Mucosal Immunity: Induction, Dissemination, and Effector Functions
Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Centre for Immune Regulation, University of Oslo, Division and Institute of Pathology, Oslo University Hospital, Rikshospitalet, Oslo, Norway
Prevention of infections by vaccination remains a compelling goal to improve public health. Most infections involve the mucosae, but the development of vaccines against many of these pathogens has yet to be successful. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion – a term coined for non-inflammatory antibody shielding of internal body surfaces – mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA) – produced by local plasma cells stimulated by antigens that target the mucosae. SIgA was early shown to be complexed with an epithelial glycoprotein – the secretory component (SC). In 1974, a common SC-dependent transport of pIgA and pentameric IgM was proposed. From the basolateral surface, pIg-SC complexes are taken up by endocytosis and finally extruded into the lumen. Membrane SC is now referred to as polymeric Ig receptor (pIgR). In 1980, it was shown to be synthesized as a larger transmembrane protein – first cloned from rabbit and then from human. Mice deficient for pIgR showed that this is the only receptor responsible for epithelial transport of IgA and IgM. In the gut, induction of B cells occurs in gut-associated lymphoid tissue, particularly the Peyer’s patches, but also in mesenteric lymph nodes. Plasma cell differentiation is accomplished in the lamina propria to which the memory/effector cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue – but by different homing receptors. Such compartmentalization is a challenge for development of mucosal vaccines.
The epithelial lining of mucous membranes is generally vulnerable and covers an area of several hundred square metres in an adult – some 200 times larger than the skin. It is apparent from everyday life that these extensive surfaces represent the most frequent portals of entry for common infectious agents, allergens and carcinogens. This is true not only for the airways and the gut but also for the conjunctiva covering the eyes as well as for the urinary and genital tracts. Mucosal infections are the major killer below the age of 5 years, being responsible for some 10 million deaths of children annually; for at least 6 million of these cases, there is no available effective vaccine. Diarrhoeal disease alone claims a toll of several million per year in the developing countries. These frightening figures document the need for a better understanding of mucosal defence mechanisms. The hope is that appropriate induction of secretory immunoglobulin A (SIgA) antibodies by mucosal vaccines can solve some of the global health problems.
Although this brief review of mucosal immunity focuses mainly on the human system, much basic information is derived from mouse experiments. However, there are important species differences, some of which will be pointed out below.
Brief history of secretory immunity
How SIgA was discovered
Research over several decades has revealed how mucosal surface epithelia are endowed with specific adaptive immune protection provided by antibodies contained in external body fluids such as tears, nasal secretions, saliva, intestinal juice and breast milk. The notion that humoral mucosal immunity is distinct from the systemic counterpart was conceived in 1919 by Besredka – the Director of the Pasteur Institute in Paris – on the basis of his experimental model for oral Shigella dysenteriae vaccination in rabbits . This concept was further supported in 1922 by the English military doctor Davies who examined antibodies in dysentery stools from soldiers stationed in Egypt . However, it took more than 35 years before antibody activities in external secretions were shown to be carried mainly by IgA as reviewed in more detail elsewhere .
IgA had been discovered in serum by Grabar and Williams as early as in 1953–54 , but Joseph F. Heremans was first to isolate and characterize this antibody isotype (1959–60). Simultaneously with Heremans’ work, it was reported that IgA was the main immunoglobulin in human milk by Gugler and von Muralt  and by a young Swedish doctor named Lars Å. Hanson . Then, Thomas B. Tomasi and his coworkers in the USA showed the predominance of IgA also in saliva and several other human secretions . The striking abundancy of IgA-producing plasmablasts and plasma cells (PC) in human gut mucosa was published by Crabbéet al.  from Heremans’ laboratory in 1965.
