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International Immunology Advance Access originally published online on January 24, 2007
International Immunology 2007 19(3):257-265; doi:10.1093/intimm/dxl142
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Secretory antibodies reduce systemic antibody responses against the gastrointestinal commensal flora

Leanne C. Sait1, Maja Galic1,2, Jason D. Price1,2,3, Kim R. Simpfendorfer1,2,3, Dimitri A. Diavatopoulos1, Tania K. Uren1,2, Peter H. Janssen1, Odilia L. C. Wijburg1,2,3 and Richard A. Strugnell1,2,3

1 Department of Microbiology and Immunology
2 Cooperative Research Center for Vaccine Technology
3 Australian Bacterial Pathogenesis Program, University of Melbourne, Victoria 3010, Australia

Correspondence to: O. L. C. Wijburg; E-mail: odilia{at}unimelb.edu.au


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The humoral response to the gastrointestinal (GI) flora was analyzed in secretory Ig (sIg)-deficient polymeric IgR (pIgR)–/– mice and otherwise congenic C57BL/6 mice. While both strains carried an ileal flora of similar size and composition, increased bacterial translocation to mesenteric lymph node was demonstrated in pIgR–/– mice. Serum IgA was greatly increased in pIgR–/– mice compared with C57BL/6 mice and reacted with commensal organisms and food. Serum IgG levels in pIgR–/– mice were increased to 6-fold above that of C57BL/6 mice and included specificities that bound to selected flora antigens. The enhanced recognition of flora antigens in pIgR–/– mice was explored using ovalbumin (OVA)-specific CD4+ T cells and feeding of low concentrations of OVA. Increased proliferation of transgenic T cells was observed in pIgR–/– mice, relative to C57BL/6 mice, suggesting elevated net uptake of protein antigens from the GI tract in the absence of sIg. These studies suggest that there is increased recognition of GI flora antigens by systemic antibodies in pIgR–/– mice, most probably as a result of increased access of antigens from the GI flora to the systemic immune compartment, and support the hypothesis that a major function of the secretory immune system is to return environmental antigens to mucosal surfaces.

Keywords: antibodies, bacterial, mucosa, rodent


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The mammalian fetus is sterile in utero. Following birth, gastrointestinal (GI) surfaces exposed to the environment are rapidly colonized by successive waves of micro-organisms (1). This flora, together with other environmental antigens, stimulates the development of the gut-associated lymphoid tissue (GALT) (2–6). The GALT, in turn, produces effector responses that protect the gut mucosa from infection. In order to maintain homeostasis in the GI tract, immune responses in the GALT must therefore distinguish between the large antigenic load of commensal micro-organisms, food-derived peptides and other environmental antigens and pathogens.

A prominent feature of the mucosal immune system is the secretion of Ig; an estimated 3 g is secreted into the human GI tract each day (7). The majority of secretory Ig (sIg) is IgA which, along with lower amounts of IgM, is secreted across the GI epithelium by the polymeric IgR (pIgR) (8–10). The large amount of Ig secreted each day, especially antigen-specific sIg, is postulated to play an important role in protection against pathogens at mucosal surfaces (8), but recent studies using sIg-deficient mice have failed to support this fundamental immunological theory (11–15). However, an important role for natural sIg in protection against bacterial infection and prevention of spread of pathogens was demonstrated recently (16). The development of the GALT, and up-regulation of sIg secretion following interaction with the GI flora, has fed the hypotheses that sIg also functions to model the size of the GI flora colonizing the mucosae, and/or limit the uptake of gut-derived antigens across the mucosa (7, 17). The observations that excreted bacteria in the fecal flora are coated with sIg (18), that sIg is induced following the bacterial colonization of the GI tract (3) and that production of sIg coincides with the halt of epithelial translocation by commensal organisms (19), all support these hypotheses. However, a previous study by our group could not detect any changes in the composition of the GI microbiota of mice that were unable to secrete Ig (20).

