Microbes and Infection, 3, 2001, 1021−1035

© 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

S1286457901014605/REV

Review

IgA responses in the intestinal mucosa against

pathogenic and non-pathogenic microorganisms
Andrew J. Macpherson*, Lukas Hunziker, Kathy McCoy, Alain Lamarre
Institute of Experimental Immunology, Universitätsspital, Schmelzbergstrasse 12, CH8091, Zürich, Switzerland

ABSTRACT – IgA is the most abundant immunoglobulin produced in mammals; most is secreted
as a dimer across mucous membranes. This review discusses the different mechanisms of induction of
IgA, and its role in protecting mucosal surfaces against pathogenic and non-pathogenic
microorganisms. © 2001 Éditions scientifiques et médicales Elsevier SAS
IgA / bacteria / viruses / intestinal mucosa

1. Introduction intestinal epithelial cells [2] and transferred to the intesti-

The intestinal immune system has the delicate task of
tolerating colonisation by a prodigious commensal flora,
whilst remaining able to mount a rapid and specific
response to invading pathogens. In mammals, commensal
bacteria colonise the intestinal tract rapidly after birth
(although the diversity and density of bacterial species
involved is reduced in the neonate during the period of
exclusive lactation [1]). Commensal bacteria are an advan-
tage to the host animal since they allow energy from
otherwise indigestible carbohydrates to be salvaged, and
the biofilms of relatively innocent microorganisms in the
intestinal mucus limit access to pathogens. However,
despite the lack of toxins and epithelial membrane attach-
ment devices in commensal organisms, they possess a
large array of immunostimulatory compounds which must
be largely confined to the lumen of the intestine if intesti-
nal and systemic immunopathology is not to ensue. This
review will focus on the role of secreted IgA in responses
to non-pathogenic intestinal bacteria and to intestinal
pathogenic microorganisms.
1.1. Secretion of IgA across intestinal mucous membranes
IgA is the most abundantly produced immunoglobulin
in mammals; it constitutes a small component of serum
immunoglobulin, and most IgA is secreted across mucous
membranes of the intestinal, respiratory, biliary and geni-
tal tracts. In the intestine, the secretory form of IgA is
synthesised by plasma cells in the lamina propria (figure
1): this is a dimer of two IgA molecules covalently linked
together through a J chain molecule between the alpha
heavy chains. The complex is then translocated through
*Correspondence and reprints.
E-mail address: amacpher@pathol.unizh.ch (A.J. Macpherson).
nal lumen (figure 2). The translocation mechanism depends
on an 80 000 Mr transmembrane glycoprotein expressed
by intestinal epithelial cells termed the polymeric immu-
noglobulin receptor (pIgR [3]). Dimeric IgA is bound to the
receptor at the basolateral surface of enterocytes by cova-
lent linkage between this protein and the CH2 domain of
one of the alpha heavy chains. Subsequently the entire
complex is endocytosed into a vesicle with the IgA
attached. The vesicle is then delivered to the apical (lumi-
nal) surface, whereupon proteolysis cuts the receptor and
releases the N-terminal part of the receptor protein (secre-
tory component), free or attached to IgA (reviewed in [4]).
This mechanism functions across the epithelial layer
throughout the small and large intestines (and other
mucous membranes), and in man approximately 3 g of IgA
is delivered each day into the intestinal lumen [5].
This transport system is more than just a means of
delivery of dimeric IgA from the basolateral surface of
epithelia into the lumen. First, it is able to transport IgA in
an immune complex with antigen so that antigens that
leak through the epithelial barrier can be cleared back into
the lumen, and monomeric IgA or IgG antibodies, which
are not themselves substrates for the pIgR, can be trans-
ported as part of these immune complexes [6]. Secondly, it
has been shown that specific IgA can even neutralise viral
replication within the epithelial cells themselves during
the transit process. The evidence for this comes from
experiments with polarised MDCK cell monolayers in
culture which had been stably transfected to express the
pIgR [7]. These were infected with Sendai virus on the
luminal surface, and anti-Sendai haemagglutinin-
neuraminidase monoclonal IgA, or irrelevant IgA was
applied to the basolateral surface (figure 3). After 4 h both
surfaces were treated with trypsin to remove adherent
antibody or virus; the incubation was then continued for a

Microbes and Infection 1021

2001, 1021-0
Review Macpherson et al.

Figure 1. Recirculation of B cells through the

lymph and the blood following triggering in
mucosal inductive sites. B1 cells from the perito-
neum are also triggered to produce IgA (indepen-
dently of help from T cells: see Section 5.3),
although whether they participate in a similar
recirculation process is unclear. The lower small
intestine and the colon contain an enormous load
of commensal bacteria which are normally not
pathogenic; this comprises about 1 000 species
with up to 1012 organisms/g of intestinal contents.

Figure 2. Transport of dimeric IgA

through the epithelial cell layer into the
intestinal lumen. In this process IgA is
covalently linked to the polymeric immu-
noglobulin receptor within the vesicular
membrane as the vesicles cross the cell. At
the apical surface IgA is released by pro-
teolysis of the receptor, part of which (secre-
tory component) remains attached to the
free immunoglobulin.