In 1961 Hanson’s studies on IgA in breast milk, which were performed in Ouchterlony’s laboratory in Göteborg, Sweden, resulted in a report describing that SIgA contains an additional antigenic epitope lacking on serum IgA . The following year this structure was also observed in IgA antibody preparations purified from milk . Tomasi’s subsequent work in Kunkel’s laboratory at the Rockefeller Institute, New York, resulted in the crucial physicochemical characterization of the additional antigenic portion of SIgA in 1965  – identified as an epithelial ∼80-kDa glycoprotein called ‘secretory piece’ and later designated secretory component (SC) by WHO or – when expressed in its elongated (∼100 kDa) transmembrane form – now generally referred to as the polymeric Ig receptor (pIgR).
How SIgA is exported
In the late 1960s and early 1970s at least six different models were proposed to explain how IgA can be selectively exported to external secretions as SIgA (Fig. 1), but they turned out to be wrong as reviewed also in this Journal some 25 years ago [12, 13]. These models all suggested that SIgA was derived from monomeric IgA that became dimerized by complexing with SC either inside or outside the epithelial cell. It was therefore a breakthrough for the visualization of the correct transport model when mucosal PC in 1973–74 were shown to produce dimeric IgA with a small associated peptide called joining (J) chain [14, 15]. Also, in 1968 it was for the first time reported that the large pentameric IgM molecules, like dimeric IgA, were translocated to external secretions by an active transepithelial mechanism . Subsequently, secretory IgM (SIgM) was shown to become associated with SC on its passage through the cytoplasm of secretory epithelial cells in a manner similar to SIgA, but without being covalently complexed – providing a less stable quaternary structure .
It was then speculated that there must be a common receptor-mediated epithelial transport mechanism for these two polymeric immunoglobulins, and SC was suggested to be a good candidate for an epithelial cell-surface receptor translocating both dimeric IgA and pentameric IgM [14, 15]. This was a unique biological concept based both on collaboration between two different cell types to produce the hybrid SIgA and SIgM molecules, and export of the product by a receptor that was sacrificed and not recycled.
An integrated function of SC and J chain as a ‘key-and-lock’ mechanism in the epithelial uptake of polymeric immunoglobulins, was firmly supported by subsequent work in our laboratory [13, 18]. Thus, the transport model published in 1974  implied that SC can act as transmembrane pIgR when expressed on the basolateral surface of secretory epithelial cells. This has been substantiated by subsequent molecular biology studies particularly by Mostov, as reviewed elsewhere [13, 20, 21].
Immunobiology of SIgA
External transport of secretory antibodies is a fundamental biological phenomenon which has been preserved and enhanced throughout the phylogeny of tetrapods , probably because it is essential for the survival of particularly the mammalian species. The SIg system represents quantitatively the most important part of the antibody-dependent defence system of the body. On the basis of immunohistochemical studies performed in our laboratory, it has been calculated that there are about 1010 IgA-producing PC per metre of bowel, whereas the total number of PC in lymph nodes, spleen and bone marrow altogether amounts to 2.5 × 1010 . This means that at least 80% of the antibody production of the body takes place locally in the gut lamina propria – mainly providing dimeric IgA ready for pIgR-mediated export [24–26]. In an adult, approximately 3 g of dimeric IgA is thus translocated to the gut lumen every day as SIgA, which is more than the total daily production of IgG in the body. This remarkable immunobiological mechanism is still fairly unknown to many immunologists.
The clinical importance of such an efficient antibody export to internal body surfaces is best appreciated in view of the paramount protective significance of the mucosal immune system. The role of SIgA antibodies in the defence against infections is highlighted by the fact that most pathogens are encountered by the mucous membranes. A substantial fraction of the almost 40,000 children under the age of 5 years dying every day succumb from mucosal infections in the gastrointestinal and respiratory tracts. The protection for the breast-fed infant provided by SIgA in mother’s milk was early shown for cholera, enterotoxic Escherichia coli and Campylobacter infections [27–29]. Moreover, breast-feeding can protect against neonatal septicemia with an odds ratio of 18 compared with infants who are not breast-fed ; and the risk of dying from diarrhoea in infancy has been reported to be at least 20 times higher without breast-feeding compared with exclusive breast-feeding in parts of the developing world . Despite the fact that the mortality of diarrhoea has shown a recent decline, it is still responsible for some 2.5 million deaths per year in children below 5 years of age, and the enormous morbidity apparently remains the same .
