Function of Mucosa-Associated Lymphoid Tissue in Antibody Formation

Abundant evidence supports the notion that human intestinal plasma cells are largely derived from B cells initially activated in gut-associated lymphoid tissue (GALT). Nevertheless, insufficient knowledge exists about the uptake, processing, and presentation of luminal antigens occurring in GALT to accomplish priming and sustained expansion of mucosal B cells. Also, it is unclear how the germinal center reaction so strikingly promotes class switch to IgA and expression of J chain, although the commensal microbiota appears to contribute to both diversification and memory. B-cell migration from GALT to the intestinal lamina propria is guided by rather well-defined adhesion molecules and chemokines/chemokine receptors, but the cues directing homing to secretory effector sites beyond the gut require better definition. In this respect, the role of human Waldeyer's ring (including adenoids and the palatine tonsils) as a regional mucosa-associated lymphoid tissue must be better defined, although the balance of evidence suggests that it functions as nasopharynx-associated lymphoid tissue (NALT) like the characteristic NALT structures in rodents. Altogether, data suggest a remarkable compartmentalization of the mucosal immune system that must be taken into account in the development of effective local vaccines to protect specifically the airways, small and large intestines, and the female genital tract.     

Conjunctiva-associated lymphoid tissue in avian mucosal immunity

Conjunctiva-associated lymphoid tissue’s (CALT) role in generating avian mucosal adaptive immunity was measured by analyzing cellular composition, expression of the polymeric immunoglobulin receptor (pIgR), and production of cytokines and antibodies in chickens ocular exposed to a replication-deficient adenovirus of serotype 5 (Ad5). These studies demonstrate that CALT contains B cells, γδ T cells, T helper, and cytotoxic T cells, and a T lymphocyte composition, which more resembles Harderian glands than spleen. CALT-derived lymphocytes contain antigen-specific, IgA-secreting plasma cells and cytokine-producing lymphocytes after ocular Ad5 vaccination. The expression of the pIgR in the CALT’s lymphoepithelium emphasizes the importance of mucosal immune protection by paraocular lymphoid tissues. The CALT immune response after ocular Ad5 boosting was influenced by prior high dose in ovo Ad5 priming. Thus, both mucosal and systemic immunization influenced Ad5-induced IFN-γ responses in CALT.     

Specific microbiota enhances intestinal IgA levels by inducing TGF-β in T follicular helper cells of Peyer's patches in mice

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-β, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the generation and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-β expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer's patches, enhancing IgA⁺ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-β and of mucosal IgA.     

Fostering of advanced mutualism with gut microbiota by Immunoglobulin A

Immunoglobulin A (IgA), the most abundantly secreted antibody isotype in mammals, not only provides direct immune protection to neonates via maternal milk but also helps program the infant immune system by regulating the microbiota. IgA continues to maintain dynamic interactions with the gut microbiota throughout life and this influences immune system homeostasis as well as other physiological processes. The secretory IgA produced independently of T-cell selection are commonly referred to as natural or innate antibodies. Our studies have shown that innate-IgA, while effective at excluding microorganisms from the gut, does not promote mutualism with the microbiota in the same way as adaptive-IgA that is selected in T cell-dependent germinal center reactions. Adaptive-IgA fosters more advanced mutualism with the microbiota than innate-IgA by selecting and diversifying beneficial microbial communities. In this review, we suggest that the diversified microbiota resulting from adaptive-IgA pressure was pivotal in promoting ecological adaptability and speciation potential of mammals.     