Lamm et al. have suggested that IgA secretion through the pIgR may expel antigens that have crossed the mucosal surface into the lamina propria (21–23). This process would prevent the generation of damaging inflammatory immune responses in genetically susceptible individuals and could explain the link between IgA deficiency and the increased risk for developing autoimmunity. This study used pIgR–/– mice, which do not secrete Ig (20, 24–26), to examine the effect of sIg on the systemic immune recognition of the GI flora.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
The generation and characterization of C57BL/6–pIgR–/– (referred to as pIgR–/–) mice have been described previously (20, 26). C57BL/6 and pIgR–/– mice were bred in the Department of Microbiology and Immunology at the University of Melbourne, Australia. OT-II transgenic mice (27) were obtained from F. Carbone (University of Melbourne). Female 6-week-old C57BL/6 and pIgR–/– mice were co-housed under conventional conditions with free access to gamma-irradiated mouse food and sterilized tap water for 4 weeks prior to analyses (20). Mice were euthanized by CO2 asphyxiation. Animal experiments were approved by the University of Melbourne Animal Experimentation Ethics Committee.

Cultivation experiments
Following euthanasia of mice, a 1-cm-long section of terminal ileum, 2 cm from the ileo–cecal junction, was removed immediately using a sterile scalpel blade. The section was opened and luminal debris was removed by four successive washes in 1 ml of PBS. Adherent bacteria were removed from the intestinal wall by homogenizing for 20 s at 2500 r.p.m. in 1 ml of PBS using a mini-beadbeater (Biospec, Bartlesville, OK, USA). The supernatant, containing bacterial cells, was collected. Serial 1/10 dilutions of the supernatant were made in PBS, and each dilution was plated onto microbiological media by spotting 20 µl in five replicates. Homogenates from mesenteric lymph node (MLN) were prepared by homogenizing MLN, removed from animals using aseptic techniques, in 3 ml sterile PBS. The enriched media horse blood agar (HBA), HBA supplemented with gentian violet and sodium pyruvate (for the isolation of fastidious organisms and streptococci), nutrient agar and eosin methylene blue (EMB) agar (differentiation of Escherichia coli) were purchased from the departmental Media Preparation Unit. de Man–Rogosa–Sharpe (MRS) medium (Lactobacillus spp.) was prepared according to the method of de Man (24). MacConkey (MAC) medium (Enterobacteriaceae) was made using MAC agar base (Life Technologies GIBCO BRL, Melbourne, Australia), brain–heart infusion (BHI) broth was made using BHI base (Oxoid, Basingstoke, Hampshire, UK) and nutrient broth (NB) was made using NB base (GIBCO BRL). Anaerobic conditions were generated using a CO2-producing BBL GasPak plus (Becton Dickinson, Sparks, MD, USA), and where stated was used in conjugation with a gas phase of 100% N2. Following incubation at 37°C, colonies were counted after 24 h (aerobic incubations) or 3 days (anaerobic incubations) and results are expressed as the mean number of colony-forming unit (CFU) per centimeter ileum.

Preparation of bacterial sonicates
A single colony from a 3-day-old pure culture of Lactobacillus sp. (isolate MRSB) or Lactobacillus sp. (isolate LBB) was inoculated into 250 ml MRS broth containing 5 mM glucose. Cultures were incubated anaerobically with nitrogen, under static conditions, for 3 days. A single colony from a 3-day-old pure culture of the Eubacterium-like organism (isolate G12) was inoculated into 250 ml BHI broth [Oxoid, containing 10% FBS (CSL, Australia)]. Liquid medium was pre-reduced for 48 h using a CO2-producing GasPak. Cultures were grown statically for 4 days under anaerobic conditions. A single colony from a 3-day-old pure culture of Bacteroides sp. strain ASF519 was inoculated into a 10-ml BHI broth, and incubated anaerobically for 2 days. Five milliliters of this culture was inoculated into 250 ml BHI broth which had been pre-reduced for 48 h using a CO2-producing GasPak. Cultures were incubated under anaerobic conditions, statically, overnight. A single colony from 1-day-old pure culture of Proteus sp. (isolate G10) was inoculated into 250 ml of NB and grown statically for 2 days, aerobically. A single colony from 1-day-old pure culture of Klebsiella sp. (isolate G6) was inoculated into 250 ml of NB and grown statically under anaerobic conditions in the presence of nitrogen for 3 days.