1022 Microbes and Infection

2001, 1021-0
IgA induction by pathogenic or non-pathogenic microorganisms
Figure 3. Set-up of the experiment by Kaetzel et al. [7], showing
that there is intracellular neutralisation of virus by specific IgA
during transport through epithelial cells. Specific IgA applied to
the basolateral surface of a polarised epithelial cell layer is able to
prevent intracellular viral replication of Sendai virus infecting
from the apical surface. Neither non-specific IgA (which is trans-
ported, but does not bind virus) nor specific IgG (which is not
transported) can do this. An in vivo version of this experiment,
carried out by Burns et al. [95] is described in Section 4.1.
further 20 h, when the apical supernatant was removed
and the cells were lysed. The titres of virus were found to
be lower both in the apical supernatants and lysates of
monolayers treated with specific IgA, compared with those
treated with non-specific IgA or specific IgG (which is not
transported through the cells because it does not bind to
the pIgR). The finding of less virus within the cells (i.e. the
lysates) indicated that specific IgA had interacted with
viral proteins during transit through the cells and inhibited
replication of the virus.
1.2. Predominant importance of IgA
as a secretory immunoglobulin
IgA is overwhelmingly the most important immunoglo-
bulin in the intestine and at other mucosal surfaces, and
relatively little (< 10%) of the other isotypes (IgM, IgG or
IgE) are locally produced or secreted unless: i) the tissues
are inflamed or diseased [8]; or ii) there is a deficiency of
IgA (found in about 1:500 Caucasian human subjects), in
which case mucosal IgM production and secretion partly
compensates for the lack of IgA [9, 10].
1.3. IgA in bile and milk
There are two other important ways in which IgA may
be delivered into the intestine other than by local mucosal
Microbes and Infection
2001, 1021-0
Review
production and transepithelial transport. The first is through
secretion into bile, which is then passed into the intestine
through the ampulla of Vater in the upper small intestine
(duodenum). To reach the bile, polymeric IgA is either
transported across hepatocytes [11, 12] (which in rodents
express the pIgR [13–15]) into biliary canaliculi, or in
humans (whose hepatocytes lack pIgR [16, 17]) transport
is across the biliary epithelia of the bile ducts and gall
bladder [18–21]. Another way in which IgA is delivered
into the intestine is through secretion of IgA into the
maternal milk, which is then fed to the infant. IgA is the
predominant immunoglobulin in milk [22]. Early in lacta-
tion most of the IgA is polymeric IgA transported from the
serum across the mammary epithelia, later local IgA-
producing plasma cells (including those induced in intes-
tinal lymphoid tissue) are found in the mammary gland
[23–25]. The possible relevance of milk IgA to the neonate
is discussed further in Section 5.2.
1.4. Serum IgA
IgA is also a component of serum, although here it is
quantitatively less than IgM or IgG and its constituent
isotypes. In humans there are two α heavy chains, giving
rise to different IgA1 and IgA2 isoforms. (Multiple α chain
isotypes are also seen in non-human primates and in
rabbits; other mammals including mice have a single α
gene.) Of these, IgA1 is almost exclusively present as a
monomeric form in human serum. The in-vivo function of
serum IgA remains very poorly understood.
In humans there is a Fcα receptor (CD89 or FcαRI) on
the surface of eosinophils, neutrophils, monocytes and
macrophages [26, 27] which can trigger responses mea-
surable in vitro, including phagocytosis [26], antibody-
dependent cellular cytotoxicity [28] and secretion of
inflammatory mediators [29]. These responses seem curi-
ous compared with the usual view of IgA as a relatively
non-inflammatory immunoglobulin, yet their significance
in vivo is hard to define because no mouse CD89 homo-
logue has yet been described. However, a transgenic
mouse in which human CD89 (together with its own
promoter and regulatory elements) has been generated in
which FcαRI expression is on the same cell types found
naturally in humans, and in vitro responses (phagocytosis
etc.) are similar. When these animals are not manipulated,
expression of the FcαRI is restricted to cells of the myeloid
lineage in the bone marrow and periphery. However, in
the presence of inflammatory mediators (such as experi-
mental treatment with granulocyte colony-stimulating fac-
tor) FcαRI was expressed on Kupffer cells in the liver [30].
These cells were then able to phagocytose bacteria coated
with serum (but not secretory) IgA. This implies that mono-
meric serum IgA may form a second-line defence system
to clear bacteria that escape intestinal mucosal barriers,
which works by coating these bacteria in the portal circu-
lation, allowing hepatocyte phagocytosis to be triggered.
Whilst this work is important as a first glimpse at the
possible purpose of having IgA in the serum, rather than
just being secreted at mucosal surfaces, whether such a
pathway is functional in normal humans or non-transgenic
animals has yet to be shown.
1023
Review Macpherson et al.