Role of secretory antibodies in the defence against infections

Adaptive immunity mediated by secretory antibodies is important in the defence against mucosal infections. Specific secretory immunoglobulin A (SIgA) can inhibit initial pathogen colonization by performing immune exclusion both on the mucosal surface and within virus-infected secretory epithelial cells without causing tissue damage. Resistance against toxin-producing bacteria such as Vibrio cholerae appears to be particularly dependent on SIgA antibodies. Like natural infections, live topical vaccines or adequate combinations of inactivated vaccines and mucosal adjuvants give rise not only to SIgA antibodies, but also to long-standing serum IgG and IgA responses. The intranasal route of vaccine application could be particularly attractive to achieve this result, but only if successful stimulation is obtained without the use of toxic adjuvants. The degree of protection after vaccination may range from complete inhibition of reinfection to reduction of symptoms. In this scenario it is generally difficult to determine unequivocally the relative importance of SIgA versus serum antibodies. However, infection models in knockout mice strongly support the notion that SIgA exerts a decisive role in protection and cross-protection against a variety of infectious agents.     

B cells and the intestinal microbiome in time,space and place

The gut immune system is shaped by the continuous interaction with the microbiota. Here we dissect temporal, spatial and contextual layers of gut B cell responses. The microbiota impacts on the selection of the developing pool of pre-immune B cells that serves as substrate for B cell activation, expansion and differentiation. However, various aspects of the gut B cell response display unique features. In particular, occurrence of somatically mutated B cells, chronic gut germinal centers in T cell-deficient settings and polyreactive binding of gut IgA to the microbiota questioned the nature and microbiota-specificity of gut germinal centers. We propose a model to reconcile these observations incorporating recent work demonstrating microbiota-specificity of gut germinal centers. We speculate that adjuvant effects of the microbiota might modify permissiveness for B cell to enter and exit gut germinal centers. We propose that separating aspects of time, space and place facilitate the occasionally puzzling discussion of gut B cell responses to the microbiota.     

Effect of Oral Vs. Parenteral Cyclophosphamide on In Vitro Iga and Igg Production by Murine Peyer's Patches and Cultured Jejunal Fragments

Abstract

The gut associated lymphoid tissue plays an important role in intestinal defenses, food allergy, oral tolerance, and certain intestinal diseases. This study describes the effect of either oral or parenteral cyclophosphamide on IgA and IgG production in the gut. Mice were treated with cyclophosphamide either IV or PO, and Peyer's patch cell cultures were established to evaluate mitogen induced production of IgA and IgG. To evaluate the effect of cyclophosphamide on the plasma cell rich lamina propria, segments of jejunum were cultured and overnight secretion of IgG and IgA were measured. We found, the secretion of IgA or IgG by jejunal fragments was not influenced by cyclophosphamide (IV or PO). Mitogen induced secretion of IgA and IgG by Peyer's patch cells was markedly decreased 24 hrs after drug administration, with significant recovery by day 7. Cell mixing experiments revealed that a single dose cyclophosphamide reduced the capacity of Peyer's patch B cells to secrete IgA or IgG when co cultured with normal T cells. This study demonstrates that a single dose cyclophosphamide can have profound effects on the gut immune system and that the drug has a similar effect when given either orally or parenterally.     

Eosinophils: important players in humoral immunity

Eosinophils perform numerous tasks. They are involved in inflammatory reactions associated with innate immune defence against parasitic infections and are also involved in pathological processes in response to allergens. Recently, however, it has become clear that eosinophils also play crucial non‐inflammatory roles in the generation and maintenance of adaptive immune responses. Eosinophils, being a major source of the plasma cell survival factor APRIL (activation and proliferation‐induced ligand), are essential not only for the long‐term survival of plasma cells in the bone marrow, but also for the maintenance of these cells in the lamina propria which underlies the gut epithelium. At steady state under non‐inflammatory conditions eosinophils are resident cells of the gastrointestinal tract, although only few are present in the major organized lymphoid tissue of the gut – the Peyer's patches (PP). Surprisingly, however, lack of eosinophils abolishes efficient class‐switching of B cells to immunoglobulin (Ig)A in the germinal centres of PP. Thus, eosinophils are required to generate and to maintain mucosal IgA plasma cells, and as a consequence their absence leads to a marked reduction of IgA both in serum and in the gut‐associated lymphoid tissues (GALT). Eosinophils thus have an essential part in long‐term humoral immune protection, as they are crucial for the longevity of antibody‐producing plasma cells in the bone marrow and, in addition, for gut immune homeostasis.     