Following cultivation, cultures were examined by phase-contrast microscopy, and also by determination of 16S rRNA gene sequences to determine or confirm the identity of the isolates as described previously (28). The GenBank nucleotide accession numbers of this gene for Lactobacillus sp. (isolate MRSB), Lactobacillus sp. (isolate LBB), Eubacterium-like organism (isolate G12), Proteus sp. (isolate G10) and Klebsiella sp. (isolate G6) are AY498750 [GenBank] , AY498749 [GenBank] , AY498748 [GenBank] , AY498747 [GenBank] and AY498746 [GenBank] , respectively. Bacterial cells were pelleted (8000 x g, 15 min) and washed twice with sterile PBS, pelleted again and stored at –70°C for further use. When required, bacterial pellets were thawed and re-suspended in 300 µl PBS. Cells were disrupted by sonication for 5 min (gram-positive strains) or for 3 min (gram-negative strains) in 20-s bursts while maintained on ice.

Preparation of food antigen
Gamma-irradiated mouse food (165 g) (Barastoc, Australia) was soaked in 600 ml sterile H2O for 24 h at 4°C. The insoluble fraction was allowed to settle and the supernatant removed. The insoluble fraction was pelleted by centrifugation (10 min at 6000 x g) and the soluble protein in the supernatant was quantified using a method based on that described by Bradford et al. (29) and stored at –20°C until further use.

Immunizations
Mice were fed ovalbumin (OVA) (Sigma) in 200 µl PBS with a 4-cm gastric gavage needle following inhalation anesthesia with Penthrane (Abbot Laboratories, North Chicago, IL, USA).

ELISA
ELISA was conducted as described in detail previously (15, 26), using 96-well Maxisorp immunoplates (Nunc A/S, Kastrup, Denmark) coated overnight at 4°C with 1 µg of bacterial sonicate or food antigen, or with unconjugated anti-mouse IgG or IgA (5 µg ml–1) (Sigma), in 50 µl in PBS. Mean and SEM values were calculated from the individual titers which were calculated from an optical density value of 3-fold above the control sample to which serum was not added.

Enzyme-linked immunosorbent spot assay
The number of antibody-secreting cell (ASC) present in lymphoid tissues was determined with the enzyme-linked immunosorbent spot (ELISPOT) technique as described in detail previously (26).

Flow cytometry
Single-cell suspensions were surface stained according to standard protocols (26). All samples were acquired and analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA, USA) using the CellQuest software program. Collected events were gated for total lymphocytes defined by forward and side scatter characteristics, and non-viable cells were excluded from analysis based on labeling with propidium iodide (PI), which was added to the samples immediately prior to acquisition.

In vivo proliferation assay
Peripheral lymph nodes were removed from OT-II mice that were the same age and sex as recipient mice. Single-cell suspensions were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) as described (30). One day after adoptive transfer of 107 CFSE-labeled OT-II lymphocytes, mice were orally administered with indicated amounts of OVA. Three days later, recipient mice were culled and single-cell suspensions from the MLN were labeled with an anti-CD4–PE antibody (PharMingen, San Diego, CA, USA) and PI. CD4-positive, PI-negative cells (1 x 106) were collected by FACS and then the number and level of fluorescence of CFSE-labeled OT-II cells were analyzed.

Statistical analysis
The non-parametric Mann–Whitney U-test was used for statistical analysis of the results. Differences were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Serum Ig phenotype of 10-week-old C57BL/6 and pIgR–/– mice
The amount of IgG and IgA in the sera of 10-week-old C57BL/6 and pIgR–/– mice was examined, as elevated levels of non-secreted isotypes might suggest increased activation of B cells. As reported previously (24–26), IgA was significantly (P < 0.001) increased in the serum of pIgR–/– mice (mean ± SD, 25 200 ± 14 300 µg ml–1 in pIgR–/– and 251 ± 106 µg ml–1 in C57BL/6 mice) (Fig. 1A). Serum IgG was also significantly (P < 0.05) elevated in the pIgR–/– mice (38 400 ± 18 500 µg ml–1 in pIgR–/– and 11 700 ± 6900 µg ml–1 in C57BL/6 mice) (Fig. 1A).


