B1 cells contribute to serum IgM,but not to intestinal IgA,production in gnotobiotic Ig allotype chimeric mice

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer's patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.     

Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy

The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels, renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.     

Migration of antigen-presenting B cells from peripheral to mucosal lymphoid tissues may induce intestinal antigen-specific IgA following parenteral immunization.

Parenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymphocytes. We have now used adoptive transfer studies to identify the cell types responsible for the generation of virus-specific Ab production by gut-associated lymphoid tissue (GALT) after i.m. immunization. Three days after i.m. immunization with rotavirus, cells obtained from the draining peripheral lymph nodes of donor mice were transferred into naive recipient mice. We found that intestinal lymphocytes produced rotavirus-specific Igs (IgM, IgA, and IgG) 2 wk after transfer of either unfractionated cells, or unfractionated cells rendered incapable of cellular division by mitomycin C treatment. Additional studies demonstrated that rotavirus-specific IgA, but not IgG, was produced by intestinal lymphocytes after transfer of purified B cells. Ig allotype analysis revealed that rotavirus-specific IgA was produced by intestinal B cells of recipient origin, suggesting that migration of Ag-presenting B cells from peripheral lymphoid tissues to GALT may contribute to the generation of mucosal IgA responses after parenteral immunization. Strategies that promote Ag uptake and presentation by B cells may enhance mucosal IgA production following parenteral immunization.     

Lymphotoxin Alpha-Deficient Mice Clear Persistent Rotavirus Infection after Local Generation of Mucosal IgA

Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα^−/−) mice that lack Peyer''s patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα^−/− and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα^−/− mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα^−/− mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.     

In vitro regulation of IgA subclass production. III. Selective transformation of IgA1 producing cells by Epstein-Barr virus

In past experiments, using limited dilution analysis, we have demonstrated that a high percentage of immunoglobulin-secreting clones derived from Epstein-Barr virus- (EBV) stimulated lymphocytes secrete IgA. To further characterize the IgA produced by these clones, the IgA subclass of supernatants from clones stimulated 4 to 6 wk previously with EBV was determined by radioimmunoassay. All of 17 IgA-producing clones secreted IgA1; none secreted IgA2. Because we have shown that surface IgM+ (sIgM+) B cells are an enriched source of IgA2 plasma cell precursors, panning techniques were used to purify sIgM+ B cells from tonsils. Of 103 clones derived from these sIgM+ B cells, 102 secreted IgA1 and only one secreted IgA2. The relative absence of IgA2-producing clones could not be attributed to an absence of EBV receptors on IgA2 cells. A mean of 84 +/- 4% of freshly isolated IgA2 B cells and 78 +/- 6% of IgA1 B cells could be stained with a monoclonal antibody binding the EBV receptor; and there was no failure of EBV to infect IgA2 plasma cells precursors. Of IgA2 plasma cells derived from peripheral blood lymphocytes stimulated 7 days previously with EBV, 54 +/- 7% were positive for the EBV nuclear antigen, compared with 54 +/- 18% of IgA1 plasma cells from the same cultures. Seven days after EBV stimulation, a mean of 25% of the total IgA plasma cells were positive for cytoplasmic IgA2, whereas by 21 days after stimulation only 7% were positive for IgA2. This shift in the proportions of IgA1 and IgA2 plasma cells could be attributed to a failure of the IgA2 plasma cell number to increase after 10 days in culture. There was no evidence for selective suppression of IgA2 production by T cells or selective lysis of IgA2 plasma cells by infectious EBV particles. These results demonstrate that although precursors for both IgA1- and IgA2-producing cells can be stimulated to differentiate in response to EBV, there is preferential transformation of IgA1-producing cells.     

