus of Ag to
rapidly generate IgA immune complexes in the lamina propria of
the intestinal mucosa. The complexes would then be available for
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endocytosis by the pIgR-expressing epithelial cells of the intestinal
lining. In preliminary experiments, 30 min after challenge was
found to be a suitable time to observe the effect. This short interval
also enabled the experiments to focus on the immediate interaction
of the immune complexes with preexisting epithelial cell surface
pIgR, independent of cellular immune events that might evolve
over time. Fig. 1 illustrates the excretory route schematically.
The biotinylated Ag was detected histochemically by its ability
to bind to avidin that was in turn complexed to biotinylated per-
oxidase. As shown in Fig. 2 (left-hand column vs other columns)
and Fig. 3 (A and B vs C and D) only mice that were immunized
and subsequently challenged i.v. with the same Ag (biotinylated
OVA) evidenced Ag in the crypt cells of the small intestinal epi-
thelium. Both the amount of Ag in the crypt cells and the percent-
age of positive mice were statistically significantly different from
two groups of control mice (Fig. 2). In one control, the mice were
mucosally immunized in the same manner with an irrelevant Ag,
BSA. In this case, the biotinylated OVA that was injected i.v.
before sacrifice was not complexed by specific IgA Ab and, there-
fore, was not transported through the intestinal epithelial cells. In
the second control, mice were immunized intragastrically with bi-
otinylated OVA but received no i.v. challenge before sacrifice.
Again, Ag could not be detected within the intestinal epithelium.
As discussed below, the results presented in Figs. 2 and 3 indicate
that Ag entered intestinal epithelial cells only from the basolateral
surface and only when complexed to specific IgA Ab.
3690 MUCOSAL IgA EXCRETORY IMMUNE SYSTEM

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FIGURE 2. Ag in intestinal crypt cells. D011.10 mice that had been
immunized intragastrically with biotinylated OVA or BSA were chal-
lenged i.v. 30 min before sacrifice with biotinylated OVA or nothing. The
ordinate shows the intensity of staining for biotinylated OVA within small
intestinal crypt epithelial cells in arbitrary units. Solid horizontal lines in-
dicate the mean intensity for the group and interrupted lines indicate the
SE. Mice in the group at the left made IgA Abs that complexed with the
challenge dose of biotinylated OVA Ag and excreted it through the mu-
cosal epithelium. The mean intensity (⫾SE) in the first group (1.4 ⫾ 0.3)
is significantly higher (F ⫽ 7.3; t ⬎3.5; p ⬍ 0.01) than in the second group
(0.2 ⫾ 0.2), and the fraction of positive mice (intensity score ⬎0) in the
first group (8/9) is significantly higher (␹2 ⫽ 3.6; p ⬍ 0.05) than in the
second group (2/9).