Experiments in neonatal rabbits have clearly documented that the most important antimicrobial factor in breast milk is SIgA , and the pIgR/SC-dependent transport model explains why colostrum and mature breast milk is such a remarkably rich source of SIgA antibodies . Importantly, it has more recently been documented in extensive epidemiological studies that breast-feeding is the most efficient feasible intervention measure to prevent deaths before 5 years of age in developing countries [35, 36]. A beneficial clinical effect is also apparent in the industrialized world, even in relation to relatively common infections or inflammatory diseases . Altogether, the mucosal immune system truly is of crucial importance for the well-being of the young as well as the adult.
Strategies of the mucosal immune system
Role of oral tolerance
It appears from the above historical account that the mucosal immune system provides a first defence line of the inner body surfaces. This reduces the need for activation of proinflammatory systemic immunity to eliminate invading infectious agents and other exogenous antigens. Thus, to maintain homeostasis, the mucosal immune system has, through evolution, developed two layers of adaptive anti-inflammatory defence: (a) immune exclusion provided primarily by SIgA antibodies to limit epithelial contact and penetration with host invasion of micro-organisms and other potentially dangerous antigens, and (b) immunosuppressive mechanisms to inhibit overreaction against innocuous luminal antigens (Fig. 2).
The latter strategy, which is referred to as ‘oral tolerance’ when induced via the gut [3, 38], depends largely on the development of regulatory T (Treg) cells in mesenteric lymph nodes to which mucosal dendritic cells (DC) carry microbial and dietary antigens and become conditioned for induction of Treg cells [39, 40]. Such mucosally induced tolerance to food and other innocuous antigens probably involves additional suppressive mechanisms, and a similar downregulatory tone of the immune system normally exists against commensal bacteria [41, 42]. Oral tolerance apparently contributes to the fact that overt and persistent hypersensitivity to food is relatively rare, although – for still unclear reasons – being on the rise in westernized societies .
The epithelial barrier
Mucosal tolerance is considered to be a robust suppressive mechanism in view of the fact that more than a ton of food may pass through the gut of an adult every year. After a meal, intact dietary antigens are taken up in the nanogram range, usually without causing harm . However, the neonatal period is critical, both with regard to infections and to priming for allergic disease, because the epithelial barrier and the immunoregulatory network are poorly developed [44, 45].
Experiments have demonstrated a crucial role of microbial colonization in establishing  and regulating  the epithelial barrier. At least in mice, the beneficial effects of commensal bacteria on the barrier function are largely mediated via pattern recognition receptors (PRR) expressed by the gut epithelium, particularly toll-like receptors (TLR) [48, 49]. Polarized epithelial cells have the ability to dampen the proinflammatory effect of PRR-mediated signals coming from the luminal side [42, 47]. However, after bacterial invasion, PRR signalling from the basolateral side results in a high level of NF-κB activation with enhanced release of defensins to combat the infection .
Secretory immunity reinforces the epithelial barrier
The mucosal and systemic immune systems differ in many structural, cellular, molecular and functional ways [40, 50]. Mucosal immunity is most abundantly expressed in the gut. As mentioned above, the intestinal mucosa of an adult contains more than 80% of the body’s activated B cells – terminally differentiated to plasmablasts and PC [24, 25]. Thus, the gut is by far the largest antibody-producing organ in the body.