Figure 1
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  		Fig. 1. (A) Total IgA and (B) IgG levels in serum of 10-week-old C57BL/6 (filled circles) and pIgR–/– (open circles) mice. The mean value for each group is represented by a solid line. (B) The percentage of B220+ cells present in naive C57BL/6 (filled columns, n = 5) and pIgR–/– mice (open columns, n = 5) was analyzed by flow cytometry. (C and D) Viable cells isolated from PP, MLN, spleen (SPL) and bone marrow (BM) of naive C57BL/6 (filled columns) and pIgR–/– mice (open columns) were assayed for IgA (C) or IgG-secreting cells (D) by ELISPOT. Shown are the results (mean ± SD) obtained in one out of three independently performed experiments with five to six animals per strain. (E) The results of ELISPOT assays are expressed as the ratio of the number of IgA (open columns) and IgG (hatched columns) ASC detected in pIgR–/– mice to the number in C57BL/6 mice. Each symbol in the graph represents the mean ratio from one out of six independently performed experiments (one out of three experiments for bone marrow analyses) in which three to six animals of each strain were used. The column represents the mean of the six experiments.

 
Flow cytometry was used to investigate whether the increased antibody levels were due to alterations in B cells numbers. Single-cell suspensions from Peyer's patch (PP), MLN and spleen were labeled with an anti-B220 antibody: there was no difference in the number of B220+ B cells in C57BL/6 mice compared with pIgR–/– mice (Fig. 1B). Furthermore, experiments in which in vivo bromodeoxyuridine incorporation was measured using flow cytometry failed to demonstrate a difference between C57BL/6 and pIgR–/– mice in the number of actively proliferating B cells (results not shown).

The number and location of IgA- or IgG-producing plasma cells were next investigated using ELISPOT method. Figure 1(C) shows that, in comparison with C57BL/6 mice, pIgR–/– mice had significantly (P < 0.01) elevated numbers of IgA-secreting cells per 106 cells in the PP, MLN and spleen and slightly increased numbers in the bone marrow. The number of IgG ASC was significantly (P < 0.01) increased in the PP of pIgR–/– mice compared with C57BL/6 mice, but not in the other lymphoid compartments (Fig. 1D). Since no difference was detected in the total number of cells per lymphoid organ between C57BL/6 mice and pIgR–/– mice (data not shown), these results indicate that in pIgR–/– mice, the absolute number of ASC is increased compared with C57BL/6 mice (data not shown).

To enable comparison of data obtained in six independently performed experiments to distinguish between C57BL/6 and pIgR–/– mice, the results are expressed as a ratio of number of ACS in pIgR–/–:C57BL/6 mice in Fig. 1(E). Although there is some variation between the experiments, the results showed a 1.5- to 2-fold increase in the number of IgA-producing cells in the MLN and PP of pIgR–/– mice compared with C57BL/6 mice, a more considerable 4-fold increase in the number of IgA-producing cells in the spleen but no difference in the number of IgA ASC in the bone marrow of pIgR–/– mice compared with C57BL/6 mice. The number of IgG ASC was increased 3.2-fold in the PP but not in the MLN, spleen or bone marrow of pIgR–/– mice compared with C57BL/6 mice.

GI flora of 10-week-old C57BL/6 and pIgR–/– mice
The ileal flora was examined to confirm that pIgR–/– mice had a similar GI flora to wild-type mice. There was no major difference in the size of the culturable fraction of the flora, using six different sets of media and cultivation conditions (Fig. 2A). A large variation in the number of CFU within both the C57BL/6 and pIgR–/– mouse phenotypes was observed. This study revealed that some animals carried no ileal bacteria that were cultured on any of the media used. Figure 2(B) shows the variation in the number of CFU recovered from the ilea of C57BL/6 and pIgR–/– mice on the medium MRS, which is semi-selective for Lactobacilli. The variation in CFU on the other media was similar (data not shown).


Figure 2
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  		Fig. 2. Estimation of the size of the ileal microbiota in 10-week-old C57BL/6 and pIgR–/– mice. (A) The ileal microbiota was cultivated from C57BL/6 (filled circles) and pIgR–/– (open circles) mice on the microbiological media MAC, EMB, MRS, HBA and HBA (gentian violet and sodium pyruvate). The number of animals used in each experiment is indicated above each bar. (B) The number of CFU per centimeter ileum in individual C57BL/6 (filled circles) and pIgR–/– (open circles) mice, cultivated on the medium MRS. (C) The number of CFU in MLN homogenates in individual C57BL/6 (filled circles) and pIgR–/– (open circles) mice cultivated on HBA.