2. The immunologic anatomy

of IgA production
2.1. IgA+ B-cell triggering in Peyer’s patches
and recirculation through the lymph and the blood
Early experiments showed that if radiolabelled large
lymphocytes from the thoracic duct lymph or mesenteric
lymph nodes of adult donors were injected intravenously
they would preferentially migrate into the intestine of adult
recipients or neonatal animals [31]. Migration could even
occur into foetal intestine grafted under the kidney cap-
sule of adult mice, showing that antigen is not required for
the homing mechanism [32]. This recirculation mecha-
nism applies to both B- and T-cell blasts, since both appear
in the recipient mucosal tissues [33]. Transfer of Peyer’s
patch cells into irradiated recipient animals showed that
these were a primary source of IgA+ B cells [34]. The
recirculation required for the induction of IgA was inves-
tigated in the classical isolated loop experiments of Gow-
ans and his colleagues. These established that IgA is
induced in intestinal Peyer’s patches and that after trigger-
ing, B cells pass through the mesenteric lymph nodes and
circulate via the lymphatic system to enter the subclavian
vein from the thoracic duct, then they home back to a
mucosal site (e.g., the intestinal lamina propria) through
the arterial blood. The key evidence for this was obtained
by constructing intestinal (Thiry Vella) loops in rats, which
retained a lymphatic and blood supply, but were discon-
Figure 4. Set-up of the experiment by Husband and Gowans
nected from the intestinal stream, with both ends having
[35] in which isolated (‘Thiry-Vella’) loops of small intestine
an external opening onto the skin (figure 4). When two
were constructed in rats. From the position marked ‘xxxx’ a
separate loops were constructed and antigen was intro-
segment of small intestine had been isolated, preserving its
duced into one of them, secretory IgA was subsequently
vascular and lymphatic supply, and both ends were connected to
observed both in the immunised loop and non-immunised
the skin of the animal. The ends of the remaining bowel were
loop, although the specific response was always greater in
anastomosed (at ‘xxxx’) to preserve intestinal continuity. The
the immunised loop [35]. Work with the powerful mucosal
effects of local immunisation into the intestine could now be
immunogen cholera toxin allowed plasmablasts to be
compared with the effects at distant sites by administering immu-
tracked as they passed through the thoracic lymph, and
nogen (cholera toxin) either into the loop, or into the main
later appeared in the intestinal lamina propria [36]. Experi-
intestine. In some animals two loops were constructed. This led
ments in rats where either Peyer’s patches or the mesen-
to the finding that local immunisation has an effect at distant
teric lymph nodes had been excised showed that Peyer’s
intestinal sites, which is an important part of the evidence that
patches are principally required for this process [35].
IgA is induced in lymphoid follicles (Peyer’s patches) in the
The existence of specialised mucosal inductive sites
intestine, and then cells recirculate through the lymph and blood
and their separation by the recirculation process from the
to diffusely populate the intestinal mucosa (and other mucosal
remainder of the mucosa was thus established. This also
tissues). See also figure 1.
provides the mucosal immune system with the ability to
induce responses at sites that are distant from the imme-
diate inductive environment of the Peyer’s patch, or even
in different mucosal tissues (leading to the concept of lymph nodes selectively home to the mammary glands
generalised functioning of mucosal tissues with some cross during lactation [24]. This appears to tailor the protection
talk between them). However, the local response of delivered to the neonatal intestine to the current antigens
mucosal immunisation is consistently greater than the sensed in the maternal intestinal immune system (see also
diffuse response [35], either as a result of some direct Section 5.2).
migration or of selective homing to the area of the intestine
2.2. The B1 and B2 lymphocytes and their origins
where induction first occurred.
The composition of milk IgA is probably an important There is a further issue concerning the immunological
example of how systemic recirculation is important fol- anatomy of mucosal antibody production, which is the
lowing B-cell induction in the intestine. Certainly milk IgA site of origin of the B cells that are induced to switch to IgA
limits the exposure of the neonate to the intestinal bacte- production. The earliest site of B-cell production is the
rial flora (which is rapidly acquired after birth) [37]. It has foetal liver, but after birth, B cells are produced both in the
been shown that milk contains IgA against antigens of the bone marrow and the pleuropericardial cavities [38]. It is
intestinal flora, and that IgA+ B cells from the mesenteric extremely controversial whether these two physical sites
1024 Microbes and Infection
2001, 1021-0
IgA induction by pathogenic or non-pathogenic microorganisms Review

represent different lineages of B cells [39], although the

progeny of bone marrow and pleuroperitoneal percursors
certainly have different levels of surface marker expres-
sion, thus the pleuropericardial cells (called B1) stain
strongly with antibodies against surface IgM, Mac-1 and
CD5, and weakly with antibodies against B220 (CD45R)
and IgD, whereas in bone marrow-derived (B2) B cells the
situation is reversed (strong B1 markers stain weakly, and
vice versa) [40–42].
Both B1 and B2 lineages contribute to IgA production in
mice, and the evidence for this is discussed below. B1 cells
also give rise to IgM that is relatively undiversified (lacking
the molecular hallmarks of having been through a germi-
nal centre reaction; mutations in the complementarity-
determining regions of the antibody variable chains [43,
44]), and the resultant IgM has specificites against micro-
bial antigens. This IgM has been considered ‘natural’
antibody, because it is produced in germ-free conditions
when the cognate antigen(s) are presumed to be absent
[45], however, there is evidence that environmental [46]
or endogenous [47] antigens are required to positively
select or expand particular B1 cell specificities. The rel-
evant questions for intestinal IgA are: 1) how much secre-
tory IgA can also be considered B1-derived antibody,
produced without germinal centre-dependent pathways Figure 5. Set-up of radiation chimaera experiment [52]. The
of IgA induction; and 2) what is its functional importance? two classes of B lymphocytes (B1 cells from the peritoneum and
B2 cells from the bone marrow) are taken from different strains of
No significant population of IgA-staining B1 cells is
inbred mice in which the IgA molecules have (slight) ‘allotypic’
found in preparations isolated from the peritoneal cavity
differences that can be detected in immunological (ELISA and
(these are IgM+ cells which have yet to switch to IgA
ELISPOT) assays. In adult mice B1 cells are absent from the bone
production), and we have very sketchy knowledge about
marrow. A mouse that has had its own lymphocytes destroyed by
where these cells are triggered. B1 cells with the charac-
irradiation is reconstituted with the two different sorts of B
teristic surface markers are not readily detected in Peyer’s
lymphocytes. Once the situation in the recipient mouse has
patches, although they can be seen in animals with a
stabilised (after 3–6 months), one can determine the relative
targeted B-cell deficiency of the transforming growth fac-
proportions of IgA that are derived from the bone marrow (B2) or
tor (TGF-β) receptor (TβRIIBcell–/–) [48]. How then do we
peritoneal (B1) subsets by assaying for the different allotypes.
know that B1 cells can end up as intestinal IgA-producing
Normally about half IgA is B1 derived. From the results of this
lamina propria cells? The answer lies in making chimaeras
experiment, where the donor and recipient animals lack T cells
from lethally irradiated animals so their own lymphocyte
(TCRβ–/– δ–/–) and only FACS purified B cells are given from the
populations are destroyed, and afterwards they are recon-
peritoneum, one can show that most of the T-independent IgA is
stituted with bone marrow cells from an animal that will
B1 derived.
produce immunoglobulins of one allotype, and with peri-
toneal cells that will produce immunoglobulins of a differ-
ent allotype (see example in figure 5) [49]. The reconsti-
tuted animal is now left to recover from the effects of B cells bearing the CD5 surface marker also constitute
manipulation, and after about 3–4 months, the allotypic 10–25% of circulating and splenic B lymphocytes in adult
markers for the different sorts of immunoglobulin can be humans, and form the majority of B cells in human foetal
analysed in the serum and the intestinal (or other) secre- spleen and in umbilical cord blood [53, 54]. They are also
tions. In wild-type mice this shows that much of the serum increased in patients with rheumatoid arthritis, in whom
IgM and about half the intestinal IgA is derived from B1 they are a source of low-affinity polyreactive antibodies
cells [50]. The functional details of the B1 contribution to [55]. Thus there is also a B1 phenotype in humans. The
IgA have been explored in MHC class II-deficient mice extent of the B1 contribution to human intestinal IgA can
that also have the xid mutation [51]. Whereas class II–/– obviously not be determined by radiation chimaera tech-
mice have a relatively normal IgA content (despite a niques. An indirect approach has therefore been taken. It
deficiency of CD4+ T cells), the presence of the xid lesion is clear that most B1-derived immunoglobulin in mice is in
abolishes peritoneal B1 cells and the intestinal IgA content an unmutated germline configuration of the constituent V,
becomes sharply reduced. We have studied mice that are (D), J and C segments [43, 44], whereas B2-derived immu-
T-cell-deficient because of targeted lesions in both the noglobulin following immunisation is characterised by
beta and delta T-cell receptor genes: radiation chimaeras multiple somatic mutations focused on the complementa-
made in these animals show directly that the remaining rity determining regions that encode the binding site of the
IgA produced in the intestine is of B1 origin [52]. variable chains. Therefore immunoglobulin variable genes