Most mucosal PC produce dimeric IgA which, along with pentameric IgM that likewise contains J chain, can be actively exported by secretory epithelia as discussed earlier. The historical account provided above shows that it took some time before it became clear that this external transport is mediated by pIgR/SC expressed basolaterally on secretory epithelial cells, with the exception of goblet cells [13, 19, 25]. When we first made a pIgR/SC knockout mouse we could exclude that other receptors were involved in this process . Thus, efficient immune exclusion depends on pIgR/SC as it is performed mainly by SIgA, and to a lesser extent SIgM, in cooperation with innate non-specific defences (Table 1, Figs. 3 and 4). In newborns and people with selective IgA deficiency, SIgM antibodies are of greater importance than in healthy adults .
Table 1. Antimicrobial effects of secretory immunoglobulin A (SIgA) antibodies.
SIgA is dimeric/polymeric, therefore exerting efficient microbial agglutination and virus neutralization
SIgA performs non-inflammatory extracellular and intracellular immune exclusion by inhibiting epithelial adherence and invasion
SIgA exhibits cross-reactive (‘innate-like’) activity and provides cross-protection in the herd
SIgA (particularly SIgA2) is quite stable (bound SC stabilizes both isotypes of IgA)
SIgA is endowed with mucophilic and lectin-binding properties (via bound SC in both isotypes and mannose in IgA2)
Immune-inductive mucosa-associated lymphoid tissue (MALT) resembles lymph nodes with B-cell follicles, intervening T-cell zones and a variety of antigen-presenting cells (APC), such as macrophages and DC (Fig. 5), but there are no afferent lymphatics . Exogenous stimuli therefore come directly from the mucosal surfaces via a follicle-associated epithelium containing specialized epithelial M cells , probably aided by DC which are activated by bacterial components and may penetrate the epithelium with their processes [54, 55].
In the intestine, induction and regulation of mucosal immunity hence takes place primarily in Peyer’s patches, together with other parts of gut-associated lymphoid tissue (GALT) such as the numerous isolated lymphoid follicles and the appendix as well as the gut-draining mesenteric lymph nodes [25, 26, 50], but also to some extent at the effector sites to which activated T and B cells home (Fig. 5). It should be noted that despite the important role of mesenteric lymph nodes in intestinal immunity, they should not be called GALT (or MALT) structures  according to the terminology recommended by the International Society for Mucosal Immunology (SMI) and the International Union of Immunological Societies (IUIS). This is so because the mesenteric lymph nodes, in contrast to GALT/MALT structures, do not sample antigens directly from the lumen through a specialized epithelium containing M cells.
Induction and homing mucosal B cells
The induction and switch process
Retinoic acid (RA) derived from vitamin A in the diet exerts a positive impact both on intestinal differentiation of naïve mucosal B cells and their gut homing as precursors for the IgA-producing PC [56, 57]. Thus, the heavy chain switching to IgA both in humans and mice is enhanced by RA, and so is the expression of integrin α4β7 and the CC chemokine receptor CCR9 (Figs. 6 and 7). The phenotype of APC in GALT, which seems to be imprinted by the action of gut bacteria on PRR, promotes RA generation by their expression of retinaldehyde dehydrogenase (RALDH); and their inducible nitric oxide synthase (iNOS) expression enhances via nitric oxide (NO) the release of the innate switch factors APRIL (A PRoliferation-Inducing Ligand) and BAFF (BlyS) from APC , and the release of the activated IgA switch factor transforming growth factor (TGF)-β from helper T cells . Furthermore, in mice it has been shown that a fraction for these follicular T helper (TFH) cells may be derived from Treg cells . There may thus be a cellular link between induction of intestinal IgA and oral tolerance.
The propensity of the mucosal immune system to generate low-affinity cross-reactive background antibodies is probably explained by the extensive innate drive imposed on it by the abundant commensal microbiota via PRR . Thus, experiments have documented a role of TLR for B-cell differentiation in GALT structures . Interestingly, human GALT follicles contain the apparatus to support both T cell-dependent and T cell-independent (not involving CD40–CD40L interactions) class switch recombination (CSR) pathways to IgA (Fig. 7). This has recently been documented by showing restricted expression of activation-induced cytidine deaminase (AID) – an essential enzyme for CSR . Also notable, the T cell-independent switch factor APRIL, which is a secreted cytokine member of the tumour necrosis factor (TNF) family, and its receptors TACI (Transmembrane Activator and CAMEL Interactor) and BCMA (B Cell Maturation Antigen) are expressed in human GALT, as well as beyond these structures. However, outside of GALT there is no co-expression of the receptors for APRIL and the switch enzyme AID .