Because the epithelium lining mucous membranes is not a per-

fect barrier, Ags can penetrate mucosal surfaces to some degree as
an ongoing process (14 –16). Our earlier experiments in vitro (8, 9)
provided initial support for the concept that IgA Abs secreted by
the local plasma cell population could provide an internal immune
barrier. In this schema, IgA Abs quickly bind Ags penetrating the
epithelium or released into the lamina propria from pathogenic
microbes. The resulting IgA immune complexes are then excreted
via the pIgR across the epithelium into the lumen.
The current experiments were designed to create soluble im-
mune complexes in mucosal lamina propria to test the hypothesis
of a local excretory function for mucosal IgA Abs in vivo. To
stimulate a potent mucosal Ab response, mice were immunized via
the gastrointestinal tract. After the specific IgA Ab response, as
reflected in the serum, reached a peak, the mice were challenged
i.v. with a large dose of Ag to ensure that the entire extracellular
fluid volume would be in Ag excess and to provide sufficient sol-
uble IgA-containing immune complexes in the lamina propria for
their excretion through mucosal epithelium to be detectable
morphologically.
The requisite immune complexes in the intestinal lamina propria
could have arisen by two mechanisms. One is diffusion of excess
free Ag from the bloodstream into the lamina propria, where it
combined with IgA Abs secreted by local plasma cells. The other
possibility is formation of IgA immune complexes in the circula-
tion and their subsequent diffusion into the lamina propria. Both
mechanisms would have been facilitated by the large excess of
injected Ag, designed to favor the formation of small immune
complexes, with a limiting complex of one molecule of Ag and one
molecule of Ab, with some Ag remaining free. Regardless, once in
the lamina propria, the IgA immune complexes would be in a
position to compete for available epithelial pIgR with background
FIGURE 3. Microscopic demonstration of Ag in intestinal mucosa. Sec-
tions of small intestine were stained by peroxidase for biotinylated OVA
(A–D) and IgA (E), and counterstained with hematoxylin. A, From a mouse
orally immunized with biotinylated OVA and subsequently injected i.v. with
biotinylated OVA, showing biotinylated OVA Ag prominently in crypt epi-
thelial cells (arrows) and to a lesser degree in the villus epithelial cells and in
the interstitial tissue of the lamina propria surrounding the crypts and in the
cores of the villi. B, As in A but at greater magnification. The biotinylated
OVA Ag at the upper arrow is in the epithelium of a crypt as it opens into the
lumen between adjacent villi. C, From a control mouse orally immunized with
irrelevant Ag (BSA) and subsequently injected i.v. with biotinylated OVA. In
the absence of specific IgA Ab the epithelium of the crypts (C) lacks biotin-
ylated OVA, although the lamina propria is prominently stained. D, As in C
but at greater magnification. E, Section stained for IgA showing IgA in plasma
cells in the lamina propria (small arrows), crypt epithelial cells (large arrow),
and to a lesser degree in the lamina propria generally. Magnifications are ⫻330
for A and C and ⫻770 for B, D, and E. M indicates external muscle layer; L,
intestinal lumen; V, villi; LP, lamina propria.
levels of free nonspecific oligomeric IgA and IgA immune com-
plexes containing naturally prevalent Ags.
For several reasons we believe the biotinylated OVA that was
detected in the small intestinal crypt cells was Ag being trans-
ported, i.e., excreted, by specific IgA Ab across the epithelium
The Journal of Immunology
from basal to apical. First, biotinylated OVA was readily detected
in the crypt cells of TCR transgenic mice that had been both mu-
cosally immunized and subsequently challenged i.v. with biotin-
ylated OVA. It was also detectable, but less prominently, in the
crypt cells of wild-type BALB/c mice that had been treated sim-
ilarly, but that, lacking the transgene, made a less vigorous IgA Ab
response (results not shown). Specific Ag was not detected in the
intestinal epithelium of mice that had been immunized mucosally
with an irrelevant Ag (BSA) before i.v. injection of biotinylated
OVA. Thus, in the absence of specific IgA Ab, biotinylated OVA
in the lamina propria was not taken up by the intestinal epithelium.
Second, mice that had been immunized intragastrically with bio-
tinylated OVA but not subsequently challenged i.v. did not evi-
dence biotinylated OVA in small intestinal crypt cells, showing
that under the experimental conditions, luminal Ag from the mu-
cosal immunization was not absorbed in detectable quantities into
the crypt cells, either nonspecifically or bound to previously se-
creted IgA Ab. Third, the architecture of the small intestine pro-
vides an internal control, namely, a gradient of decreasing transit
of IgA across the lining epithelium along the crypt-villus axis. This
parallels the expression of epithelial pIgR, the transporter of IgA,
which is also most prominent in the crypt cells, diminishing toward
the tips of the villi (17–19). Consistent with this gradient in IgA Ab
transport, biotinylated OVA Ag was detected much more promi-
nently in crypt than in villus epithelial cells. This marked decrease
in amount of Ag in the epithelium proceeding toward the tips of
the villi, in turn, argues against epithelial uptake due to nonspecific
events and staining artifacts.
Specific IgM Ab, also present after immunization (data not
shown) and also capable of binding to epithelial pIgR, could con-
tribute to uptake of Ag into intestinal epithelial cells. Quantita-
tively, however, mucosal IgA production greatly exceeds that of
mucosal IgM; moreover, the transport of IgA appears to be favored
under in vivo conditions (20).
Mucosal surfaces, especially in the gastrointestinal tract, are
constantly exposed to foreign substances, and secreted IgA Ab in
the lumen has long been known to provide an immunological bar-
rier to limit the penetration of Ags into mucous membranes. From
previous experiments in vitro (8, 9) and the present experiments in
vivo, we propose that mucosal IgA Abs in the lamina propria ad-
ditionally provide a backup, internal barrier beneath the epithelium
that can trap Ags missed by the initial IgA barrier in the lumen. For
example, Ags could by-pass luminal IgA Ab either during the early
stages of a mucosal Ab response when there was insufficient Ab to
prevent the absorption of all the Ag present or even in the face of
an established response if there was sudden exposure to a partic-
ularly large quantity of luminal Ag. Moreover, Ags will also be
released into the lamina propria from microbial pathogens during
infections of the mucosae. Therefore, we believe that to some ex-
tent foreign Ags are a regular presence in mucosal lamina propria.
Regardless of the origins of particular Ags at those sites, given the
ongoing production of IgA by the numerous local plasma cells,
IgA Abs are in a position to bind and efficiently transport Ags out
of the body proper and into the lumen. Potentially IgA Abs could
even excrete particles as large as intact viruses (21).
Removal of a variety of Ags from the lamina propria via the
same mechanism that is used for transporting free IgA across mu-
cosal epithelium would serve to limit the amounts of Ag reaching
the circulation, where Ags would also be more likely to be bound
by Abs of the more abundant and more phlogistic IgG class. In this
way, the excretory function of IgA could help to prevent diseases
that result from circulating immune complexes. A particularly rel-
evant example is IgA nephropathy, the most common form of glo-
3691
merulonephritis, which is thought to result from abnormal regula-
tion of the immune response to mucosal infections (22).
In addition to extracellular locales, IgA Abs are also capable of
binding to Ags inside epithelial cells during pIgR-mediated trans-
port of free IgA. IgA Abs acting intracellularly have been shown
to neutralize viruses (23–26) and to block their apical to basal
transcytosis (27). Thus, overall, IgA Abs appear to be capable of
mediating an integrated, multilayered mucosal defense system
(28). The first layer, in the lumen, is exclusion of Ag by secreted
free IgA. The second layer, within the lining epithelial cells, allows
for inhibition of intracellular pathogens like viruses. The third
layer, as demonstrated in the current work, is the lamina propria
from which IgA Abs can directly excrete Ags into the lumen.
Acknowledgments
We thank Dr. Kenneth M. Murphy, Washington University for the
transgenic mice.
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