Microbes and Infection 1025

2001, 1021-0
Review
from human CD5+ cells have been examined to determine
whether they have somatic mutations or remain in germ-
line configuration to see if they have been through an
antigen-driven process. Problems with this approach are:
i) that comparisons are usually made with database V, D,
(J), C sequences from different individuals, and polymor-
phisms may be incorrectly assigned as mutations; and ii)
that analysis of cDNA clones even in sorted populations of
B1 cells may be biased by higher mRNA expression levels
in the contaminating B2 cells (e.g., reference [56] gives
examples of these problems).
Sequencing of rearranged IgA immunoglobulin V
regions has been carried out from intestinal biopsies to
look at mutational frequencies using both a reverse tran-
scription PCR and rearranged DNA approach. In adults
there certainly seems to be a high mutational frequency
[56, 57], which is less in children [58], although insuffi-
cient material from young children has been studied at the
single cell level. Whilst it is possible that B1 lymphocytes
make a similar contribution to intestinal IgA in both
humans and mice, this has not been resolved.
3. Mechanisms of IgA induction
in model systems
Early experiments with nude mice (which have a very
restricted population of T cells) showed that much of the
IgA production was dependent on T-cell help [59, 60]. As
shown below for both intestinal pathogens and commen-
sal bacteria, this is only part of the story, and there is also
an important T-independent pathway of IgA induction.
There are four different experimental approaches, dis-
cussed in the following sections, that have been taken to
study the mechanisms of IgA induction further.
3.1. IgA switching and secretion in cell culture
The cytokine requirements and the details of the
IgM→IgA switching mechanism have been addressed by
experiments on B-cell cultures in vitro. These have used B
cells purified ex-vivo free from other cell types (derived
either from the spleen or Peyer’s patches), or B-cell lines,
which have been cultured in media supplemented with
lipopolysaccharide as a non-specific stimulant and vari-
ous combinations of cytokines. The object has been to
look at the proportion of B cells that switch to IgA produc-
tion. This approach showed that TGF-β and interleukin
(IL)-4 promoted the switch from IgM→IgA surface expres-
sion and subsequent IgA secretion in splenocytes that
were depleted of T cells [61, 62]. The effect was increased
in the presence of IL-2, but IL-2 had little effect on its own
[61]. Once the switch IgM→IgA has taken place, IL-5 [62,
63] and IL-6 [63, 64] then act to enhance secretion of IgA.
IL-10 also synergises with TGF-β to increase the efficiency
of IgA switching [65].
3.2. Cells required for the IgM→IgA switch process
Experiments have also been carried out to attempt to
reconstitute the different cell types that are required (in
addition to B cells) to allow the IgA switch to take place.
For this, combinations of B, T and antigen-presenting cells
1026
Macpherson et al.
isolated from intestinal lymphoid follicles or other lym-
phoid tissues have been cultured in vitro to determine
whether the origins of accessory cells have a critical
bearing on IgA induction. This has shown that the natural
source of Peyer’s patch B [66], T and dendritic cells [67] is
more efficient for IgA production than when these cells are
derived from a non-mucosal site (such as the spleen). The
relative importance of having just one of these compo-
nents (B, T or dendritic cells) from the Peyer’s patches has
also been addressed (i.e. Peyer’s patch T cells cultured
with splenic B and dendritic cells or other permutations):
the interpretation of the results of different experiments is
complicated by technical considerations of cell purifica-
tion, but B cells probably become committed to IgA switch-
ing within the Peyer’s patches as a result of the unique
cytokine environment (especially TGF-β) generated by the
T, antigen-presenting and stromal cells there [66].
3.3. Requirements for IgA induction in vivo
To understand the functional requirements for different
cell types, cytokines and accessory molecules in vivo,
gene-targeted mice have been studied. To measure spe-
cific IgA induction in these strains, extensive use has been
made of ’oral adjuvants’, of which the most popular has
been cholera toxin. When given into the stomach of mice
in three successive doses (of 15 µg) at 10-day intervals,
cholera toxin does not cause diarrhoea, but both intestinal
and serum IgA are induced against cholera toxin itself
[68]. It is termed an oral adjuvant because when a soluble
protein is given orally with the cholera toxin, a similar
response is mounted against this protein, whereas nor-
mally giving a soluble protein alone into the intestine
would not immunise but rather induce tolerance to subse-
quent systemic immunisation protocols [69].
It is important to realise that this method of IgA induc-
tion may be rather a special case, because serum IgG and
IgE are also induced [68, 70]. Nonetheless, the model has
been a powerful tool to demonstrate that a memory IgA
response can be seen in mice 2 years after the initial
immunisation with cholera toxin [71], and it has been a
good system to look at the requirements for induction of
IgA in vivo in different gene-targeted mice. It is also
possible to study functional protection from cholera toxin
in experiments in which a segment of mid small intestine is
isolated in anaesthetised mice by ligatures tied around the
bowel about 6–8 cm apart [72]. If cholera toxin is then
injected into the lumen of this isolated segment, after 4 h
the fluid accumulation/cm length of the segment can be
measured. The fluid accumulation response is consider-
ably reduced in animals where specific secretory IgA has
been induced previously by immunisation with cholera
toxin, but if IgA secretion is defective (for example in
J-chain-deficient mice) protection is impaired [73].
Oral immunisation with cholera toxin is CD4+ T-cell
dependent in vivo (since it is defective in CD4–/– mice [72]
and MHC class II-deficient mice [51]). There is also a
requirement for IL-4, as responses to cholera toxin are also