Thus, previous claims about B-cell switch to IgA in human lamina propria appear questionable, and the same is true for an extrafollicular switch from IgA1 to IgA2 which has recently been proposed . The possibility remains, however, that the unique B1 cell population generated in the mouse omentum  may provide a substantial fraction of lamina propria PC and be subjected to IgA switch either in the peritoneal cavity or in the lamina propria of this species [50, 63]. Another and perhaps more likely possibility is nevertheless that T cell-independent switch to IgA mainly takes place in the numerous isolated lymphoid follicles in the distal gut , and that APRIL outside of MALT structures mainly promotes the survival of PC in mucosae similarly to the role of BAFF in the bone marrow .
Quite recently, the long-lasting debate [25, 26] about the role of vasoactive intestinal polypeptide (VIP) in IgA induction also appears to be clarified (Fig. 7). An IgA-inducing protein (IGIP) first identified in the bovine species has now been characterized in humans – with somewhat different properties ; its CD40L-stimulated expression in DC was found to be 35-fold enhanced by VIP, and IGIP was directly shown to induce CSR to IgA in naïve (IgM+IgD+) B cells. However, a problem with all the experimental studies on IgA-promoting factors operating in GALT, such as TGF-β, RA and IGIP, is that they have not been tested for J chain-inducing properties, which are a prerequisite for production of dimeric IgA. Although there is considerable knowledge about the regulation of J-chain expression in mice, such knowledge is lacking in humans; and the factors responsible for the high level of J chain in GALT-derived B cells are not known in any species [21, 26].
Mucosal vaccination challenges
Although immunological memory for high-affinity IgA antibody production is generated after proper mucosal priming , this may be masked by a self-limiting SIgA response shielding the inductive lymphoid structures, particularly the Peyer’s patches of GALT . Additional challenges for mucosal vaccine application is the choice of immunogen formulation for best response and the regionalization of the mucosal immune system with regard to migration of memory/effector B cells to various effector sites [67–69]. Nasal vaccines that target nasopharynx-associated lymphoid tissue (NALT) of Waldeyer’s ring and cervical lymph nodes elicit both regional mucosal and systemic immunity (Fig. 8) but do not regularly furnish the small intestine with activated B cells [26, 70]. Such disparity of mucosal B-cell homing is masked in the intestinal lumen of rodents where much of the SIgA in the upper part of the gut is derived from bile .
Ethical constraints restrict direct tracking of immune-cell migration throughout the human body in vivo. We therefore used deletion of the IgM heavy-chain constant-gene (Cμ) segment as a marker to provide a dispersal signature of an effector B-cell subset (IgD+IgM−CD38+) induced selectively in human NALT represented by palatine tonsils and adenoids . By DNA analysis, the Cμ deletion identified dissemination of such plasmablasts and their PC progeny to peripheral blood, lymph nodes, and bone marrow, as well as to mucosae and glands of the upper airways. Also the endocervix was often positive while the small intestine was mainly negative, as could be expected from the identified homing-molecule profile of the marker cells – with relatively low levels of integrin α4β7 and the CCR9 (α4β7int./lowCCR7highCCR9lowCCR10+ CD62Lhigh). Of further importance, the circulating cells abundantly expressed CD62L (L-selectin) and CCR7, which provided a mechanism for integration of respiratory and systemic immunity. Importantly, lactating mammary glands depend on CCR10, at least in mice , and apparently receive precursors for IgA+ PC both from GALT and NALT (Fig. 8), as reflected in breast milk SIgA antibody activities .