Immunologic studies in bronchoalveolar fluid in a patient with allergic bronchopulmonary aspergillosis

Bronchoalveolar lavage was performed in a patient during an acute stage of allergic bronchopulmonary aspergillosis (ABPA) and repeated 17 mo later during a remission stage and cessation of corticosteroid therapy. Measurement of protein concentrations (albumin, IgG, IgA, and IgM) by laser nephelometry and crossed immunoelectrophoresis indicates that transudation of proteins occurs from the circulation into the lung compartment during the acute stage of ABPA. Furthermore, comparing total Ig concentrations and titers of ELISA IgG, IgA, and IgM antibodies against Aspergillus fumigatus with the corresponding values of serum measurements indicates a preferential local production of IgA antibodies and IgM to a lesser extent. No indications were found for a local production of either IgG, total IgE, or IgE against A. fumigatus. During remission of the ABPA, titers of antibodies in the bronchoalveolar fluid (BAF) decreased to normal levels except IgA against A. fumigatus that remained elevated. The elevated titers of IgA in the BAF and IgE antibodies in the serum against A. fumigatus together with the elevated numbers of granulocytes in the BAF during the remission stage indicate that the immunologic situation is not yet normalized.     

Systemic immunization against IgA in immunoglobulin deficiency.

The presence of serum IgM and IgG antibodies against IgA is common among individuals with IgA deficiency. The route of immunization is still unknown, but it is possible that immunization occurs through the gut. We analysed anti-IgA antibody production in gastrointestinal lavage, saliva and breast milk from patients with IgA deficiency. In no case was there any evidence of local production of anti-IgA antibodies. Immunization may thus be due to exposure to endogenous IgA and therefore represent a 'true' autoimmune phenomenon which may possibly be involved in the pathogenesis of the disease.     

The anatomical basis for the immune function of the gut

Summary The barrier function of the gut wall can be divided into different histotopographically defined lines of defence. These consist not only of lymphoid cells but also of goblet cells, entero-endocrine cells, macrophages and mast cells. Subsets of lymphoid cells are found preferentially within the epithelium (T suppressor) or in the lamina propria (T helper). Most plasma cells produce IgA. Peyer's patches are described in detail as typical organized lymphoid structures of the gut. In man, they are present well before birth and are found in large numbers even in old age. They are not only typical for the ileum but are also present in the duodenum and jejunum. The four compartments in Peyer's patches, i.e. follicle, corona, interfollicular area and the dome, are defined by the typical localization of lymphocyte subsets and by their different functions. Typical features of the epithelium of the dome are the lack of villi and goblet cells and the presence of specialized epithelial cells (M cells) which are important for the uptake of particulate antigen from the gut lumen. Precursor cells of IgA producing plasma cells leave the intestinal wall via the lymphatics and return preferentially to the gut mucosa, and this is summarized by the term gut-associated lymphoid tissue (GALT). Other organs with mucous membranes, such as mammary and salivary glands, bronchial and genital tract, are also included in this circulatory route and this is expressed by the term mucosa-associated lymphoid tissue (MALT). Mast cells in the gut mucosa can be classified as connective tissue or mucosa mast cells. These differ in their sensitivity to formaldehyde as a fixative, contain different granules and mediators, have different origins, and show major differences in the effectiveness of antiallergic compounds on the stabilizing of the cell membrane. Mucosa mast cells have also been demonstrated in the human gut. The histotopographical relationship of many cell types such as goblet and M cells in addition to cells of the immune system such as lymphoid cells, macrophages and mast cells, is essential in the understanding of the barrier function of the gut wall.     