 
Increased bacterial translocation to MLN in pIgR–/– mice
To further investigate whether the lack of sIg in pIgR–/– mice results in increased translocation of bacteria across the epithelium, viable counts of homogenates of MLN of C57BL/6 and pIgR–/– mice were obtained after a 3-day culture on HBA under anaerobic conditions. The results in Fig. 2(C) show that increased numbers of bacteria were detectable in the MLN of pIgR–/– mice compared with C57BL/6 mice (P < 0.05).

Identification of the dominant members of the commensal flora
Bacterial colonies were selected from different media and pure cultures derived by subculturing these on fresh plates of the same media. Twenty-three isolates were identified by comparative analysis of partial 16S rRNA gene sequences. The isolates derived from media that yielded high mean culturable numbers were members of the genera Pasteurella (two isolates), Lactobacillus (12 isolates), Klebsiella (one isolate), Staphylococcus (one isolate) and Enterococcus (one isolate), and also a member of a Eubacterium-like group (one isolate). The isolates derived from media that yielded lower mean culturable numbers, EMB and MAC, which favor the growth of members of the family Enterobacteriaceae, were members of the genera Escherichia (three isolates), Klebsiella (one isolate) and Proteus (one isolate).

Reactivity of serum Ig with the GI flora
The elevated dimeric serum IgA in pIgR–/– mice (26) is thought to be an antibody that would be destined for secretion if the pIgR was intact and therefore is representative of the specificity of sIg at mucosal sites. The reactivity of serum IgA in C57BL/6 and pIgR–/– mice with dominant members of the commensal flora as well as food was measured by ELISA. The pIgR–/– mice showed binding of serum IgA to all the antigens that were tested (Fig. 3A). In contrast, all serum IgA titers to the tested antigens in C57BL/6 mice were below the detection limit. Some pIgR–/– mice exhibited elevated serum IgA titers specific for all the commensal strains tested, suggesting that some pIgR–/– mice have increased responsiveness to the GI flora relative to other pIgR–/– mice (data not shown). These data indicate that the high molecular weight serum IgA circulating in pIgR–/– mice can bind to food and bacterial antigens.


Figure 3
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  		Fig. 3. Reactivity of serum Ig in C57BL/6 and pIgR–/– mice against selected antigens. (A) Serum IgA titers against Bacteroides sp. (ASF 519), Klebsiella sp. (isolate G6), Eubacterium-like organism (isolate G12), Lactobacillus sp. (isolate LBB), Lactobacillus sp. (isolate MRSB), Proteus sp. (isolate G10) and soluble food antigen in C57BL/6 (filled circles) and pIgR–/– (open circles) mice were determined by ELISA. (B) The serum IgG titers against the same set of antigens. The value of each individual animal is represented by the symbols, the mean value for each group is represented by a solid line and the detection limit of the assay is indicated by the dashed line. **P < 0.05.

 
As immune responses to antigen can result in increased levels of antigen-specific IgG, the reactivity of IgG to antigens from the GI flora and food in pIgR–/– and C57BL/6 mice was examined. An increase in antigen-specific serum IgG was measured in pIgR–/– mice relative to the C57BL/6 mice (Fig. 3B). This increase was reflected in the overall response to each of the antigens tested, where pIgR–/– mice had increased mean IgG titers against each antigen, and was statistically significant (P < 0.001) for the antigen preparations from Klebsiella sp. (isolate G6), Lactobacillus sp. (isolate LBB) and Lactobacillus sp. (isolate MRSB).