Protective intestinal anti-rotavirus B cell immunity is dependent on alpha 4 beta 7 integrin expression but does not require IgA antibody production

Rotavirus (RV) is the main cause of severe gastroenteritis in young children; protection has been correlated with intestinal Ab responses. Using a mouse model of RV infection and beta(7)-deficient (beta(7)(-/-)) mice, which do not express alpha(4)beta(7) integrin, we demonstrated the importance of alpha(4)beta(7) integrin in B cell-mediated anti-RV immunity. beta(7)(-/-) mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. alpha(4)beta(7)(-/-) immune B cells did not resolve RV infection when adoptively transferred into RV-infected Rag-2-deficient mice. Fewer RV-specific B cells were found in the intestine of Rag-2-deficient mice transferred with beta(7)(-/-) B cells compared with wild type. The absence of alpha(4)beta(7) expression and/or a lower frequency of IgA-producing cells among transferred beta(7)(-/-) B cells could have accounted for the inability of these cells to resolve RV infection following passive transfer. To distinguish between these possibilities, we studied the importance of IgA production in RV infection using IgA-deficient (IgA(-/-)) mice. IgA(-/-) mice depleted of CD8(+) T cells were able to clear primary RV infection. Similarly, adoptive transfer of immune IgA(-/-) B cells into chronically infected Rag-2-deficient mice resolved RV infection. We further demonstrated in both wild-type and IgA(-/-) mice that, following oral RV infection, protective B cells reside in the alpha(4)beta(7)(high) population. Our findings suggest that alpha(4)beta(7) integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA.     

Targeting of secretory IgA to Peyer's patch dendritic and T cells after transport by intestinal M cells

In addition to being instrumental to the protection of mucosal epithelia, secretory IgA (SIgA) adheres to and is transported by intestinal Peyer's patch (PP) M cells. The possible functional reason for this transport is unknown. We have thus examined in mice the outcome of SIgA delivered from the intestinal lumen to the cells present in the underlying organized mucosa-associated lymphoreticular tissue. We show selective association of SIgA with dendritic cells and CD4(+) T and B lymphocytes recovered from PP in vitro. In vivo, exogenously delivered SIgA is able to enter into multiple PP lining the intestine. In PP, SIgA associates with and is internalized by dendritic cells in the subepithelial dome region, whereas the interaction with CD4(+) T cells is limited to surface binding. Interaction between cells and SIgA is mediated by the IgA moiety and occurs for polymeric and monomeric molecular forms. Thus, although immune exclusion represents the main function of SIgA, transport of the Ab by M cells might promote Ag sampling under neutralizing conditions essential to the homeostasis of mucosal surfaces.     

IgA plasma cells express the negative regulatory co-stimulatory molecule programmed cell death 1 ligand and have a potential tolerogenic role in the intestine

To maintain immune homeostasis in the intestine, the intestinal immune system has evolved several tolerogenic mechanisms toward intestinal microflora and food antigens. Although programmed cell death-1 (PD-1) protein has been implicated in immunological tolerance in the intestine and gut-associated lymphoid tissues (GALTs), distribution of its ligands PD-L1 and PD-L2 in the small intestine lamina propria (LP) are unknown. We investigated PD-L1 expression in intestinal LP and found that IgA plasma cells (PCs) were major PD-L1 expressing cells. PD-L1 expression levels on IgA PCs were higher than that on IgG PCs in peripheral lymphoid tissues. IgA PCs expressed antigen-presenting molecule MHC class II and co-stimulatory molecules CD80, CD86, and PD-L2. IgA PCs isolated from intestinal LP exhibited antigen presentation activity, and in the presence of TGF-β induced FoxP3⁺ regulatory T cells, but not IFN-γ⁺ Th1 cells, from naïve T cells. Thus, IgA PCs in the intestine may be involved in an immune regulatory role in the intestinal immune system.     

Downregulated expression in high IgA (HIGA) mice and the renal protective role of meprinbeta