IgG and IgA subclass mRNA-bearing plasma cells in periodontitis gingival tissue and immunoglobulin levels in the gingival crevicular fluid

The humoral immune response, especially the production of IgG and IgA, is considered to have a protective role in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. In order to determine local IgG and IgA production, we investigated the presence of human IgG and IgA subclass mRNA-bearing plasma cells within periodontal tissue by in situhybridization using digoxigenin-labelled oligonucleotide probes in 24 gingival biopsy samples (pocket depth>5 mm) which were obtained from eight patients with adult periodontitis. Furthermore, we examined IgG and IgA subclass proteins and digested IgA1 Fab portions in the gingival crevicular fluid (GCF), corresponding to the sites from which the tissues were taken, by ELISA. IgG and IgA subclass mRNA-expressing cells were detected in all serial formalin-fixed/paraffin-embedded gingival tissue sections sampled. Plasma cells showed strong cytoplasmic staining with a high contrast and a good retention of morphology with these probes. IgG1 mRNA-expressing cells were predominant (mean 63%) and IgG2 mRNA-expressing cells were present at around 23% of total IgG plasma cells, while IgG3 and IgG4 mRNA-expressing cells were present to a much lesser extent (3% and 10%, respectively). Similar proportions of IgG subclass proteins in GCF were detected, which were also consistent with ‘normal’ serum levels. In terms of IgA subclass, IgA1 mRNA-positive cells were predominant (mean 65.1%, P<0.001). In contrast, IgA2 protein in the GCF samples were detected at higher concentrations than IgA1 (P<0.001). The ratio of total IgG to IgA mRNA-positive plasma cells was ≈7.5:1. There was a good correlation between the amounts of IgG subclass proteins in GCF and the number of IgG subclass mRNA-positive cells in the same sites, but not between IgA subclass proteins and the number of IgA subclass mRNA-positive cells. These data suggest that IgG and IgA subclass proteins can be locally produced in the periodontitis gingiva. In addition, as we detected IgA1 Fab fragments in GCF, this is further confirmation that secreted IgA1 protein in GCF may be digested by periodontal bacteria.     

Serum and salivary IgA antibody responses to Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans in orofacial granulomatosis and Crohn's disease

Orofacial granulomatosis (OFG) is a condition of unknown aetiology with histological and, in some cases, clinical association with Crohn's disease (CD). However, the exact relationship between OFG and CD remains uncertain. The aim of this study was to determine whether OFG could be distinguished immunologically from CD by comparing non-specific and specific aspects of humoral immunity in serum, whole saliva and parotid saliva in three groups of patients: (a) OFG only (n = 14), (b) those with both oral and gut CD (OFG + CD) (n = 12) and (c) CD without oral involvement (n = 22) and in healthy controls (n = 29). Non-specific immunoglobulin (IgA, SigA, IgA subclasses and IgG) levels and antibodies to whole cells of Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans were assayed by enzyme-linked immunosorbent assay (ELISA) in serum, whole saliva and parotid saliva. Serum IgA and IgA1 and IgA2 subclasses were raised in all patient groups (P < 0.01). Salivary IgA (and IgG) levels were raised in OFG and OFG + CD (P < 0.01) but not in the CD group. Parotid IgA was also raised in OFG and OFG + CD but not in CD. The findings suggest that serum IgA changes reflect mucosal inflammation anywhere in the GI tract but that salivary IgA changes reflect involvement of the oral cavity. Furthermore, the elevated levels of IgA in parotid saliva suggest involvement of the salivary glands in OFG. Serum IgA antibodies to S. cerevisiae were raised markedly in the two groups with gut disease while serum IgA (or IgG) antibodies to C. albicans were elevated significantly in all three patient groups (P < 0.02). No differences were found with antibodies to S. mutans. Whole saliva IgA antibodies to S. cerevisiae (and C. albicans) were raised in the groups with oral involvement. These findings suggest that raised serum IgA antibodies to S. cerevisiae may reflect gut inflammation while raised SIgA antibodies to S. cerevisiae or raised IgA or IgA2 levels in saliva reflect oral but not gut disease. Analysis of salivary IgA and IgA antibodies to S. cerevisiae as well as serum antibodies in patients presenting with OFG may allow prediction of gut involvement.     