Increased GI uptake of protein antigens
Previous studies of pIgR–/– mice have suggested an increased ‘leakiness’ in the GI tract, which resulted in the presence of increased levels of systemic proteins, such as IgG, in the GI tract lumen (24, 25). In this study, the translocation of protein from the GI lumen into the systemic compartment and the resulting induction of immune responses were measured using an in vivo T cell proliferation assay modified by the administration of a limiting amount of antigen. The amount of the protein antigen OVA able to stimulate adoptively transferred, CFSE-labeled OVA-specific TCR transgenic T cells (OT-II) in the MLN was titrated. No differences were observed in distribution of OT-II cells in recipient pIgR–/– and C57BL/6 mice, and the absolute numbers of cells in each tissue was equivalent between the two strains of mice. Relatively high (i.e. >5 mg) oral doses of OVA resulted in proliferation of OT-II cells in mice of both phenotypes whereas oral doses of 1 or 2 mg of OVA stimulated proliferation of OT-II cells in the MLN of pIgR–/– but not in C57BL/6 mice (Fig. 4A). OVA-specific proliferation was analyzed further by comparing the number of divided OT-II cells as a proportion of the total OT-II cells present in the cell suspensions (divided + undivided). Proliferation of OT-II cells in the spleen was only observed after feeding a high dose (10 mg) of OVA (Fig. 4B). The percentage of proliferating cells was higher (P = 0.04) in spleens of pIgR–/– mice (58–98%, mean 76.6%) compared with C57BL/6 mice (43–63%, mean 54.6%). In the MLN, no difference in proliferation of OT-II cells in C57BL/6 and pIgR–/– mice was observed after feeding 10 mg OVA (Fig. 4C).


Figure 4
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  		Fig. 4. Increased proliferation of adoptively transferred OT-II cells following feeding of OVA in the MLN of pIgR–/– mice. (A) Populations of CFSE-labeled, adoptively transferred OT-II cells in the MLN of C57BL/6 and pIgR–/– mice 3 days after feeding 0, 1, 2 or 10 mg of OVA. Proliferating OT-II cells are indicated by the solid line marked as CFSElow. Samples with >10% of OT-II cells in this gate were considered positive. Shown is the result obtained in one representative out of two samples. (B and C) Proliferation of CFSE-labeled OT-II cells in the spleen (B) and MLN (C) of C57BL/6 (filled circles) and pIgR–/– (open circles) mice fed 10 mg of OVA, expressed as the frequency of proliferated OT-II daughter cells (cells within the CFSElow gate) as a percentage of the total CFSE-labeled OT-II cell population in the cell suspensions. (D) Proliferation of CFSE-labeled OT-II cells in the MLN of C57BL/6 (filled circles) and pIgR–/– (open circles) mice fed with 1 mg of OVA. The C57BL/6 (filled triangle) and pIgR–/– (open triangle) mice fed 1 mg of OVA shown in part (A) are also shown, as are C57BL/6 (filled square) and pIgR–/– (open square) mice that were fed no OVA (PBS controls). Symbols represent individual mice, and the dashed line in panels B–D indicates 10% proliferation.

 
Using a small dose of antigen (1 mg OVA), the observed level of proliferation was small but measurable (Fig. 4D). A positive result (>10% of OT-II cells proliferated) was recorded for a greater number of pIgR–/– mice (8/10) compared with C57BL/6 mice (3/9). The number of proliferating OT-II cells was also greater (P < 0.05) in the pIgR–/– mice (6.7–33.9%, mean 19.9%). C57BL/6 mice showed limited proliferation, with only 5.6–12.1% (mean 8.8%) of adoptively transferred OT-II cells proliferating.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The experiments described in this study tested the hypothesis that sIg reduces systemic immune reactivity with the GI flora. In this role, sIg either prevents access of antigens from the GI flora across the mucosal surface of the GI tract or returns translocated antigens from the GI flora back to the gut lumen, acting as an immunological ‘blanket’ and/or ‘antigen sink’ to prevent the generation of immune responses against commensal organisms, or their antigens, which cross the epithelial barrier. Observations that dimeric IgA, complexed with antigen, can be transported across epithelial cell monolayers both in vitro (21) and in vivo (22) support the ‘antigen sink’ hypothesis. While the mechanism by which sIg recycles soluble antigens across the GI epithelium has been established, the relevance of this process to antigens arising from the GI flora is less well defined. A number of experimental observations of the interaction between sIg and the GI flora support the idea that a degree of control of the commensal flora is mediated by sIg (18, 19, 31–33). In particular, the secretion of IgA into the gut lumen of monoassociated mice (i.e. animals carrying a single gut species) correlates with the halt in bacterial translocation into the MLN (19). Furthermore, the secretion of Ig into the GI tract has been proposed as a mechanism via which a ‘suitable’ commensal flora is retained (7, 17).