The vulnerable neonatal period
Development of secretory immunity
IgA-producing PC are generally undetectable in the mucosae before 10 days of age, but thereafter they increase rapidly. However, IgM-producing PC often remain predominant up to 1 month . Usually, intestinal IgA increases little after 1 year, although a much faster establishment of secretory immunity is often seen in developing countries with a heavy microbial load . The mucosal PC development thus appears to reflect the progressive microbial stimulation of MALT [26, 44]. Accordingly, only occasional traces of SIgA and SIgM occur in intestinal juice during the first postnatal period, whereas some IgG is often present, reflecting paracellular ‘leakage’ from the lamina propria, which after 34 weeks of gestation contains readily detectable maternal IgG . In addition, some IgG may be actively exported by epithelial FcRn . More importantly, both RA from vitamin A and buturate derived from microbial fermentation of oligosaccharides in food (and breast milk), as well as certain microbial TLR ligands, can upregulate the epithelial pIgR/SC expression and thereby enhance the active SIgA export . This can add to the cytokine-stimulated enhancement of pIgR/SC expression [21, 26].
Uptake of SIgA antibodies from breast milk via the human neonatal gut mucosa is negligible, however, and probably of no immunological importance except perhaps in preterm infants . So-called gut closure normally occurs mainly before birth, but the mucosal barrier may be inadequate up to 2 years of age. Although the mechanisms involved remain poorly defined, SIgA from breast milk and the development of the infant’s immune system are two related variables in this process [39, 45].
Promotion of homeostasis
Animal experiments have suggested that SIgA-containing immune complexes may be taken up via M cells of GALT and guide the induction of the breast-fed neonate’s immune system to a homeostatic response . Altogether, therefore, it is not surprising that recent meta-analyses show that breastfeeding protects against allergic disease and several other immune-mediated disorders, driven by exogenous factors in developed societies . The same is true for coeliac disease [39, 77]. Notably in this context, pIgR knockout mice that lack secretory antibodies not only show reduced resistance to pathogens such as Vibrio cholera  and Salmonella typhimurium  but they also have increased uptake of antigens from commensal bacteria and food [51, 80]. Their decreased epithelial barrier function leads to a hyperreactive immune system and the mice show predisposition for systemic anaphylaxis after sensitization; this development is, however, counteracted by enhanced oral tolerance as a homeostatic back-up mechanism .
Many variables influence mucosally induced tolerance and productive IgA-dependent secretory immunity. Some of these variables are reciprocally modulated to achieve homeostasis [39, 40]. Increased epithelial permeability is an important primary or secondary event in the pathogenesis of many diseases, including allergy, coeliac disease, and inflammatory bowel disease. The barrier function is determined by the individual’s age (e.g. preterm versus term infant); genetics; mucus; interactions between mast cells, nerves, and neuropeptides; concurrent infection; and the mucosa-shielding effect of SIgA provided by breast milk or produced by the infant’s gut. The remarkable output of SIgA during feeding serves as an optimally targeted passive immunization of the breast-fed infant’s gut, and also may serve as a positive homeostatic feed-back loop.
Many studies indicate that allergy is associated with delayed or impaired development of the IgA system . This is not surprising because secretory immunity is of such great importance for the intestinal barrier function. SIgA not only maintains mutualism with the indigenous microbiota  but also forms the first line of defence against commensals and pathogens as well as other harmful agents (Fig. 3). In addition, epithelial integrity depends on interaction with microbial components from the environment and particularly from the indigenous microbiota, both by direct engagement of epithelial PRR and induction of mucosal tolerance via different immunosuppressive mechanisms, including tolerogenic APC and Treg cells . The SIgA system and the Treg cells seem to be integrated in an effort to preserve the vulnerable mucosal barrier (Fig. 2).
The author is grateful to Hege Eliassen for excellent secretarial assistance. Studies at LIIPAT were supported by the Research Council of Norway, the Norwegian Cancer Society, the University of Oslo, and Oslo University Hospital.