Mucosal Immune System in Health and Disease

The mucosal immune system is characterized predominantly by the secretory antibody response and gut-associated lymphoid tissue, cellular part of the mucosal immune system. The secretory antibody system depends on local production and selective epithelial transport of secretory IgA and IgM. Furthermore, secretory antibodies and interactions between the intestinal epithelium and T cells are involved in the mucosal down-regulation of the systemic immune system. Neuropeptides play a crucial role in the regulation of mucosal immune responses. It is possible that impairment of the mucosal immune response contribu tes to the pathogenesis of various intestinal diseases, such as inflammatory bowel disease. Until recently, however, mucosal immunity received relatively little attention from both basic and clinical scientists. Further research on mucosal immunity seems to have promise in helping to provide new understanding of the immune mechanisms and pathogenesis of several gastrointestinal and systemic diseases.     

A simple method for determining polymeric IgA-containing immune complexes

A simplified assay to measure polymeric IgA-immune complexes in biological fluids is described. The assay is based upon the specific binding of a secretory component for polymeric IgA. In the first step, multimeric IgA (monomeric and polymeric) immune complexes are determined by the standard Raji cell assay. Secondly, labeled secretory component added to the assay is bound to polymeric IgA-immune complexes previously fixed to Raji cells, but not to monomeric IgA immune complexes. To avoid false positives due to possible complement-fixing IgM immune complexes, prior IgM immunoadsorption is performed. Using anti-IgM antiserum coupled to CNBr-activated Sepharose 4B this step is not time-consuming.

Polymeric IgA has a low affinity constant and binds weakly to Raji cells, as Scatchard analysis of the data shows. Thus, polymeric IgA immune complexes do not bind to Raji cells directly through Fc receptors, but through complement breakdown products, as with IgG-immune complexes. Using this method, we have been successful in detecting specific polymeric-IgA immune complexes in patients with IgA nephropathy (Berger's disease) and alcoholic liver disease, as well as in normal subjects after meals of high protein content. This new, simple, rapid and reproducible assay might help to study the physiopathological role of polymeric IgA immune complexes in humans and animals.     

Development of gut immunoglobulin A production in piglet in response to innate and environmental factors

The current review focuses on pre- and post-natal development of intestinal immunoglobulin A (IgA) production in pig. IgA production is influenced by intrinsic genetic factors in the foetus as well as extrinsic environmental factors during the post-natal period. At birth, piglets are exposed to new antigens through maternal colostrums/milk as well as exogenous microbiota. This exposure to new antigens is critical for the proper development of the gut mucosal immune system and is characterized mainly by the establishment of IgA response. A second critical period for neonatal intestinal immune system development occurs at weaning time when the gut environment is exposed to new dietary antigens. Neonate needs to establish oral tolerance and in the absence of protective milk need to fight potential new pathogens.     

Effect of Oral Vs. Parenteral Cyclophosphamide on In Vitro Iga and Igg Production by Murine Peyer's Patches and Cultured Jejunal Fragments

The gut associated lymphoid tissue plays an important role in intestinal defenses, food allergy, oral tolerance, and certain intestinal diseases. This study describes the effect of either oral or parenteral cyclophosphamide on IgA and IgG production in the gut. Mice were treated with cyclophosphamide either IV or PO, and Peyer's patch cell cultures were established to evaluate mitogen induced production of IgA and IgG. To evaluate the effect of cyclophosphamide on the plasma cell rich lamina propria, segments of jejunum were cultured and overnight secretion of IgG and IgA were measured. We found, the secretion of IgA or IgG by jejunal fragments was not influenced by cyclophosphamide (IV or PO). Mitogen induced secretion of IgA and IgG by Peyer's patch cells was markedly decreased 24 hrs after drug administration, with significant recovery by day 7. Cell mixing experiments revealed that a single dose cyclophosphamide reduced the capacity of Peyer's patch B cells to secrete IgA or IgG when co cultured with normal T cells. This study demonstrates that a single dose cyclophosphamide can have profound effects on the gut immune system and that the drug has a similar effect when given either orally or parenterally.     