reduced in IL-4–/– mice; interestingly, where cholera toxin
is used as an adjuvant in IL-4–/– mice for oral immunisation
with a soluble protein, the response to the soluble protein
itself is completely absent [74]. Immunisation is also inef
Microbes and Infection
2001, 1021-0
IgA induction by pathogenic or non-pathogenic microorganisms
fective in CTLA4-Hγ1 transgenic mice: these express a
CTLA4 protein construct under the control of the immu-
noglobulin heavy chain promoter which blocks the CD28-
CD80/86 costimulation signals between antigen-
presenting cells and T cells [75]. Despite this,
paradoxically, the intestinal IgA content in both CD4–/–
and CTLA4-Hγ1 mice is normal, indicating that induction
of IgA (by other antigens) may be independent of these
T-B-cell interactions.
The in vivo importance of TGF-β, IL-2 and IL-10 have
been more difficult to assess, because mice with targeted
deletions at these loci have generalised (TGF-β [76]) or
intestinal (IL-2, IL-10) chronic inflammation [77, 78],
which interferes with assessment of unmanipulated IgA
levels and the results of immunisation protocols. How-
ever, an inducible targeted deletion of TGF-β receptor II
confined to B cells has recently been reported, these mice
do not have generalised systemic or intestinal inflamma-
tion, but there are low IgA levels combined with a consid-
erably expanded population of peritoneal B1 cells and
hypertrophied Peyer’s patches [48]. Whether the intestinal
secretory IgA is also reduced by this selective induced
lesion in TGF-β signalling (as found in TGF-β–/– mice [79])
has not yet been reported; if so the activation of Peyer’s
patches and the peritoneal B1 compartment may represent
compensatory changes as a result of increased exposure to
antigens at the mucosal surfaces in the absence of secre-
tory IgA function.
3.4. Mechanisms of in-vivo induction of IgA
to intestinal pathogens or non-pathogenic
commensal intestinal bacteria
Experiments have also addressed the mechanisms of
IgA induction to microbial pathogens or non-pathogenic
intestinal commensal intestinal flora. It is now clear that
although IgA induction by cholera toxin is very dependent
on T-cell help, cognate T-cell help is certainly not obliga-
tory for IgA induction by pathogens (Section 4.1) or non-
pathogenic commensal intestinal bacteria (Section 5.3).
Of course, this is an explanation of the paradox in CD4–/–
and CTLA4-Hγ1 animals described above, where T-cell
help or co-stimulatory responses are defective, yet the
unmanipulated content of intestinal IgA is normal.
Other strong antigens (recombinant Salmonella,
ISCOMS) can also induce IgA in IL-4–/– mice [80, 81]. In
vivo investigations of isolated deficiency of cytokines that
act in vitro to enhance IgA secretion after the switch
process has taken place shows no defect in IL-5–/– mice,
but conflicting results with IL-6–/– mice [82, 83].
4. Protection by IgA
from intestinal infections
IgA has been shown to neutralise viruses in culture, and
to form part of the protective response in vivo. The success
of mucosal vaccination against polio with live attenuated
virus has been one of the great achievements of modern
healthcare; it was clear from early studies that the route of
immunisation was critical, secretory IgA was induced far
more effectively after mucosal rather than parenteral vac-
Microbes and Infection
2001, 1021-0
Review
cination [84] and it depends on mucosal exposure to
vaccine [85]. A requirement for mucosal vaccination for
secretory immunity was also found for rubella [86].
4.1. Rotaviruses
Rotaviruses have been studied extensively in relation to
the functional importance of the mucosal IgA response
(reviewed in [87]). These cause the death of 800 000
infants per year worldwide, and the impetus to develop a
vaccine led to development of animal models. Rotavirus
infections in calves and piglets were used as models of
human disease, but had the disadvantage that germ-free
conditions were often required. Rotavirus was also shown
to cause epidemic diarrhoea in young mice (postnatal
days 4–14). Certain strains are also capable of infecting
adult mice; although they do not get diarrhoea, there is
subclinical fluid malabsorption at the time of peak viral
load [88]. The mouse rotavirus model has thus allowed the
immune mechanisms resulting in viral clearance to be
explored in detail.
Clearance of virus after primary infection, and subse-
quent protection against reinfection (seen for at least
1 year) correlates well with the production of mucosal and
serum IgA [88, 89]. BALB/c or C57BL/6x129 strains of
severe combined immunodeficiency (SCID) or RAG–/–
mice (both of which lack both B and T cells) become
persistently infected, so adaptive immunity induced fol-
lowing infection is clearly important for viral clearance
[90, 91]. However, innate mechanisms also play a role, as
40% of C57BL/6 SCID mice can also clear the virus. The
relative importance of the T- and B-cell responses in the
adaptive response has been extensively examined by infec-
tion of strains: i) after immunodepletion of T or B cells [92];
ii) following transfers into (SCID) animals [90]; by studies
in mice: iii) deficient in B cells (JH–/– mice [91, 92]); or iv)
selectively deficient in IgA [93]); v) those deficient in both
CD4 and CD8 T cells (nude mice or T-cell receptor β–/–
δ–/– [94]); or vi) deficient in cytotoxic T cells alone (β2
microglobulin–/– mice or mice depleted of CD8+ cells [91,
94]). The overall conclusions from these experiments are
that B cells which permit antibody production are protec-
tive both in the initial infection and in rechallenge, whereas
CTL deficiency delays viral clearance.
As described in Section 3, the induction of IgA has
usually been thought highly dependent on the presence of
specific T-cell help. Thus it was a surprise when both nude
mice (having a very restricted T-cell compartment) and
T-cell receptor β–/– δ–/– mice (which lack T cells) were
capable of viral clearance even on the C57BL/6x129
background, where innate mechanisms are relatively inef-
fective [94]. Moreover, both IgM (known to be T-cell-
independent) and small amounts of specific IgA were
produced in the serum and secretions of these mice. Since