This study discusses the critical role of the metalloproteinase meprinbeta in the progression of glomerulonephritis. Using a microarray technique, the gene expression profiles in glomeruli isolated from high serum IgA (HIGA) mice with a purity of 97% or greater were examined. HIGA mice are a valid model of human IgA nephropathy (IgAN), with the typical pathological features of this condition, including a consistently high serum IgA level as well as dominant mesangial IgA deposition and mesangial enlargement. Among the many upregulated/downregulated genes after the development of IgAN, the downregulation of meprinbeta was intriguing. The expression level of the meprinbeta gene at 40 weeks of age was 52% of that observed at 8 weeks of age (prior to the development of IgAN), although in the control BALB/c mice, a 2.19-fold elevation was seen. These results were also confirmed by semi-quantitative RT-PCR and immunostaining analyses. As meprinbeta is a subunit of metalloproteinase meprins (meprin A, meprin B) and meprins are capable of proteolytically degrading extracellular matrix (ECM) components and proteolytically processing bioactive peptides, the downregulation of meprinbeta may contribute to the progression of glomerulonephritis and the eventual glomerular scarring. This working hypothesis was examined using an in vivo meprinbeta inhibition study. The inhibition of meprins by actinonin exacerbated some parameters of renal injury in mice afflicted with anti-glomerular basement membrane (anti-GBM) antibody-associated nephritis. These in vitro and in vivo results suggest that meprinbeta may play a protective role against the progression of renal injury through the degradation of ECM and bioactive peptides.     

BAFF induces a hyper-IgA syndrome in the intestinal lamina propria concomitant with IgA deposition in the kidney independent of LIGHT

BAFF is a peripheral B cell survival factor and can mediate antibody (Ab) class switching. Over-expression of BAFF in mice results in B cell hyperplasia, elevated serum immunoglobulin (Ig), spontaneous germinal centre (GC) reactions and mild glomerulonephritis (GN). Here we show that, in addition to driving excessive levels of serum IgA, BAFF over-expression results in increased IgA levels within the intestinal lamina propria (LP) and deposition of IgA immune complexes in the renal glomerular mesangium. LIGHT has been previously shown to mediate a similar phenotype via signaling through the lymphotoxin-beta receptor (LTbetaR). We evaluated if LIGHT and BAFF cooperate in the etiology of a hyper-IgA syndrome in BAFF-overexpressing transgenic (BAFF-Tg) mice. We find that LIGHT-deficient BAFF-Tg mice exhibit similar levels of IgA in the serum, gut and kidney and develop nephritis to the same degree as LIGHT-sufficient BAFF-Tg mice. Therefore, in the context of BAFF over-expression, LIGHT is dispensable for the generation of a hyper-IgA syndrome accompanied by nephritis.     

An important role for polymeric Ig receptor-mediated transport of IgA in protection against Streptococcus pneumoniae nasopharyngeal carriage

The importance of IgA for protection at mucosal surfaces remains unclear, and in fact, it has been reported that IgA-deficient mice have fully functional vaccine-induced immunity against several bacterial and viral pathogens. The role of respiratory Ab in preventing colonization by Streptococcus pneumoniae has now been examined using polymeric IgR knockout (pIgR(-/-)) mice, which lack the ability to actively secrete IgA into the mucosal lumen. Intranasal vaccination with a protein conjugate vaccine elicited serotype-specific anti-capsular polysaccharide Ab locally and systemically, and pIgR(-/-) mice produced levels of total serum Ab after vaccination that were similar to wild-type mice. However, pIgR(-/-) mice had approximately 5-fold more systemic IgA and 6-fold less nasal IgA Ab than wild-type mice due to defective transport into mucosal tissues. Wild-type, but not pIgR(-/-) mice were protected against infection with serotype 14 S. pneumoniae, which causes mucosal colonization but does not induce systemic inflammatory responses in mice. The relative importance of secretory IgA in host defense was further shown by the finding that intranasally vaccinated IgA gene-deficient mice were not protected from colonization. Although secretory IgA was found to be important for protection against nasal carriage, it does not appear to have a crucial role in immunity to systemic pneumococcus infection, because both vaccinated wild-type and pIgR(-/-) mice were fully protected from lethal systemic infection by serotype 3 pneumococci. The results demonstrate the critical role of secretory IgA in protection against pneumococcal nasal colonization and suggest that directed targeting to mucosal tissues will be needed for effective vaccination in humans.     

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.     

Impaired vitamin A-mediated mucosal IgA response in IL-5 receptor-knockout mice.