It is believed that increased levels of serum dIgA in pIgR–/– mice, which is produced by increased numbers of mucosally derived IgA ASCs in the spleen, MLN and mucosa [(26) and this study], would normally be secreted via the pIgR (26). Similarly, serum IgM, which is also secreted via the pIgR, is elevated in pIgR–/– mice (24–26). Serum IgG levels were also elevated in pIgR–/– mice and it has been shown by others that this elevation may increase with age (25). Since IgG is only secreted in minor amounts and via a pIgR-independent mechanism (34), the elevated serum IgG in pIgR–/– mice, produced by increased numbers of IgG ASC in the PP and spleen, is suggestive of increased immunization from ‘environmental’ antigens.

Data presented in a previous study (20) showed that the composition of the flora colonizing the terminal ileum of sIg-deficient pIgR–/– mice was similar to that of C57BL/6 mice, suggesting that sIg does not function to select suitable colonizing organisms. Here, we conclude that sIg does not appear to control the size of the flora, at least of the terminal ileum, although a high variability between animals of the same phenotype was observed. In some animals of either genotype, no ileal flora was cultured. It is likely that the flora of the terminal ileum of these mice was dominated by organisms that would not be cultured using the techniques described in this paper, e.g. strictly anaerobic Bacteroides spp., or so-called unculturable species (35). In contrast to our findings, others have shown that at least in activation-induced cytidine deaminase (AID)–/– mice, which lack hypermutated IgA, the commensal flora is overgrown by segmented filamentous bacteria (36, 37). In those studies, however, the ileal flora from the entire small intestine was obtained by washing the intestinal walls in PBS, whereas in this study, only the adherent bacteria in washed, homogenized terminal ileum were analyzed. Furthermore, it cannot be excluded that differences in housing conditions may have affected the composition and size of the ileal flora. In our studies, pIgR–/– and C57BL/6 mice were mixed and co-housed for 4 weeks before analysis to avoid any skewing in the commensal flora due to housing. Furthermore, Suzuki et al. (36) reported a change in flora composition in 5- and 16-month old AID–/– mice, whereas we analyzed the commensal flora of 10-week-old mice. It will be of great interest to determine whether the commensal flora of pIgR–/– mice alters with aging.

Since sIg does not appear to control either the composition (20) or the size of the ileal flora, any change in the immune recognition of ileal flora antigens is likely due to increased exposure. Indeed, we were able to demonstrate that in pIgR–/– mice, increased numbers of bacteria are translocated to the MLN. Further examination of the reactivity of serum Ig with the GI flora revealed that the increased serum IgA in pIgR–/– mice was reactive with all antigen preparations tested, albeit at differing levels in individual animals. The varied reactivity of salivary sIgA with the oral commensal flora has been demonstrated previously (32) and Ig produced by gut-derived B lymphocytes is specific for commensal bacteria (38, 39). The reactivity of serum IgA of pIgR–/– mice with commensal isolates, compared with that of serum IgA from C57BL/6 mice, supports the hypothesis that the accumulated IgA in the serum of pIgR–/– mice is unsecreted dIgA. Recently, we demonstrated that IgA accumulated in the serum of the pIgR–/– mice reacts with flora antigens in a cross-reactive manner (16), suggesting that the antigen-binding capacity at the mucosal surface of pIgR–/– mice is reduced. This will increase access to the GI epithelium of both flora and food antigens, and/or inhibit their re-translocation back to the lumen after gaining entry across the epithelium.

We further confirmed that there was increased access of GI antigens to the systemic immune system in pIgR–/– mice by demonstrating elevated anti-flora IgG in the serum of pIgR–/– mice. These flora-specific IgG responses were measured against a background of elevated specific serum IgA in the pIgR–/– mice which will compete with IgG for epitope binding in the ELISAs. Assuming the relative affinities of the IgA and IgG antibodies were similar, this competition could serve to significantly decrease the ELISA titers of the IgG antibodies. Therefore, the IgG titers calculated in the pIgR–/– mice are probably significantly underestimated. As each antigen preparation used is a composite of antigens that could be common to many bacterial species, the identity of any individual bacteria that may have gained increased access to the systemic compartment could not be determined from these experiments. Increased serum reactivity to members of the aerobic commensal flora in IgA–/– mice and a strain of E. coli in pIgR–/– mice has also been demonstrated by others (24, 40).