A comparative study of gut‐associated lymphoid tissue in calf and chicken

The calf contains two types of Peyer's patches (PPs): jejunal and ileal. The ileal PP has been thought to be equivalent to the bursa of Fabricius (BF) as a central lymphoid organ. The morphologies of ileal and jejunal PPs in the calf were compared with those of the BF and the caecal tonsil (CT) in the chicken. Immunoglobulin G–positive (IgG⁺) cells appear in the follicles of them all and exhibited a dendritic appearance after birth. We investigated whether the IgG in these follicles was produced in situ. IgG‐producing cells were detected in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. CD4⁺ cells were distributed in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. The data suggest that Ig class switching occurs in both jejunal PP follicles and CT follicles, but does not occur in either the ileal PP follicles or the bursal follicles. Because CD4⁺ T cells would be prerequisite for Ig class switching in these follicles, IgG⁺ cells of the follicular medullas in the ileal PP and the BF would trap immune complexes from the gut lumen. The primary B‐cell repertoire might be selected by gut‐derived antigens in the ileal PP and the BF before seeding the periphery. Anat Rec 266:207–217, 2002. © 2002 Wiley‐Liss, Inc.     

Helicobacter pylori infections in IgA deficiency: lack of role for the secretory immune system

During the last years considerable attention has been focused on the possibility of the development of an oral vaccine against Helicobacter pylori. However, Helicobacter infection is known to be life-long, despite a vigorous immune response, and the hypothesis that an increased local production of secretory IgA in the gastric mucosa, due to vaccination, should protect against the colonization of the bacterium may therefore be questioned. In this study, when comparing the seropositivity and titre against H. pylori in IgA-deficient patients and age-related normal blood donors, it appears that lack of secretory IgA does not seem to have any major influence on the prevalence of the infection, nor is it reflected in titres of specific IgG antibodies. These results may argue against a pivotal role for IgA in the defence against Helicobacter, and raise questions about current strategies for the development of an oral vaccine against H. pylori and may point to a need for alternative therapeutic strategies.     

Deletion of the α immunoglobulin chain membrane-anchoring region reduces but does not abolish IgA secretion

Class switching and plasma cell differentiation occur at a high level within all mucosa-associated lymphoid tissues. The different classes of membrane immunoglobulin heavy chains are associated with the Igα/Igβ heterodimer within the B-cell receptor (BCR). Whether BCR isotypes convey specific signals adapted to the corresponding differentiation stages remains debated but IgG and IgA membranes have been suggested to promote plasma cell differentiation. We investigated the impact of blocking expression of the IgA-class BCR through a 'αΔtail' targeted mutation, deleting the Cα immunoglobulin gene membrane exon. This allowed us to evaluate to what extent class switching and plasma cell differentiation can be concurrent processes, allowing some αΔtail(+/+) B cells with an IgM BCR to directly differentiate into IgA plasma cells and yield serum secreted IgA in spite of the absence of membrane IgA(+) B lymphocytes. By contrast, in secretions the secretory IgA was very low, indicating that J-chain-positive plasma cells producing secretory IgA overwhelmingly differentiate from previously class-switched membrane IgA(+) memory B cells. In addition, although mucosa-associated lymphoid tissues are a major site for plasma cell accumulation, αΔtail(+/+) mice showed that the gut B-cell lineage homeostasis is not polarized toward plasma cell differentiation through a specific influence of the membrane IgA BCR.