protection from virus correlated with T-cell-independent
IgA production, induction of some protective IgA can
probably occur in response to pathogens independently of
T-cell help.
To look at the protective effect of secreted IgA per se,
Burns et al. investigated mice that carried a ’backpack’
hybridoma secreting monoclonal IgAs directed against
one of the outer rotaviral capsid proteins haemagglutinin
1027
Review
(VP4) or glycoprotein (VP7), or the inner capsid protein
VP6 [95]. Two monoclonal antibodies to VP6 secreted
from backpack tumours were able to protect BALB/c mice
from rotaviral infection, or to clear the virus from prein-
fected SCID mice. Strangely, these monoclonal antibodies
were not neutralising in vitro (i.e. they do not protect cells
in culture from becoming infected with virus as seen with
some antibodies against VP4 or VP7). Moreover, they were
not active in vivo when present on the luminal side of the
gastrointestinal tract, so the in vivo effect in the mucosa
(which presumably depends on intracellular transport of
IgA across epithelial cells by the pIgR) works through an
intracellular mechanism. The probable physiological
importance of IgA in protecting animals from mucosal
infections was reinforced by the observation that a back-
pack hybridoma secreting large amounts of IgG antibody
that did effectively neutralise in vitro (against VP7) could
neither prevent nor resolve rotavirus infection in vivo.
However, whilst IgA is sufficient for protection, it is not
obligatory, and other mechanisms (probably mainly IgM)
can compensate for its absence in IgA–/– mice [93].
4.2. Other mucosal infections
The role of secreted IgA has also been studied in
relation to mucosal infection with influenza. Whilst the
influenza models depend on respiratory tract challenge
with pathogen, they are discussed here because of the
relevance of potential functional protection by IgA.
After transferring polymeric or monomeric monoclonal
IgA, or monomeric monoclonal IgG, each specific for the
haemagglutinin of PR8 influenza virus, Renegar and Small
were able to show that polymeric IgA was selectively
transported into nasal secretions and bile, and that poly-
meric IgA (but not IgG) transfer protected mice against
subsequent nasal challenge with live virus. This protection
could be abrogated by intranasal administration of anti-
IgA antiserum [96]. Similarly, mice that had recovered
from an influenza infection 4–6 weeks previously could
be reinfected intranasally if the nasopharynx was treated
with anti-IgA [97]. Studies of cross-protection between
different strains of influenza infecting mice through a nasal
route also correlate better with sIgA production than cyto-
toxic T-cell reactivity [98, 99]; CD8+ CTLs do contribute to
recovery from infection, but are not strong mediators of
resistance to reinfection [100–103].
Despite the data above that show that that passive
transfer of immune immunoglobulins can protect against
subsequent influenza infection, it has not been straightfor-
ward to show increased susceptibility to (intranasal) influ-
enza infection in mice that are selectively deficient in IgA
[104] or J chain [103] as a result of a targeted genetic
lesion. This appears to be partly due to the experimental
protocols used: in the protection study of the IgA–/– mice
[104], the influenza subunit vaccine was given with chol-
era toxin; when this strong mucosal adjuvant is omitted,
the protection is much weaker in IgA–/– than in wild-type
animals [105].
Backpack models have been used with other experi-
mental pathogens to show that specific IgA produced in a
hybridoma is protective against challenge; these include
1028
Macpherson et al.
Vibrio cholera [106], Shigella (through the bronchial
mucosa [107]) and the low-grade pathogen Helicobacter
felis [108].
Potentially therefore, IgA in sufficient quantities can be
shown to be protective in at least some models. However,
evidence from the strain of mice which selectively lack
IgA (but not other immunoglobulin isotypes) owing to a
targeted genetic lesion, suggests that IgA is often not
obligatory for mucosal protection.
5. Interactions between IgA
and the commensal intestinal flora
5.1. Effects of colonisation of intestines
with commensal bacterial flora on intestinal IgA
As noted at the start of this review, the intestinal tract of
mammals is colonised by a dense bacterial flora of (usu-
ally) non-pathogenic organisms, which confer advantages
to the host in terms of metabolism of otherwise indigest-
ible carbohydrates, and thus allow the host animal to
salvage energy from the diet. The presence of the com-
mensal flora also competes with pathogenic organisms,
and limits their ability to colonise/infect the intestinal
tract. The commensal flora has a remarkable density: there
are 1012 bacteria/g faeces in the colon, and approximately
103 known species, with anaerobes predominating.
A useful tool to understand the impact of the non-
pathogenic commensals on the immune system has been
the use of germ-free (gnotobiotic) mice. A colony of germ-
free animals can initially be set up by delivering the pups
through Caesarian section and hand-rearing them in a
sterile isolator. Such hand-rearing is extremely demand-
ing, but subsequently it is much easier to interbreed the
offspring within the isolator, keeping the conditions
(including the food and water) sterile. Germ-free animals
can be moved between isolators to separate some animals
from the sterile breeding colony for experiments, such as
recolonisation of the intestine with defined bacteria. Also,
different strains of mice can be derived germ-free without
the need for hand-rearing, by transferring embryos from
that strain into a germ-free recipient foster-mother.
Germ-free mice have extremely small Peyer’s patches
and a very small number of lamina propria IgA-producing
plasma cells (figure 6). The number of CD4+ T cells in the
intestinal lamina propria, and αβ T-cell receptor CD8+
cells in the intraepithelial compartment is also reduced,
although the γδ T-cell receptor CD8+ intraepithelial com-
ponent is essentially normal.
Cebra and his colleagues have carried out experiments
to define the sequence of changes in the mucosal immune