To clarify actions of vitamin A on mucosal immunity associated with interleukin-5 (IL-5), we examined effects of vitamin A on mucosal IgA level in IL-5 receptor alpha-chain-knockout (IL-5Ralpha(-/-)) mice. Daily supplementation of retinyl acetate (1 mg/mouse) increased Th2 cytokine levels and a number of their positive cells in the small intestinal mucosa of IL-5Ralpha(-/-) mice, as observed in wild-type or IL-5Ralpha(+/-) mice. Wild-type and heterozygous mice increased the IgA level and a number of IgA-containing cells in the mucosa in response to the vitamin A treatment, but not in IL-5Ralpha(-/-) mice. Retinyl acetate increased anti-cholera toxin (CT) IgA level in the mucosa of wild-type mice, improving their survival rate after an exposure to 0.4 mg of CT. However, retinyl acetate failed to induce resistance to CT toxicity in IL-5Ralpha(-/-) mice. Our results suggest that IL-5 may play an important role in an action of vitamin A on mucosal IgA system.     

Sphingosine 1-phosphate regulates the egress of IgA plasmablasts from Peyer's patches for intestinal IgA responses

It is well established that Peyer's patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM(+)B220(+) B cells to IgA(+)B220(+) B cells, but recovered on IgA(+)B220(-) plasmablasts for their emigration from the PPs. Thus, IgA(+)B220(-) plasmablasts migrated in response to S1P in vitro. Additionally, IgA(+) plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA(+) plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.     

IgA response to symbiotic bacteria as a mediator of gut homeostasis

Colonization of germ-free mice with a normal gut microbiota elicits bacteria-specific IgA antibody responses. The effects of these responses on microbial and host biology remain poorly defined. Therefore, we developed a gnotobiotic mouse model where the microbiota is reduced to one bacterial species, and the antibody repertoire to a single, monoclonal IgA against the bacterium's capsular polysaccharide. Bacteroides thetaiotaomicron was introduced into germ-free wild-type, immunodeficient Rag1(-/-), or Rag1(-/-) mice harboring IgA-producing hybridoma cells. Without IgA, B. thetaiotaomicron elicits a more robust innate immune response and reacts to this response by inducing genes that metabolize host oxidative products. IgA reduces intestinal proinflammatory signaling and bacterial epitope expression, thereby balancing suppression of the oxidative burst with the antibody's negative impact on bacterial fitness. These results underscore the adaptive immune system's critical role in establishing a sustainable host-microbial relationship. Immunoselection of bacterial epitope expression may contribute to the remarkable strain-level diversity in this ecosystem.     

Pathogenic Role of a Proliferation-Inducing Ligand (APRIL) in Murine IgA Nephropathy

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) superfamily. Despite advances in clinical and genetic studies, the details of the pathological roles of APRIL in IgA nephropathy (IgAN) remain to be fully defined. The present study aimed to further assess the pathological role of APRIL using a mouse model of IgAN. Mice with IgAN designated “grouped ddY” (gddY) were intraperitoneally administered an anti-APRIL monoclonal antibody (anti-APRIL Ab) or control IgG (Control Ab) twice each week for 2 weeks starting during the early stage of IgAN (6–7 weeks of age). Urinary albumin, serum IgA, and glomerular IgA deposition were evaluated. We further assessed the inflammatory responses during treatment by measuring the levels of the chemokine fractalkine (FKN) and its receptor CX3CR1 as well as the level of peripheral blood monocytosis. Anti-APRIL Ab treatment significantly decreased albuminuria and tissue damage combined with decreases in serum IgA levels and deposition of glomerular IgA. In contrast, the abundance of IgA⁺/B220⁺ or CD138⁺/B220⁺ B cells in the spleen and bone marrow, respectively, was unchanged. Treating gddY mice with anti-April Ab reduced the overexpression of FKN/CX3CR1 in the kidney and the increase in the population of circulating Gr1⁻/CD115⁺ monocytes. The size of the population of Gr1⁻/CD115⁺ monocytes correlated with renal FKN and urinary albumin levels. Moreover, mice treated with anti-APRIL Ab exhibited reduced progression of IgAN, serum IgA levels, and glomerular IgA deposition as well as an attenuated inflammatory process mediated by FKN-associated activation of monocytes. To the best of our knowledge, this is the first study to implicate the APRIL signal transduction pathway in the pathogenesis of nephrogenic IgA production. Moreover, our findings identify APRIL as a potential target of therapy.     