We next obtained direct evidence that increased amounts of antigen from the GI lumen are able to translocate the epithelium in pIgR–/– mice to interact with specific immune cells. Increased proliferation of adoptively transferred OT-II T cells in response to the oral administration of a limiting dose of OVA was observed. The subtlety of the increase in OVA translocation in the pIgR–/– mice is evident in the observations that oral administration of non-limiting OVA doses did not trigger increased OT-II proliferation in MLN from pIgR–/– mice relative to C57BL/6 mice [this study and (26)]. The likely reason for this is that administration of non-limiting OVA doses saturates the proliferation of antigen-specific cells in the PP and MLN, obscuring the difference in antigen uptake between pIgR–/– and C57BL/6 mice. However, in contrast to our previous observations (26), we did observe increased proliferation of OT-II cells in the spleen of pIgR–/– mice compared with C57BL/6 mice, suggesting that increased amounts of OVA may have been translocated beyond the MLN in pIgR–/– mice. On the other hand, the possibility that the increased proliferation of OT-II cells in the spleen is the result of increased migration of antigen-presenting cells from the PP and/or MLN to systemic sites, or increased migration of OT-II cells, activated in the PP and/or MLN, cannot be excluded. The discrepancy between our earlier observations and those reported here is most likely due to the amounts of OVA fed to the mice [20 mg in (26) versus 1 mg in this study], but may also be explained by the use in this study of a more sensitive technique to detect OT-II cells undergoing division.

We have recently reported enhanced susceptibility of naive pIgR–/– mice to infection with Salmonella typhimurium via the fecal–oral transmission route (16). However, despite the increased exposure of the immune system to GI luminal antigens in pIgR–/– mice, no signs of inflammation or reduced mucosal tolerance has been observed in these mice (26). In addition, no auto-antibodies were detectable in the serum of either strain of mice (results not shown). It is possible though that the lack of sIg in these mice may impact on the mucosal homeostasis when the mice get older and we are currently investigating this hypothesis. Loss of sIg function can result in the loss of mucosal and systemic homeostasis, evident by the link between IgA deficiency and the increased likelihood of developing autoimmunity (41), inflammatory bowel disease (42, 43) or celiac disease (44). At present, there is no causal relationship between autoimmune disease and IgA deficiency. As with pIgR–/– mice studied here, only a subset of individuals may exhibit the increased antigen translocation through the epithelium that could serve to trigger or drive autoimmune responses. Observations of IgA-deficient humans have demonstrated increased circulation of serum immune complexes containing orally ingested antigen (41) which may contribute to the development of disease. The presence of increased systemic IgG, which had reactivity with the GI flora in pIgR–/– mice, the increased translocation of bacteria to the MLN and the augmented uptake of orally administered antigen, demonstrated that sIg plays a role in the immune exclusion of antigens from the GI flora. Further experiments are required to determine whether the increased translocation of these antigens can contribute to the loss of mucosal or systemic homeostasis.


   Acknowledgements
 
This work was supported by grants from the NHMRC. O.L.C.W. is a C. R. Roper Fellow at the University of Melbourne. The authors would like to thank A/Prof. I. van Driel (Department Biochemistry and Molecular Biology, Bio21 Institute at the University of Melbourne) for analysis of mouse serum for the presence of auto-antibodies.


   Abbreviations
 
AID, activation-induced cytidine deaminase
ASC, antibody-secreting cell
BHI, brain–heart infusion
CFSE, carboxyfluorescein diacetate succinimidyl ester
CFU, colony-forming unit
ELISPOT, enzyme-linked immunosorbent spot
EMB, eosin methylene blue
GALT, gastric-associated lymphoid tissue
GI, gastrointestinal
HBA, horse blood agar
MAC, MacConkey
MLN, mesenteric lymph node
MRS, de Man–Rogosa–Sharpe
NB, nutrient broth
OVA, ovalbumin
PI, propidium iodide
pIgR, polymeric IgR
PP, Peyer's patch
sIg, secretory Ig

   Notes
 
Transmitting editor: D. Tarlinton

Received 4 July 2006, accepted 13 December 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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