system when germ-free mice are recolonised with a single
species of Gram-negative aerobic bacteria [109]. Their
results are illustrated in figure 7. Initially, there was very
low production of intestinal IgA, and the recolonising
commensal bacteria were not successfully confined to the
intestine. Organisms could be cultured from the spleen
and mesenteric lymph nodes for up to 100 days, having
penetrated or ’translocated’ from the intestinal lumen to
underlying tissues. The reduction and disappearance of
Microbes and Infection
2001, 1021-0
IgA induction by pathogenic or non-pathogenic microorganisms
Figure 6. Histology of intestinal sections from germ-free mice
and mice with a normal (specific pathogen-free, SPF) intestinal
bacterial flora. A) IgA-secreting cells are very sparse in germ-free
mice compared with B) SPF mice. C) The Peyer’s patches of
germ-free mice are macroscopically very small: microscopically
they have relatively few germinal centres and IgA+ B cells; D)
SPF Peyer’s patch shown for comparison. E) The number of CD4+
cells in the lamina propria is also less in germ-free mice compared
with F) SPF mice, but the numbers of CD8+ intrapepithelial cells
in germ-free animals (G), are comparable with those with an SPF
flora (H).
translocation coincided with the appearance of secreted
intestinal IgA, so it is possible that IgA, once induced,
prevents the commensal bacteria from leaving the lumen
of the intestine and reaching extraluminal tissues. Whilst
this is probably broadly correct (see Section 5.3 below),
IgA is not the only factor that limits the translocation of
commensal bacteria, as other changes also occur after
recolonisation, including increased content of lamina pro-
pria CD4 and αβTCR CD8 cells, and increased macro-
phage activation, so the exact contribution of secreted
intestinal IgA in preventing the penetration of commensal
organisms from the lumen of the bowel has not been
formally shown. However, we have found that spontane-
ous priming of the systemic immune system by commen-
Microbes and Infection
2001, 1021-0
Review
Figure 7. Set-up of the experiment by Shroff et al. [109] in
which germ-free mice were recolonised by a single Gram-negative
commensal organism Morganella morganii. The mice were kept in
an isolator throughout to avoid contamination with other organ-
isms. Initially the bacteria penetrated into the body and could be
cultured from the mesenteric lymph nodes and the spleen. How-
ever, once IgA had been induced (and other adaptive changes had
occurred in the mucosal immune system), the bacteria were
successfully confined to the intestine.
sal bacteria, which depends on penetration of the com-
mensal organisms, is much more likely in IgA–/– animals
[52].
5.2. Functional effects of milk IgA
on the neonatal mucosal immune system
In another series of experiments carried out by Cebra
and his colleagues, the immune value of the mothers’ milk
was determined. IgA in milk is initially derived from the
serum, and later is produced by plasma cells within the
mammary tissue. IgA in the milk can also be shown to bind
to commensal bacteria [37], and it is clear that during the
period of exclusive lactation the number of species of the
commensal flora and their density are restricted, with a
normal adult flora composition being rapidly established
once other nutritional sources (formula milk/weaning to
solids) are introduced. To look at the impact of immuno-
globulins in the milk on the development of the mucosal
immune system, the results of immunoincompetent (SCID)
dams nursing immunocompetent (scid/+) pups were com-
pared with immunocompetent dams nursing immuno-
competent pups. Since SCID dams have the innate immune
components present in milk, these experiments reveal the
1029
Review
effects of adaptive components, principally the immuno-
globulins. Those pups nursed by SCID mothers showed
early induction of germinal centres in the Peyer’s patches
at postnatal day 15, rather than day 21 when the nursing
mother was immunocompetent [37].
Using this system the responses of neonatal mice to
infection with reovirus were also studied [110]. They
found that the neonatal mice were quite able to mount
specific mucosal responses to reovirus (from postnatal day
10), and to generate specific IgA in the Peyer’s patches.
Moreover, when litter swap experiments were carried out,
an immunocompetent pup lactating (since birth) on a
non-immune nurse dam mounted a mucosal response to a
reovirus infection whether or not it had been born to a
reovirus immune (immunocompetent) mother. The reverse
situation, where an immunocompetent pup was lactated
by an immune dam, did not mount a response, whether or
not it had been born to an immune dam. Therefore the
adaptive immunity of milk (in mice) is critical in conferring
immune protection against oral challenge with reovirus
independently of placental immunoglobulin transfer.
In mice, transfer of maternal IgG continues from birth
by uptake from the milk in the duodenum (which is largely
complete by postnatal day 10) [111, 112]. (This mecha-
nism plays a much smaller role in humans, although some
colostral IgG can probably be transferred through the
neonatal intestine [112,113]). Whilst adaptive responses
are therefore the key to explaining the premature induc-
tion of the natural responses (presumably to commensal
bacteria) in the mucosal immune system, and protection
from infection in neonatal mice, one cannot say what role
IgA and IgG have individually in the effects. However, the
resources to resolve this experimentally are available.
In humans (where maternofoetal immunoglobulin trans-
fer is largely placental, so benefits of lactation are on
limiting the exposure of the neonate to commensal bacte-
ria or to pathogens) it is clear that breast milk can contain
antibodies to pathogenic intestinal bacteria recently
encountered by the mother. Epidemiological studies have
consistently demonstrated that the benefits of breast-
feeding are enormous, and that breast-fed infants in devel-
oping countries are protected from death from infectious
causes (especially dehydrating diarrhoea), although a com-
ponent of this benefit is improved nutrition [114, 115]. In