Toll‐like receptor 5 is not essential for the promotion of secretory immunoglobulin A antibody responses to flagellated bacteria

Toll‐like receptor 5 recognizes bacterial flagellin, plays a critical role in innate immunity, and contributes to flagellin‐specific humoral immunity. Further, TLR5‐expressing dendritic cells play an important role in IgA synthesis in the intestine; however, the contribution of TLR5 to antigen (Ag)‐specific mucosal immunity remains unclear. Thus, whether TLR5 is essential for the induction of intestinal secretory (S)IgA antibody (Ab) responses against flagellin and bacterial Ags attached to the bacterial surface in response to an oral flagellated bacterium, Salmonella, was explored in this study. Our results indicate that when TLR5 knockout (TLR5^?/?) mice are orally immunized with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella‐Tox C), tetanus toxoid (TT)‐ and flagellin (FliC)‐specific systemic IgG and intestinal SIgA Abs are elicited. The numbers of TT‐specific IgG Ab‐forming cells (AFCs) in the spleen and IgA AFCs in the lamina propria (LP) of TLR5^?/? mice were comparable to those in wild‐type mice. rSalmonella‐Tox C was equally disseminated in TLR5^?/? mice, TLR5^?/? mice lacking Peyer's patches (PPs), and wild‐type mice. In contrast, TLR5^?/? PP‐null mice failed to induce TT‐ and FliC‐specific SIgA Abs in the intestine and showed significantly reduced numbers of TT‐specific IgA AFCs in the LP. These results suggest that TLR5 is dispensable for the induction of flagellin and surface Ag‐specific systemic and mucosal immunity against oral flagellated bacteria. Rather, pathogen recognition, which occurs in PPs, is a prerequisite for the induction of mucosal immunity against flagellated bacteria.

     

Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.     

Human IgA activates the complement system via the mannan-binding lectin pathway

The recently identified lectin pathway of the complement system, initiated by binding of mannan-binding lectin (MBL) to its ligands, is a key component of innate immunity. MBL-deficient individuals show an increased susceptibility for infections, especially of the mucosal system. We examined whether IgA, an important mediator of mucosal immunity, activates the complement system via the lectin pathway. Our results indicate a dose-dependent binding of MBL to polymeric, but not monomeric IgA coated in microtiter plates. This interaction involves the carbohydrate recognition domain of MBL, because it was calcium dependent and inhibited by mannose and by mAb against this domain of MBL. Binding of MBL to IgA induces complement activation, as demonstrated by a dose-dependent deposition of C4 and C3 upon addition of a complement source. The MBL concentrations required for IgA-induced C4 and C3 activation are well below the normal MBL plasma concentrations. In line with these experiments, serum from individuals having mutations in the MBL gene showed significantly less activation of C4 by IgA and mannan than serum from wild-type individuals. We conclude that MBL binding to IgA results in complement activation, which is proposed to lead to a synergistic action of MBL and IgA in antimicrobial defense. Furthermore, our results may explain glomerular complement deposition in IgA nephropathy.     

Phenotypic Analysis of a Population of IgA+ Cells in the Follicle-Associated Epithelium of Mouse Peyer's Patches

The follicle-associated epithelium (FAE) selectively transports prions, viruses, pathogenic bacteria, commensal microflora, and even secretory IgA (SIgA)-immune complexes from the intestinal lumen to underlying gut-associated lymphoid tissues like Peyer’s patches. The FAE consists of a single layer of columnar epithelial cells that includes enterocytes and M (microfold) cells, intermingled with dendritic cells (DCs), macrophages, and naïve and memory B and T lymphocytes. In this report we describe a population of IgA⁺ cells that reside within and immediately below the FAE in mouse Peyer’s patches. Immunofluorescence microscopy analysis indicated that the FAE-associated IgA⁺ cells were negative for surface antigen markers specific for B cells (B220), T cells (CD3), DCs (CD11c), and plasma cells (CD138). The IgA⁺ cells were also negative Ki-67 and IRF4, indicating that they are not mature B cells or plasma cells. The IgA⁺ cells were, however, often found in close proximity to DCs, leading us to speculate that the population of IgA⁺ cells in the FAE constitutes an atypical subset of B cells involved in mucosal antigen surveillance and/or immune recall.