developed countries, where nutrition from natural and
formula milk is similar, there is also an association with
reduced gastrointestinal illness [115]. There is also some
evidence that the benefits of adequate breast-feeding can
persist many years after weaning, with significant reduc-
tions in childhood respiratory illness and other allergic
disorders [116–118]. Exclusively breast-fed infants have a
different composition of the commensal intestinal bacte-
rial flora compared with exclusively or partially formula-
fed infants [1] (as simple examination of the nappies will
confirm). The details of the in vivo mechanisms by which
innate and adaptive responses in the milk influence the
intestinal flora of the neonate and impact on later mucosal
and systemic immunity remain unclear and need further
study.
1030
Macpherson et al.
5.3. Intestinal IgA directed against
the commensal intestinal bacterial flora
Despite the immense load of commensal intestinal
bacteria, normal mice are systemically immunologically
ignorant of these organisms when kept in a pathogen-free
environment. We showed this by studying serum IgG-
binding to proteins of Enterobacter cloacae (the principal
aerobe in the intestinal flora): whilst normal mice had no
binding IgG, this was easily induced 14 days after an
intravenous infection with 106 cultured bacteria. Some of
this protection is due to the barrier of secreted IgA, because
IgA–/– mice are much more likely to have spontaneous
priming of a systemic immune response (i.e. without intra-
venous infection) despite being kept in pathogen-free con-
ditions [52].
Although wild-type mice have no serum IgG- (or serum
IgA-) binding to commensal bacteria, Western blot experi-
ments show that intestinal secretory IgA is spontaneously
induced by the presence of commensal organisms in the
intestine. Thus germ-free mice do not bind proteins of
E. cloacae (even allowing for the reduced intestinal IgA
content). That intestinal secretory IgA is responsive to
changes in the intestinal flora can also be shown by
colonising the intestines of mice with bacteria that express
a novel protein in vivo; following colonisation sIgA bind-
ing to the new commensal bacterial protein is induced
[52].
Experiments with cholera toxin (described in Section
3.3) have shown that induction of specific intestinal IgA is
very dependent on T-cell help, yet the total content of IgA
is not reduced in circumstances where T-cell help or
costimulation are lacking, when cholera toxin induction is
ineffective. Using mice with targeted lesion of the β and δ
chains of the T-cell receptor (TCRβ–/– δ–/–), we showed that
intestinal IgA content was reduced, but only to about a
quarter of wild-type levels. In agreement with this, the
induction of intestinal IgA was found to be unimpaired in
tumour necrosis factor receptor (TNFR)-I–/– mice, which
are characterised by almost no Peyer’s patches, and those
that are present lack the normal follicular structure and
germinal centres. Moreover, the ability of the T-cell-
deficient (or TNFR-I–/–) mice to mount intestinal sIgA adap-
tive responses to changes in the antigenic content of the
intestinal flora was unimpaired. Thus IgA is only partly
dependent on T-cell help for induction, and in particular
the responses to the commensal intestinal flora are much
more T-independent than responses to cholera toxin. This
appears to make sense, since protection from the intestinal
flora would presumably require a broad-based secretory
IgA response, whereas the production of neutralising activ-
ity against intestinal bacterial toxins needs a narrow speci-
ficity, high-affinity IgA.
It has been known that IgA hybridomas from B1 cells
produce antibodies that bind to intestinal bacterial deter-
minants [119]. Using the radiation chimaera approach
described in Section 2.2, and illustrated in figure 5, we
showed that much of the T-independent intestinal IgA was
of B1 lymphocyte origin. The T-independent intestinal IgA
derived from B1 cells is not ’natural’ antibody in the sense
that it is present in the absence of intestinal bacteria
themselves; T-cell-deficient mice recolonised with bacte
Microbes and Infection
2001, 1021-0
IgA induction by pathogenic or non-pathogenic microorganisms
ria producing a novel protein in the intestine in vivo mount
an IgA response to that protein, so as with some serum
B1-derived antibodies [46, 47, the T-independent system
is moulded (positively selected) by changes in the intesti-
nal flora. The fact that this occurs even in mice which
already have a flora indicates that this is not just a non-
specific immunostimulatory effect of the presence of intes-
tinal bacteria per se, but the system can respond to alter-
ations in the content and diversity of the intestinal flora.
The precise role of these adaptive IgA responses in provid-
ing a barrier against penetration of commensal organisms
from the intestine into the body remains to be determined.
6. Conclusions
Although we now know that IgA can be induced both
by T-dependent and T-independent mechanisms, and it
can bind bacteria of the commensal intestinal flora, we
still know comparatively little about its influence on the
density and composition of the flora. This is not an easy
area to address, because intestinal bacteria exist in mucus
biofilms adjacent to the epithelial cell layer as well as the
free-living (planktonic) forms present in the intestinal
lumen. It is the organised biofilm colonies that are likely to
be the most relevant for stimulation of the mucosal and
systemic immune systems, and penetration into the body
when the intestinal barrier is breached. The fact that the
bacterial flora is limited in neonates by adaptive and
innate immune factors in maternal milk has poorly under-
stood consequences on the developing immune system,
but epidemiological evidence in formula-fed infants in the
developing and developed world suggests that adaptive
immunity transmitted from the intestine to the mammary
gland may have significant (and possibly long-term) con-
sequences for human health.
Acknowledgments
We would like to thank Professors Rolf Zinkernagel and
Hans Hengartner for their encouragement and support.
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